biotecnologia

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Exploring Environmental Issues:

Introduction: Biotechnology Background Information for Educators and Students

Has biotechnology been a part of your day today? Have you used any organisms in part or whole, alive or dead, to make something that has helped you get through your day? How about a piece of bread, some yogurt, or piece of cheese? All of those food products were created using processes of biotechnology that have been practiced for thousands of years. According to the United Nations Convention on Biological Diversity, biotechnology is defined as Any technological application that uses biological systems, living organisms or derivatives thereof, to make or modify products or processes for specific use.1 The word “biotechnology” usually conjures images of modern techniques and topics of controversy such as cloning, stem cell research, and genetically modified organisms; however, the practice of manipulating organisms to create a product has long been used in human societies.

What are the historical uses of biotechnology? Food has been one of the primary areas where organisms have been put to work to create an edible product for human consumption. Fungi have been injected into “bleu” cheeses to help them develop the tastes and characteristics found in varieties such as roquefort and gorgonzola. Yogurt is a food that contains millions of living bacteria busily fermenting milk, while yeast will do most of the work of making bread, beer, and wine through the anaerobic fermentation of sugar. Waste management is another area of biotechnology that has seen many traditional methods carried into the 21st century nearly intact. It was common practice early in history to allow naturally occurring soil organisms to break down the agricultural waste, sewage, and manure so nutrients would be returned to the ecosystem. Although much more

is now understood about the chemistry and biology of decomposition, modern methods of sewage treatment still use local organisms for reducing waste products to less hazardous or more useful substances. Direct methods of artificial selection, which are considered biotechnological practices, have been used in farming and animal husbandry and involve human intervention in breeding for selection of the most desirable traits while using organisms chosen from the current generation. More than 7,000 years ago, humans cultivated a plant that is the ancestral species to modern day corn. Two thousand years after southern Mexico inhabitants began choosing, eating, and replanting the seeds of the best fruits from this plant, the fruiting body, the cob, was only two to three centimeters long.2 It would take another 4,000 years before the corn cob would be as long as we see it today and for it to have the large kernels that are the product of generation after generation of artificial selection. Artificial selection depends on stepwise genetic modifications in successive generations of a particular population with the goal of making certain physical traits appear in the offspring or to make certain physical traits more pronounced. Although the process of artificial selection is gradual, it has allowed humans to target a specific genetic trait or group of traits and to breed a species until those characteristics are produced in the offspring.

What are the current uses of biotechnology? Advances in the field of genetics added new tools and techniques to the pursuit of biotechnology, thereby opening the door to a type of genetic modification called genetic engineering. After James Watson, Francis Crick, and Rosalind Franklin resolved the molecular structure of DNA in 1953, information about base pairing and about principles of genetics led scientists to understand the process by which traits were inherited. This

Introduction: Biotechnology Background Information for Educators and Students © American Forest Foundation

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knowledge generated procedures to find genes and eventually to manipulate individual genes for inclusion or exclusion in an organism.3 This type of biotechnology is termed “genetic engineering “ because it allows scientists in a laboratory setting to design and produce organisms with specific combinations of DNA. Although there is no doubt that centuries of artificial selection have resulted in genetic modification of modern crop and livestock species relative to their wild progenitors, the term “genetically modified organism” is generally used to refer to the products of genetic engineering and is, therefore.

synonymous to many people with genetically engineered organism (GEO) or transgenic organism. However, we will make the distinction explicit by using genetically engineered organism(s), or GEO, to refer to transgenic individuals. There are three overlapping divisions of biotechnology: (a) agricultural biotechnology, (b) industrial biotechnology, and (c) pharmaceutical biotechnology. Environmental biotechnology is a fourth category that is included in developments that have occurred in all three areas (see Figure I-1, also Table I-1).

Figure I-1. Divisions of Biotechnology

Agricultural Biotechnology

Environmental Biotechnology

Industrial Biotechnology

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Pharmaceutical Biotechnology

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Table I-1. Examples of Biotechnology Applications in Selected Fields of Use Agricultural Biotechnology Modification through cross-breeding and artificial selection Insertion of genes to resist herbicide applications and pest damage

Industrial Biotechnology Pharmaceutical Biotechnology Use of organisms in sewage Mass production of penicillin and treatment cortisone Use of agricultural and urban waste Diagnostic testing for antibodies to limit or replace petroleum products

Insertion of genes to tolerate environmental stress Insertion of genes, such as Vitamin A, to increase nutrition of staple foods Methods to detect unsafe levels of pesticides on food

Use of plant starch to make acetone during World War II Breakdown of industrial and hazardous waste Testing of groundwater, soil, and air for contamination Use of genetically engineered bacteria to produce food supplements

How does biotechnology affect me? You may not be aware of some ways your life has been affected directly and indirectly by the use of living organisms to produce a product. The water you drink has been treated using processes that take advantage of microorganisms. The air you breathe may be cleaner as a result of advances in waste cleanup that are facilitated by plants, invertebrates, and soil organisms. Of the cotton grown in this country, 80 percent comes from genetically engineered seeds, so it is likely that you are wearing pants produced by biotechnology and that you probably ate some food today that contains genetically engineered soybean, cottonseed, corn, or canola oil. In addition to the ways biotechnology has touched aspects of your daily lives, there are specialized areas of science where advances in biotechnology may have a greater effect on your life in the future. Botanists and wildlife management scientists are now using genetic analysis to preserve gene pools, to create seed banks, and to make better choices regarding cross-breeding to increase the genetic diversity of fragile populations of plants and animals. Forestry management scientists have developed fast-growing species of trees that are engi-

Human protein production for hemophilia and diabetes Vaccines to offer disease resistance More than 125 approved medicines Use of genetically engineered bacteria to produce enzymes

neered with environmental tolerance to insects or to poor soils so firewood may become more readily available worldwide and so trees can be used for erosion control in decimated areas. Engineered plants and microbes will play an important role in providing the materials for more efficient production of biofuels from plant and urban waste. Biotechnology plays a large role in creating vaccines, medicines, and medical treatments to reduce pain and suffering worldwide, and there is hope that greater achievements will be made in this field in the future.

What are the risks of biotechnology? Although humans have been manipulating the genetic makeup of individuals and populations for more that 6,000 years, there has been growing concern about the risks, benefits, and ethics of those practices as advances in biotechnology techniques have occurred and have resulted in products from genetic engineering. Genetic modifications introduced by genetic engineering technologies can differ in both nature and scope from those that have been introduced by artificial selection. Thus, the concerns regarding ethical and environmental issues surrounding biotechnology and GMOs are generally focused on geneti-

Introduction: Biotechnology Background Information for Educators and Students © American Forest Foundation

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cally engineered organisms rather than on crops and livestock produced by artificial selection. Moreover, opposition, fear, and doubt have been expressed from different groups worldwide. Genetic modification using artificial selection and controlled breeding practices takes a long period of time with the forces of nature as a key player in the success of each new venture. Genetic engineering is a technique that is on a much reduced time scale, and the organisms produced have much less influence by the natural environment or ecosystem constraints. No international laws or oversight committees currently exist to regulate the field of genetic engineering, even though stem cell research, cloning, and the use of genetically engineered organisms as food products have caused debate in many communities, countries, and governments. Individual countries are creating the rules and procedures about how to handle each area of science as concerns are brought to government officials. Have you ever heard or read about certain practices and had concerns yourself? What are the doubts and fears you have heard expressed about genetic engineering?

There is concern that modern methods of biotechnology involving genetic engineering allow humans to make changes in organisms without being able to assess the risks of those actions. Issues such as (a) a reduction in biodiversity, (b) creation of invasive species, (c) gene transfer, and (d) genetic resistance of insects to crops with an overuse of insect resistant genes—as well as effects that cannot be foreseen—concern people inside and outside the scientific community. How can you tell if the risks regarding genetic practices are scientifically based? How can you tell if the advantages of a particular practice outweigh the costs? The following four activities are aimed to help you—as a teacher—learn about the traditional and modern techniques that fall under the label of biotechnology. With information rooted in scientific facts and direct experiences, you may feel more familiar with the terminology and concepts when reading current events, joining in discussions, and making decisions.

Box I-1. What Is a hybrid? By definition, hybrids are the offspring of a cross between inherently unlike parents.4 Gregor Mendel used this definition in its most conservative form to describe offspring derived from cross-breeding lineages of plants that were true-breeding for a particular trait. The offspring plants were the same species as the parent plants; however, unlike their parents, the hybrids contained different alleles for certain traits he was studying. This interpretation of the term means that hybrids are any offspring that contain genetic rearrangements, whether the result of a naturally occurring incident or a human-manipulated incident. The word “hybrid” is also used to mean new varieties or species that occur from natural or artificially selected sexual reproduction of two separate taxa. For example, domestic dogs and coyotes can interbreed and produce viable, fertile offspring with genetic information from two separate species. Modern genetic techniques have taken hybrids one step further using genetic engineering to insert a gene from one organism into another organism. When DNA is cut and then sealed back together to include a new segment of DNA, it is said to have undergone recombination. Bioengineered hybrids are recombinant hybrids. For example, many seed varieties of agricultural crops now contain a bacterial gene, called Bt, that gives the plant resistance to certain insects. This type of hybrid offspring, termed a transgenic organism, can incorporate and express genes from a species in another kingdom, or in this case, another domain.

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Box I-2. What Is a Species? The answer to this question is trickier than you might think. The Biological Species Concept, as defined by Ernst Mayr, states that species are “groups of actually or potentially interbreeding natural populations, which are reproductively isolated from other such groups.”5 The term reproductive isolation can be clarified when applied to animals that are able to reproduce, but often do not. Reproductive isolation may be due to (a) gamete incompatibility; (b) mating behaviors: (c) morphology; or (d) spatial, temporal, or other limitations. Even though there are many cases where this definition must be explicitly stated, the terminology is appropriate when considering most animal species. However, problems arise when applying this definition to organisms outside the animal kingdom. Many plants, fungi, and bacteria interbreed with other species readily, thus producing fertile offspring that may start a new lineage or may breed back to one parent species or the other. Species, such as bacteria, that reproduce by exchanging portions of their genome complicate the definition of a species because individual lineages are constantly evolving. Scientists have produced many other definitions of the word “species” using measures (such as ecological or morphological factors) that are applicable to the taxa they study. Controversies and ethics around manipulating genetic lineages sometimes become even more complicated because the definition of a species is still ambiguous.

endnotes 1. Convention on Biological Diversity, “Article 2. Use of Terms,” United Nations Environmental Programme, New York, 1992. cbd.int/convention/articles.shtml?a=cbd-02. 2. Janet Raloff. “Corn’s Slow Path to Stardom,” Science News 143, no. 16 (1993). 3. Stanley N. Cohen, Annie C. Y. Chang, Herbert W. Boyer, and Robert B. Helling, “Construction of Biologically Functional Bacterial Plasmids in Vitro,” Proceedings of the National Academy of Sciences of the United States, 70, no. 11 (November 1973): 3,240-3,244. 4. David Hartl. Genetics, 3rd ed. (Boston: Jones and Bartlett Publishers, 1994). 5. E. Mayr, Systematics and the Origin of Species, (New York: Columbia University Press, 1942). 6. D. Rowland Burdon and William J. Libby, Genetically Modified Forests: From Stone Age to Modern Biotechnology (Durham, NC: Forest History Society, 2006.)

Introduction: Biotechnology Background Information for Educators and Students © American Forest Foundation

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Figure I-3. Timeline of Genetic Discovery

Mendel

Mendel’s experiment 1866 DNA isolated 1869 Chromosomes described 1902–1904

“Transforming principle” transmits traits 1928

Basic law of population genetics 1908

Chromosomes

Mendel’s work discovered 1900

Transmission Cytogenetics Genetics

Agriculture 13,000– 15,000 years ago

DNA building blocks described 1929 Human chromosome number is 48 1933

Clues to DNA structure and function discovered 1952 Watson and Crick deduce DNA structure 1953 Sickle cell mutation described 1956

Human chromosome number is 46 1956

First human chromosome abnormality identified 1959

Molecular Genetics

DNA infects, not protein 1950

DNA Structure and Function

“Transforming principle” is DNA 1944

Mechanism of DNA replication demonstrated 1958

Genetic code deciphered 1961–1963 Recombinant DNA work begins 1972

“Humulin” arrives, first recombinant DNA drug 1978

Human genome map of markers 1980 Huntington disease marker discovered 1983

Biotechnology

DNA Mapping and Sequencing Tools

Ways to sequence DNA invented 1977

PCR invented 1985 Idea for human genome project 1986

Cystic fibrosis gene found 1989 HGP begins 1990 Expressed sequence tags invented 1991 Huntington disease gene identified 1993 First genome sequenced: Haemophilus influenzae 1995 SNP project starts 1998 First gene therapy death 1999 Golden rice invented 2000

First human chromosome sequenced 1999 First draft of human genome sequence 2000

Genomics

Yeast genome sequenced 1996

Sequencing Genomes

DNA chips invented 1995

Medical Genetics

Single Gene Discoveries

Duchenne muscular dystrophy gene 1987

Source: Genetic Epidemiology Master of Science, Washington University School of Medicine, St. Louis, MO. biostat.wustl.edu/~treva/genetics-timeline.htm. 6



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Activity 1: Biotechnology and You In this activity, students will explore artificial selection, as well as learn how advances in science are allowing increasingly specific methods of genetic manipulation in organisms (genetic engineering). Students will explore the risks and benefits of genetic engineering and concerns that affect what we eat and wear. Subjects: Biology, AP Biology, Environmental Science, AP Environmental Science Concepts: 1.4, 1.6, 2.2, 2.8, 2.10, 3.5, 3.11, 4.1, 4.7, 5.4, 5.6 Skills: Classifying and Categorizing, Compare and Contrast, Decision Making, Determining Cause and Effect, Discussing, Inferring, Interpreting, Organizing Information, Problem Solving, Reasoning, Representing, Researching Materials: pinto beans, rulers, calculators (optional), bags or bowls (for pinto beans), scissors, clear tape, copies of student pages, transparency, overhead projector Time Considerations: Preparing the Activity Part A: 30 minutes Part B: 60 minutes Part C: 30 minutes Part D: 30 minutes Part E: 30 minutes Part F: 15 minutes Doing the Activity Part A: One 50-minute period Part B: Two 50-minute periods Part C: Two 50-minute periods Part D: One 50-minute period Part E: One or Two 50-minute periods Part F: Two 50-minute periods

Objectives: Students will (a) model artificial selection through a simulation; (b) simulate genetic engineering through insertion of novel genes into a model of a plasmid; (c) recognize and detect bias in writing; and (d) explore the benefits, risks, and risk management strategies for genetically engineered products. Assessment Opportunities: ƒ To evaluate the students’ knowledge of artificial selection: – Ask your students to identify the selective force in the bean activity. The selective force is your students; they are choosing which beans “survive” and will be allowed to “reproduce.” ƒ To evaluate students’ knowledge of genetic engineering: – Have students compare and contrast artificial selection and genetic engineering. Students should realize that both are forms of genetic modification Selection can modify traits that are controlled by many genes, while genetic engineering works best on traits controlled by a single gene. This factor is one that limits the genetic engineering of crops and forest trees for improved productivity and stress resistance. Such complex traits are affected by many genes; in field testing, it has not proven easy to use single genes to make improvements that show increased value. ƒ To evaluate students’ understanding of the use of plasmids in transformation: – Ask your students to explain what a plasmid is, where it is naturally found, and why it is useful for this type of activity. Plasmids are small, circular, extra-chromosomal pieces of DNA that are typically double-stranded and are found in

Activity 1: Biotechnology and You © American Forest Foundation

bacteria. Because plasmids replicate autonomously and do not undergo recombination, they can pass on the inserted gene without interruption. ƒ To evaluate students’ understanding of the use of enzymes in the transformation process: – Ask your students to identify which enzymes are represented by the scissors and tape in the activity that uses Student Page: Paper Plasmid Construction and to explain their respective functions. The students may use pictures, diagrams, or text in their answers. Scissors represent restriction enzymes (used to cut DNA at specific sites), and the tape represents ligase (used to join strands of DNA that have double-stranded breaks). ƒ To evaluate students’ understanding of risks vs. benefits associated with genetically engineered organisms: – Ask your students to write a list that contains at least three risks and three benefits of genetically engineered organisms. Next, have the students write a brief report (one page) on whether they would purchase clothing that has been produced from genetically modified cotton. Ask them to address at least two of the risks or benefits that they listed. Remind them that although there is no right or wrong answer, they should be able to articulate why they have taken a specific stance. ƒ To evaluate students’ understanding of containment and escape issues of genetically modified organisms: – Ask your students to choose a genetically modified plant that is currently grown and to discuss the specific ways in which potential escape of that organism or gene has been addressed.

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background Humans have been genetically modifying organisms for thousands of years. So why does there seem to be such a controversy over genetically engineered organisms (GEOs) in today’s world? The answers to that question are varied and complex. In this activity, students will explore (a) the distinction between artificial selection and genetic engineering; (b) the ways some scientists use genetic engineering to modify agricultural crops; and (c) several of the scientific, economic, environmental, and ethical considerations that must be addressed when assessing GEOs. What does it mean to genetically modify an organism? Artificial selection is one example of how an organism can be genetically modified. When people choose to breed only dogs that have a specific phenotypic trait, those people are ultimately creating a group of organisms that possess a specific trait, and the results are often called a breed. Labradors make great retrievers, collies make great herders, and Dobermans are good guard dogs.

There are also many different examples of artificial selection when it comes to food. A classic example is corn. The corn that we eat today (Zea mays ssp. mays) was developed from a wild grass, teosinte (Zea mays ssp. parviglumis). The ancestor to today’s variety was much smaller in size, with hard, indigestible kernels. Today’s corn has bigger, more easily digested kernels.1 When the modification involves altering DNA, it is referred to as genetic engineering, which is a form of genetic modification that is a more precise process than traditional artificial selection and is based on phenotypes because it deals directly with DNA. Genetic engineering involves recombinant DNA (rDNA)—DNA that has been altered, usually using gene splicing. Genetic engineering became possible in the middle of the 20th century when advanced technology allowed researchers to learn much more about the genetic code and how it worked. In 1953, James Watson and Francis Crick published their findings in the journal Nature about the double helix structure of DNA.2 In 1983, Kary Mullis

Box 1.1 Did You Know? The first commercially available and genetically engineered pet is the GloFish®.1 This fish has been genetically engineered with a protein that causes it to fluoresce, or glow, all the time! By inserting a gene normally found in marine organisms (such as jellyfish and sea anemones) into a zebra fish, scientists were able to create a fish that glows all the time. Although the fish was originally developed for use in genetic studies in the laboratory, researchers are now looking to use these fish as bio-monitors to detect environmental pollutants.2 Along the way, someone realized there was a demand in the pet market for this novel aquarium critter. Even though the GloFish can be legally sold in the United States (the Food and Drug Administration deemed there were no significant differences with respect to safety between the glowing fish and its traditional counterpart3), some countries, including Australia, Canada, and most of those in Europe, have banned the sale of this genetically engineered organism.2 1. GloFish fluorescent website at www.glofish.com. 2. Z. Gong, B. Ju, and H. Wan, “Green Fluorescent Protein (GFP) Transgenic Fish and Their Applications,” Genetica 111 (2001): 213–25. 3. J. Knight, “GloFish Casts Light on Murky Policing of Transgenic Animals,” Nature 426 (2003): 372. 8



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Box 1.2 Policies Governing Labeling of Genetically Engineered Organisms Genetically engineered organisms are becoming increasingly prevalent in today’s society, especially in agriculture. With the use of genetic engineering comes an entirely new set of rules and regulations, not only in the United States but also in countries throughout the world. In our increasingly globalized society, the economic, social, and environmental implications of how individual countries choose to regulate such crops can be far-reaching. Consider the United States and the European Union (EU). The EU has stringent rules when it comes to labeling food originating from organisms that have been genetically engineered, whereas the U.S. rules are much less stringent.1 To learn more about how decisions governing food labeling are made, check out the World Health Organization’s website at www.who.int/foodsafety/biotech/en/. 1. “U.S. vs. EU: An Examination of the Trade Issues Surrounding Genetically Modified Food,” Pew Initiative on Food and Biotechnology, December 2005, www.pewtrusts.org/our_work_report_detail.aspx?id=24138.

revolutionized the field of genetics by creating a process known as the polymerase chain reaction, or PCR, (eventually winning the Nobel Prize for his discovery).3 PCR enables scientists to make millions of copies of a single region (a specific gene, for example) of a genome. This copying, or amplification technique, enables researchers to study a specific region of a genome in detail. By 2001, a group of more than 200 researchers published a paper announcing that they had sequenced (that is, determined the order of nucleotides or base pairs, or ACGT) the entire human genome (more than 2.9 billion base pairs).4 So why are GEOs so controversial? The answer is complex and has its roots in two very different areas: science and ethics. Bioethics is a subset of ethics that deals with issues that arise out of advances in biology, medicine, and technology. There is no comprehensive list of the ethical concerns, but some of the ethical issues that are often discussed revolve around religion (genetic engineering is viewed by some as akin to “playing God”), concern for animal welfare (some people view genetic engineering techniques as painful invasions), concern for human health (how well scientists understand the long-term implications of consuming genetically engineered food), and concern for the environment (whether or not a GEO might transfer its genes to the wild).5,6 Activity 1: Biotechnology and You © American Forest Foundation

Scientific controversy over GEOS centers around human health concerns, as well as environmental concerns.7 For example, inserting genes for pest resistance into plants raises questions about how the products of those genes might affect humans and other nontarget organisms, such as butterflies, when and if they are consumed. Environmentally, there is concern about the escape of transgenes into plant populations that have not been genetically engineered (see the case study of StarLink™ corn in part E). Those concerns need to be evaluated against the potential benefits of transgenic organisms, such as reduced pesticide use, higher yield, more nutritious food, bioremediation, and production of pharmaceuticals. Many people are concerned that the scientific technology that allows us to modify and change living organisms is increasing so fast that society is unable to stay on top of such issues with respect to debating and developing ethical and moral guidelines for using the techniques. Another concern is that although there are formal agencies that regulate the use of GEOs in our society (Food and Drug Administration, U.S. Department of Agriculture, Environmental Protection Agency),8 no agency is specifically set up to evaluate the ethical implications.

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With all this controversy over genetic engineering, one might automatically assume that there are uniform guidelines that regulate GEO production. But that is not the case and, again, the reasons are complex. Because much of the science is so new, there are few long term studies that have specifically addressed many of the concerns surrounding GEOs, such as their effect on human health and the environment. In addition, different countries have adopted different laws and regulations to deal with GEOs. In a world where commerce is becoming increasingly globalized, those differences can have enormous economic, political, and environmental effects.9 As with many issues, the controversy surrounding biotechnology is complex. Learning more about the science, including the different reasons and ways that organisms are genetically engineered, will allow you and others to make more informed decisions. Critically evaluating all information is important when examining issues. Part C of this activity allows students to critically evaluate information from several sources with differing points of view (bias). Part D of this activity introduces students to different reasons for genetically modifying crops and allows them to explore both the risks and benefits of genetic engineering. Part F of this activity encourages students to investigate the use of genetically modified cotton for clothing. Biotechnology is a global issue—with global implications. For example, 75 percent of processed food in America contains ingredients derived from a genetically engineered organism, and those foods are often sold in the world market.10 Additionally, more than 20 different countries worldwide are growing genetically engineered crops.11 Learning about the scientific, environmental, and ethical issues surrounding GEOs will help students make informed decisions in this area of biotechnology. .

endnotes

3. Mullis Kary, F. Faloona, S. Scharf, R. Saiki, G. Horn, and H. Erlich, “Specific Enzymatic Amplification of DNA in Vitro: The Polymerase Chain-Reaction,” Cold Spring Harbor Symposia on Quantitative Biology 51 (1986): 263–73. 4. J. C. Venter, M. D. Adams, E. W. Myers, P. W. Li, R. J. Mural, G. G. Sutton, H. O. Smith, et al., “The Sequence of the Human Genome,” Science 291, no. 5507 (2001): 1304–51. 5. “Exploring the Moral and Ethical Aspects of Genetically Engineered and Cloned Animals,” Pew Initiative on Food and Biotechnology, January 2005, www.pewtrusts.org/ uploadedFiles/wwwpewtrustsorg/Summaries_-_ reports_and_pubs/PIFB_Moral_Ethical_ Aspects_GE_and_Cloned_Animals.pdf. 6. M. Marvier, “Pharmaceutical Crops Have a Mixed Outlook in California,” California Agriculture 61, no. 2 (2007): 59–66. 7. O. V. Singh, S. Ghai, D. Paul, and R. K. Jain, “Genetically Modified Crops: Success, Safety Assessment, and Public Concern,” Applied Microbiology Biotechnology 71 (2006):598– 607. 8. “Issues in the Regulation of Genetically Engineered Plants and Animals,” Pew Initiative on Food and Biotechnology, April 2004, www.pewtrusts.org/our_work_report_detail. aspx?id=17976. 9. “U.S. vs. EU: An Examination of the Trade Issues Surrounding Genetically Modified Food,” Pew Initiative on Food and Biotechnology, December 2005, www.pewtrusts.org/uploadedFiles/ wwwpewtrustsorg/Reports/Food_and_ Biotechnology/Biotech_USEU1205.pdf. 10. Ibid. 11. “Global Status of Commercialized Biotech/GM Crops 2006: Executive Summary,” International Service for the Acquisition of Agri-Biotech Applications, 2006, www.isaaa.org/Resources/ Publications/briefs/35/executivesummary/ default.html.

1. Fred Pearce, “Going Bananas,” New Scientist, January 18, 2003, p. 27. 2. J. D. Watson and F. H. C. Crick,, “A Structure for Deoxyribose Nucleic Acid,” Nature 171 (1953): 737–38. 10

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Part A: Artificial Selection Students will explore the differences between artificial selection and genetic engineering. They will then model the process of artificial selection using a simulation involving pinto beans of varying sizes.

getting ready ƒ Make enough copies of Student Page: Bean Activity for each student. ƒ Prepare one bag of 200 beans for each pair of students. Label it “New Beans” (pinto beans work well because they can be quite variable in size [a 1-pound bag is enough for about six groups], but other beans (e.g., lima beans) may work if their sizes vary). ƒ Each student should have a ruler to record his or her results. ƒ A calculator for each group is optional. ƒ Students should sit facing each other with a flat desk or table in between them. ƒ Supply each student group with one extra bag or bowl (labeled “Discarded Beans”) for the discarded beans.

doing the activity 1. Begin this activity by asking students to list organisms that have been genetically modified by humans. They will likely come up with a list that includes examples of organisms that have been modified through both artificial selection and genetic engineering. If they do not, shape the discussion by suggesting some examples of both types.

Genetically modified organisms include various crops (corn from teosinte is an excellent example), dogs, ornamental plants, and trees. GEOs include crops (Bt-resistant corn and cotton), Flavr-Savr tomato, animals (GloFish®), and bacteria (used to produce insulin and many other proteins).

Activity 1: Biotechnology and You © American Forest Foundation

2. Explain to the students that in this activity they will be exploring a specific type of genetic modification called artificial selection. Ask them to come up with a definition of artificial selection. If they are having trouble, remind them of the examples they have just listed in step 1. They should end up with an understanding that artificial selection involves breeding organisms according to their desired phenotypic traits. 3. Go back to the list of organisms the students came up with in step 1, and ask them to identify and circle those that were modified using artificial selection. They should realize that not all the organisms on their list have been circled. Explain to them that genetic modification can be accomplished through different methods and that artificial selection is only one of the methods. At this point, you can introduce the term genetic engineering and explain that the remaining items on their list fall under that category. 4. Tell the students that you are going to have them perform a simulation that models artificial selection, and explain that this activity is an analogy. After briefly outlining the simulation, ask the students to think throughout the simulation how the different parts relate to the concept of artificial selection. Explain that each bean represents an allele for bean size. In this simulation, big beans represent alleles for big bean plants, and small beans represent alleles for small bean plants. Remind students that each organism has two alleles and contributes one of the alleles to the next generation. It is also important to note that many genes affect a single trait and that this example of a single allele representing plant size is greatly simplified. 5. Have the class break up into pairs. For each pair of students, pass out a bag of beans (each bag should contain at least 200 beans), and one “Discarded Beans” bag or bowl. Each student should have a ruler and a copy of Student Page: Bean Activity.

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6. Have each student pick 10 beans out of the bag labeled “New Beans.” Using the ruler, have each player measure the length of each of his or her beans in millimeters and record that information on the student page in the column labeled “Length at Beginning of Simulation (mm).” After they have measured all 10 of their beans, ask the players to each calculate the average size of his or her 10 beans and to record that information in the last row of the column. 7. Next, have the players lay out their beans in front of them, in a line, in a random order about 1 inch apart. The line of beans from player 1 should line up with the beans from player 2 (see Figure 1.1). The line of beans represents 10 organisms with two alleles each. Explain to the class that the goal is to use artificial selection to increase the average size of the beans in their possession. Each pair of beans represents a single breeding organism, with each bean representing an allele.

Figure 1.1. Pair of Beans, Each Representing Two Alleles of a Single Organism. 8. Round 1: Taking turns, have each player select a pair of beans. Remind students that their goal is to end up with larger beans. They cannot mix and match between pairs; they must select two beans that are across from each other (see Figure 1.1; selection may result in their choosing a really big bean that is paired with a really small bean). After each player has selected three pairs, have them put the remaining unselected beans in the “Discarded Beans” bag (those beans will no longer be used). The pairs of beans that each student has selected (representing six alleles) are going to be used in

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the next round. Explain that this round of the activity represents the generation time for beans. Generation time is the time it takes an organism to grow and reproduce, and the time varies for different organisms. The generation time for a pinto bean is 2.5 months. 9. Have each student randomly select four new beans from the “New Beans” bag (not the “Discarded Beans”). Ask them to mix those beans in with the selected beans from the previous round. Each student should now have 10 beans (6 from the previous round and 4 new beans), which they should place in a line, in random order. They should then repeat the selection activity, where each student takes a turn at selecting pairs of beans (representing an organism with two alleles). Remind students that after each round of choosing three pairs each, they should put the beans they did not choose in the “Discarded Bean” bag and should keep their selected beans for the next round. 10. Students should repeat step 9 until they have completed 10 rounds. After rounds 5 and 10, students should measure each of their six remaining beans, calculate the average bean size, and record their data on their papers (add the length of the six remaining beans and divide by six). This result is an indication of the average bean size in the population that they have created through their selection process. 11. Have each student subtract the initial average bean size from the final average bean size. The result represents the net increase (or decrease) in bean size for each person. Have each person record this value on a chart on the board. 12. Once all the students have recorded their values, ask them to contemplate causes for the variation. They should come up with reasons, such as different sizes of beans in their bags (representing different genetic material), dif-

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ferences among students in their ability to discriminate among beans of different sizes (representing differences among people in characterizing phenotypic traits), and differences as a result of random chance (a big bean randomly paired with a small bean). You can use corn as an example of a genetically modified crop to relate this activity to the real world. 13. As a follow-up to the activity, use the following set of questions to generate discussion among the students. The questions will allow them to solidify their understanding of the analogy of the bean selection simulation to the process of artificial selection. ƒ This activity is intended to model a real-world process. In this analogy, what are the beans meant to represent in real life? The beans are meant to represent alleles for bean size. Each bean represents a single allele donated by either the male or the female. ƒ What is the addition of new beans to your line supposed to represent? It is meant to represent the addition of new alleles into the population. In artificial selection, this determination is done by bringing new plants or animals into your breeding population. ƒ Why must you choose a pair of beans rather than just one? One of the beans represents an allele from the mother, and the other bean represents an allele from the father. The pair of beans that is chosen, therefore, reflects a new individual, which has two alleles: one from each parent.

Activity 1: Biotechnology and You © American Forest Foundation

ƒ Although analogies help us understand new ideas, they are often imperfect. What are some ways in which the bean analogy does not accurately reflect reality? In reality, no single allele codes for size. It is a complex process, influenced by many genes at many loci, as well as by environmental conditions. Also in this analogy, the generation time was very fast, so students were able to see the results of their selections almost immediately. In reality, the generation time for bean plants would be several months. ƒ Describe the trend in the class data of average bean size. Students should notice that in general the average bean size has increased. There may be some students whose average bean size stayed the same or even decreased. This is to the result of random chance and reflects the importance of collecting more than one set of data. ƒ Ask the students how they think the results would differ at 100 rounds? 1,000 rounds? The more rounds they play, on average, the more dramatic the difference between the beginning average bean size and the final average bean size. Eventually, however, the difference in size will stop changing as they reach the size limit of the population (i.e., their maximum average bean size can never exceed the largest bean in the population).

endnotes 1. N. M. Fedoroff, “Prehistoric GM Corn,” Science 302, no. 5648 (2003): 1158–59.

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STUDENT P AGE Bean Activity In this activity, you will be using beans to model the process of artificial selection. 1. Pick 10 beans from your bag labeled “New Beans.” Measure the length of each bean using your ruler and then calculate the average size of all 10 beans. Hint: You calculate the average by adding up all the lengths and dividing by the total number of beans. Remember that in this simulation, the first round has 10 beans; however, after the 5th and the 10th rounds, you are measuring only 6 beans. Record Bean Length

Bean

Length at Beginning of Simulation (mm)

Length after 5 Rounds

Length after 10 Rounds

1 2 3 4 5 6 7 8 9 10 Average length 2. Lay your beans out in front of you. Line them up randomly. In other words, you do not want to try to line them up according to their color or their length. You partner will do the same so that you each have a line of beans in front of you. Your beans and your partner’s beans should be parallel (in line with) each other. Your row of beans

Your partner’s row of beans

Figure 1.1a. Pairs of Beans, Each Representing Two Alleles of a Single Organism 3. Players take turns picking the pairs of beans from the lines in front of them until each player has chosen three pairs. The goal is to pick the biggest pair. But you cannot mix and match among pairs! For example, if the biggest bean is paired with the smallest bean, you have to choose both. It is up to you to decide the best strategy. For example you could pick a pair with the biggest bean, even if it is paired with the smallest bean. Or you could pick a pair that has two medium beans. After each player has chosen three pairs, have one player take the remaining unselected pairs and put them in the bag labeled “Discarded Beans.” Those beans will no longer be used in the simulation.

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project learning tree Exploring Environmental Issues: BioTechnology © American Forest Foundation

STUDENT P AGE Bean Activity (continued)

This circle represents a pair of beans. In this simulation, a pair represents one bean from your line and one bean from your partner’s line.

Figure 1.1b. Pairs of Beans, Each Representing Two Alleles of a Single Organism 4. Round 2: Each player should pick four new beans from the bag labeled “New Beans.” Again, each player should randomly line up his or her 10 beans, parallel to the partner’s beans.

Repeat the instructions in step 3. Do this until you have completed five rounds (a new round begins every time you select new beans).

5. After five rounds, each player needs to measure the six remaining beans in his or her row and to record the information on his or her data table (remember to do this BEFORE you pick the new beans for the next round). Calculate the average bean size.

Activity 1: Biotechnology and You © American Forest Foundation

15

Part B. Genetic Engineering Students will identify the variety of organisms that have been genetically engineering by humans. They will then mimic the process of genetic engineering by transforming a plasmid with two novel genes: one for pest resistance and one for fluorescence.

getting ready ƒ Make enough copies of Student Page: Transformation for each student. ƒ Make enough copies of the following student pages for each group (three to four students per group). Making each of the following student pages a different color will facilitate the activity: – Student Page: Paper Plasmid Construction – Student Page: Genes for Insertion into Plasmid – Student Page: Restriction Enzymes – Student Page: Instructions – Obtain one pair of scissors and clear tape for each group.

doing the activity In this activity, students will simulate the insertion of a reporter gene (GFP) and a pest-resistance gene (Bt gene) into a tobacco plant. 1. Begin this activity by reminding students of the list of organisms they developed in the beginning of part A. Explain that in this activity they are still focusing on genetically modified organisms but will explore those GEOs that have been modified using genetic engineering instead of artificial selection and that will go directly to step 2. If you have not already completed part A with your students, you can guide the discussion using the following: ƒ Ask your students to come up with a list of organisms that have been modified by humans. Write their examples on the board as they come up with them. They will likely come up with a list that includes examples of organ16



isms that have been modified through artificial selection and genetic engineering. If they do not, shape the discussion by suggesting some examples of both types. Examples of organisms modified through artificial selection may include various crops (corn from teosinte is an excellent example), dogs, ornamental plants, and trees. Organisms modified through genetic engineering may include crops (Bt-resistant corn and cotton), Flavr-Savr tomato, animals (Dolly, the cloned sheep), and bacteria (used to produce insulin and many other proteins). ƒ At this point, introduce the terms artificial selection and genetic engineering. Artificial selection is based on phenotypic selection of organisms, whereas genetic engineering is more specific than artificial selection in that it usually deals directly with DNA. ƒ Go back to the list of organisms that the students came up with in step 1, and ask them to identify those that were modified using artificial selection (by circling them) and those that were modified using genetic engineering (by underlining them). They should realize that genetic modification can be accomplished through several different routes, namely, artificial selection and genetic engineering. At this point, introduce the term transgenic, and explain that it refers to organisms that have been genetically modified through the process of genetic engineering. The class should now be familiar with the definition of genetic engineering and can go directly to step 3. 2. As a class, have the students develop a definition for genetic engineering. Their definition should touch on the fact that genetic engineering involves inserting or deleting DNA. At this point, you can introduce the term transgenic and let them know it refers to organisms that have been genetically modified through the process of genetic engineering. 3. Ask students what they know about transgenic crops. The following questions can be used to lead the discussion so that students mention

project learning tree Exploring Environmental Issues: BioTechnology © American Forest Foundation

some of the costs and benefits associated with transgenic crops. ƒ What are some examples of transgenic crops? ƒ Why are the specific advantages of the crops mentioned? ƒ What are the risks of transgenic crops? 4. Once the students have an understanding of the costs and benefits of transgenic crops, pass out Student Page: Transformation to each student. Have each student read the background information to himself or herself. Initiate a discussion in which students can ask questions about the information they have just read. Note: The use of GFP to monitor an accidental release of a transgenic organism is technically possible and has been used in research settings. It has not, however, been used as yet by farmers for this purpose in a nonresearch setting.1 5. Have one student volunteer to read the scenario from Student Page: Transformation. This information will set the stage for the paper plasmid activity that follows. 6. After the students have read the Student Page: Transformation for the activity, you should initiate a discussion. You will want to give the students an opportunity to discuss why some farmers would choose to use transgenic crops and why some would choose to grow organic crops. Divide the class into two groups, and ask one group to represent the farmer who is growing transgenic crops and one group to represent the organic farmer. Have each group come up with the list of the costs and benefits of each type of crop. Bring the class back together, and then have the groups share their lists with each other. 7. Explain to the students that they will now investigate in more detail how scientists have created the transgenic plant by modeling the steps used to create the plasmid that contains the genes that will be inserted into the crops.

Activity 1: Biotechnology and You © American Forest Foundation

8. Divide the students into small groups (two to four students each). Each group should receive a copy of the following student pages: ƒ Student Page: Paper Plasmid Construction ƒ Student Page: Genes for Insertion into Plasmid ƒ Student Page: Restriction Enzymes ƒ Student Page: Instructions In the following steps, students will assemble their circular plasmid and will cut out the genes they will insert. After identifying the correct restriction enzyme, they will use scissors to mimic the actions of the restriction enzyme, cutting both the plasmid and the genes to be inserted. They will then insert the genes into the plasmid. The final product will be a genetically modified plasmid that now contains two novel genes: one for pest resistance (Bt gene) and one for green fluorescence (GFP gene).

helpful hints Explain that the DNA fragments are copies of the two genes (Bt resistance and GFP) that were generated by scientists through the use of the polymerase chain reaction with a special protocol that generates “sticky ends” (overhanging base pairs at each end of the DNA fragment). The sticky ends are necessary because they allow the two genes to be connected (or ligated) to form a single piece of DNA. This piece of DNA will eventually be inserted into the plasmid and, after the transformation process, will allow the plants to resist certain pests (caused by the Bt gene) as well as to fluoresce (caused by the presence of the GFP gene). Student Page: Restriction Enzymes lists the three restriction enzymes from which students must choose, as well as details the specific sequences where each one cuts the DNA. Tell students that they need to identify where to cut the plasmid so they can insert the Bt and GFP genes. There are several different cutting sites, so remind the students of what they need to consider, including the following: ƒ The restriction enzyme should cut the plasmid only once, but it should cut the genes of interest twice. 17

Students should have identified HindIII as the best choice. It cuts the plasmid only once and cuts the genes on both ends. EcoR1 also cuts the plasmid only once, but the sequence does not appear on the genes. Pst1 does not have a restriction site on either the plasmid or the genes. ƒ The restriction enzyme should produce the appropriate sticky ends that will allow it to incorporate the DNA fragment containing the genes of interest. Ask the students if they can explain why many restriction enzyme cutting sites are palindromes. A palindrome is a word or phrase that reads the same in either direction (e.g., the words “civic,” “level,” “ radar,” and “testset” or the phrase “a man, a plan, a canal: Panama”). Students should understand that the sequence will be read the same when the two strands of DNA are separated and will be read from the 5-foot end to the 3-foot end. ƒ Explain the origin of restriction enzymes. Restriction enzymes are naturally produced in bacteria as a defense mechanism. Restriction enzymes chop up DNA that can invade bacteria, thus serving a protective role. ƒ Remind the students that they are using scissors to mimic the action of the restriction enzyme they have chosen and are using tape to mimic the action of the enzyme DNA ligase. 9. The students have now created a plasmid that contains the genes for Bt resistance and fluorescence. The next step in this process would be to insert the plasmid into the crops. This step will allow the crops to be both pest resistant and identifiable as transgenic. Use the following questions to assess the student’s understanding of the paper plasmid activity: ƒ In this activity, you used scissors and tape to represent the tools used in genetic engineering. What tools are those items supposed to represent?

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Scissors represent restriction enzyme, and the tape is supposed to represent the enzyme ligase. ƒ In this activity, we inserted two new genes into the plants: one for fluorescence and one for a pest resistance. What was the purpose of the gene for fluorescence? The fluorescence is a “reporter” gene and allows us to see if a particular plant has been genetically engineered. In this scenario, fluorescence was desired because the organic farmer wanted a way to be able to see if any of the genetically engineered seeds ended up in her fields. Although the average farmer does not currently use fluorescence for this purpose, the technique is routinely used in this way on a smaller scale in the lab or in experimental plots. ƒ Would it have been possible for a plant to have been transformed by the Bt gene and not by the GFP gene? Why or why not? Because you inserted both genes into a plasmid, the genes would be transformed together. This transformation is because plasmids are circular pieces of DNA that are taken up by plant cells in their entirety. It is not possible for only part of a plasmid to enter a cell and still be functional. ƒ What are some other types of genes that are being inserted into plants? In addition to genes for pest resistance, genes for herbicide tolerance are transformed into crops. This process allows farmers to spray herbicides onto the crops to kill weeds, leaving the crops unaffected.2 Genes for increased nutrition are also used to transform crops, such as in the case of Golden Rice (genes that increase the amount of vitamin A are inserted).

project learning tree Exploring Environmental Issues: BioTechnology © American Forest Foundation

endnotes 1. C. N. Stewart, “Monitoring the Presence and Expression of Transgenes in Living Plants,” Trends in Plant Science 10, no. 8 (2005): 390– 96. 2. O. V. Singh, S. Ghai, D. Paul, and R. K. Jain, “Genetically Modified Crops: Success, Safety Assessment, and Public Concern,” Applied Microbiology and Biotechnology 71 (2006): 598–607.

Enrichments ƒ Ask the students to research and write (or draw) an outline that details the next steps necessary to complete the transformation process (transformation of plants using the plasmids). A useful resource that gives an overview of this process can be found at http://ppge.ucdavis.edu/ Transformation/transform1.cfm. ƒ Classroom kits are available that contain all the necessary materials to conduct a transformation activity in your classroom. Refer to the “Ordering Supplies” appendix for ordering information.

Activity 1: Biotechnology and You © American Forest Foundation

ƒ Have students check Career Connection: Bioinformatics. The field of bioinformatics centers on the vast amounts of data that are being produced in scientific labs today. For example, determining the genetic sequence of the genomes of organisms (from flies to dogs and humans) is made possible by the use of computer programs that generate, store, and manage extremely large amounts of nucleotide data (the human genome, for instance, is made up of more than three billion base pairs). Computer programs that can simulate complex molecular reactions (such as the folding of proteins) allow scientists to model interactions and reactions that could not otherwise be visualized. Bioinformaticists can also specialize in conducting the complex statistical analyses required by scientists to interpret the data they generate. In addition to understanding the science behind the data, biostatisticians have a strong background and interest in computer programming, software analysis, and database management. Additional information about the field of bioinformatics can be found at www.ncbi.nlm.nih.gov/ About/primer/bioinformatics.html.

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STUDENT P AGE Transformation Transgenic plants (plants that have had genes from another type of organism inserted into them) are commonplace in agriculture today. More than 75 percent of the processed food in the United States is derived from genetically engineered organisms.1 Pest resistance is one of the most common traits introduced into plants through genetic engineering. Other examples of transgenes include genes for increased growth, longer shelf life, and increased flavor. Although there are benefits to transgenic plants such as increased crop yield or increased pest resistance, which were just mentioned, there are also concerns about the safety of those plants. One concern is the possibility that genetically engineered plants may breed with their traditional counterparts. Although it is possible to genetically test individual plants to determine if they have been genetically engineered, it is not economically feasible to test every plant from an entire field of crops. How would farmers know for sure that the plants they grow each year were, or were not, genetically engineered? Scientists have come up with a possible solution to this problem. Insertion of a reporter gene along with the gene that contains the desired transgenic trait (such as the ability to resist a certain pest species) would ensure that all plants that were genetically engineered would also produce a discernable phenotypic trait.2 A reporter gene is a gene that produces some sort of phenotypic signal.

Scenario: Scientists are inserting a gene for pest resistance (Bt gene) into soybeans. The Bt gene naturally occurs in a species of bacteria. It codes for a protein that is toxic to certain insects. By inserting this gene into a crop, a farmer can make that crop resistant to insects. This transgenic plant containing the Bt gene will be sold to a group of farmers whose fields are next door to a farmer who grows only organic crops. The organic farmer has expressed concern that some of the transgenic soybeans from his neighbor will accidentally be sown in his organic fields. To mitigate this problem, the farmer planting the transgenic crop has agreed to use a species that contains a reporter gene in addition to the Bt pest resistance gene. In this activity, you will help the scientists design a plasmid that will contain the gene for pest resistance (Bt gene), as well as a reporter gene (green fluorescent protein gene). Once you have inserted the genes of interest in your plasmid, your research and development team will take over and continue the transformation process that will insert the plasmids into bacteria known as Agrobacterium tumefaciens. Plants will then be infected with the transformed bacteria, thus delivering the genes for pest resistance and green fluorescent protein into the soybeans.

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STUDENT P AGE Transformation (continued) Often, reporter genes involve a trait such as antibiotic resistance that allows scientists in the lab to select successful transformants by growing them in the presence of antibiotics that will kill any plants that did not successfully incorporate the desired trait. But another type of reporter gene can cause an organism to glow in the presence of ultraviolet light. This type of trait is visible to the naked eye and would allow farmers to know, simply by looking at a plant at night using an ultraviolet light, whether or not it has been genetically engineered. The type of gene that researchers use for this detection is one that codes for (that is, has a DNA sequence that directs the production of a specific protein) a green fluorescent protein and is referred to as a GFP gene. The most common source of this gene is marine organisms such as jellyfish that produce the protein naturally.2 Endnotes: 1. “Exploring the Moral and Ethical Aspects of Genetically Engineered and Cloned Animals,” Pew Initiative on Food and Biotechnology, January 2005, http://pewtrusts.org/uploadedFiles/wwwpewtrustsorg/ Summaries_-_reports_and_pubs/PIFB_Moral_Ethical_Aspects_GE_and_Cloned_Animals.pdf. 2. C. N. Stewart, “Monitoring the Presence and Expression of Transgenes in Living Plants,” Trends in Plant Science 10, no. 8 (2003): 390–96. 

Activity 1: Biotechnology and You © American Forest Foundation

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STUDENT P AGE Paper Plasmid Construction 1. Cut out each strip of DNA on the dotted lines. 2. Tape the strips together in any order (but make sure the letters are facing the same way). 3. This is your paper representation of a plasmid. A plasmid is a circular piece of DNA that is extrachromosomal (that is, is not part of the DNA found in an organism’s nuclear DNA). C

G

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project learning tree Exploring Environmental Issues: BioTechnology © American Forest Foundation

STUDENT P AGE

Genes Genes Genes Genes Genes Genes Genes Genes Genes

C T A A G C T T A A T T C G T G A C C T G T A C G T A C G G A T T T T G A C G T G A C A A G C T T T

G A T T C G A A T T A A G C A C T G G A C A T G C A T G C C T A A A A C T G C A C T G T T C G A A A

Genes for Insertion into Plasmid

Activity 1: Biotechnology and You © American Forest Foundation

This strip of DNA contains copies of both the GFP gene (bold and on dark gray) and the Bt resistance gene (bold and in light gray). The two genes were generated using polymerase chain reaction and pieced together using an enzyme known as ligase. Cut out the strip of DNA along the dotted line. This fragment of DNA will be the one you will insert into the plasmid. Note that the sequences have been greatly reduced in length for this simulation. The true gene sequences would be hundreds of base pairs in length.

Bt gene

green fluorescent protein gene (GFP)

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STUDENT P AGE Restriction Enzymes The following diagram (Figure 1) illustrates the specific sequences recognized by three different restriction enzymes: EcoR1 (pronounced “eco ar one”), HindIII (pronounced “hin dee three”), and Pst1 (pronounced “pee ess tee one”). The cutting site is indicated with scissors. Each of these sequences is a palindrome. (Hint: The words “civic,” “level,” and “radar” and the phrase “a man, a plan, a canal: Panama” are palindromes.) Why do you think this sequence occurs?

"

A T

Pst1

C G

A T

T A

T A

C G

EcoR1 recognizes and cuts at the sequence “G A A T T C”

A T

G C

C G

T A

T A

HindIII recognizes and cuts at the sequence “A A G C T T”

T A

G C

C G

A T

G C

Pst1 recognizes and cuts the sequence “C T G C A G”

" "

HindIII

A T

"

G C

"

EcoR1

"

Figure 1

Because each of the three restriction enzymes cuts within the recognition sequence, “sticky ends” are produced. A sticky end is a single strand of overhanging base pairs that allow two sequences to be aligned and joined by the enzyme ligase (see Figure 2). Your job is to decide which restriction enzyme you would use to cut out the genes that will be inserted into your plasmid and to cut your plasmid to allow the insertion. You will use the same restriction enzyme for both. Keep the following in mind when choosing your restriction enzyme:

Enzyme cuts 5' G A A T T C 3' 3' C T T A A G 5'

5' G 3' 3' C T T A A 5'

5' A A T T C 3' 3' G 5'

Figure 2. Example of an EcoR1-Cutting Site that Produces “Sticky Ends”

• You want to cut the plasmid in only ONE location. • You will need the restriction enzyme to cut both ends of the genes you will insert. This change will create sticky ends that will allow the insert to be ligated into the plasmid.

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project learning tree Exploring Environmental Issues: BioTechnology © American Forest Foundation

STUDENT P AGE Instructions 1. Cut out the strips of DNA from the page labeled “Paper Plasmid Construction.” Tape them together (in any order, as long as the letters face the same way) to form a circle. This circle represents your plasmid DNA. 2. Now, cut out the piece of paper that represents the genes you are going to insert from the page labeled “Genes for Insertion into Plasmid.” This paper represents the two genes that you are going to insert into your plasmid. 3. Next, you need to identify which restriction enzyme to use that will produce the correct cutting pattern. You want the restriction enzyme to cut the plasmid only once, but it needs to cut the piece of DNA that contains the genes you want to insert twice. Below, circle the restriction enzyme that is the best choice, and circle the cutting site on both the plasmid and gene. Use Student Page: Restriction Enzymes to learn more about how restriction enzymes work.

EcoR1

G A A T T C

C T T A A G

HindIII

A A G C T T

T T C G A A

Pst1

C T G C A G

G A C G T C

4. Once you have identified the appropriate restriction enzyme, use your scissors to cut both your plasmid and your gene at the restriction enzyme cutting sites (follow the dotted lines on the restriction enzyme you circled above). Your results should look like the pictures below.

5. Once you have made the appropriate cuts with scissors, you are ready to insert the genes into the plasmid. Tape the genes into the plasmid, making sure to match up the corresponding base pairs (remember, A pairs with T, G pairs with C). You final product should be a complete plasmid with two novel genes inserted.

Activity 1: Biotechnology and You © American Forest Foundation

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Part C: Risks and Benefits of Genetically Engineered Organisms Students will evaluate the risks and benefits associated with genetic engineering. They will evaluate information from different sources and will learn to perceive, identify, and evaluate bias in information sources.

Getting ready ƒ Make enough copies of the following student pages for each student (each student page should be on uniquely colored paper if possible). If you have multiple classes, you can laminate the copies (or put them in clear plastic paper protectors). Then you can use them repeatedly so that you need to make only as many as you would need to accommodate your largest class. Students can even write on them with markers, and you can clean them with ethanol (or water if the marker is water soluble). – Student Page: Genetically Engineered Organisms – Perspective A (����������������� industry perspective: pro-biotechnology) – Student Page: Genetically Engineered Organisms – Perspective B (environmental group perspective: anti-biotechnology) – Student Page: Genetically Engineered Organisms – Perspective C (���������������� third-party perspective: independent, verifiable information) – Student Page: Detecting Bias

doing the activity 1. Ask students if they have taken any risks today? To initiate the discussion, you might ask how they traveled to school, what they have eaten, if they drank water from a tap, and so forth. Then ask them to define a risk. 2. Next, divide the class into groups. We suggest keeping group sizes to four or below to encourage participation from all group members. Give everyone in one group copies of Student Page: Perspective A, everyone in the second group copies of Student Page: 26

Perspective B, and everyone in the third group copies of Student Page: Perspective C. If you have more than 12 students, some topics can be covered by multiple groups. Have each group assign the role of reporter and recorder to members of the group. 3. Ask each group to read through the information on its student page and to make a list of the risks and benefits of the genetically engineered organisms that are presented in the information. Make sure to remind the students who have been assigned the role of recorder to write down the information. 4. Have the reporter from each group write its list on the board. Have each group make a separate list so that you end up with three lists. 5. Ask all the students to take a moment and to read through the lists on the board. Ask them if they notice any differences or similarities among the lists. They should notice that some of the lists contain mainly risks, one contains both risks and benefits, and one contains mainly benefits. 6. Ask the students why they think their lists were so different. During this discussion, they should determine that the lists they received contained different information. 7. Pass out the remaining student pages so that each group has access to all three. Give students time to read through the two new student pages. Ask each student to mark which student page he or she finds the most persuasive and why. 8. Challenge the students to come up with suggestions of how to detect and evaluate bias in articles. Write their suggestions on the board. Discuss any apparent bias found in each student page. 9. Pass out Student Page: Detecting Bias. Ask the students to identify any methods for detecting bias that are not represented on the list they made in step 8.

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10. Ask the students if they have changed their opinion about which article was most persuasive. Why or why not? 11. Ask the students which student page seems the least biased and to explain why. 12. Ask each group to identify the potential sources of each student page. Have the reporter from each group verbally share the group’s thoughts with the class. Reveal the actual sources of the data to the students. Student Page: Perspective A was developed by a group composed of professionals from leading biotechnology companies; Student Page: Perspective B was written by the environmental advocacy group Green Peace; and Student Page: Perspective C was written by a group consisting of scientists, religious leaders, and academics who had no personal financial stake in the agriculture biotechnology industry.1

endnotes 1. A. Tegene, W. E. Huffman, M. Rousu, and J. F. Shogren, “The Effects of Information on Consumer Demand for Biotech Foods,” Technical Bulletin No. 1903, U.S. Department of Agriculture, April 2003, www.ers.usda.gov/ publications/tb1903/.

enrichment Ask the students to consider the risks and benefits of genetically engineered organisms from a global perspective. For example, the need for droughtresistant species in a developing country might be more compelling than the need for the same type of crops in the United States. How might different cultures and different political systems affect how one might perceive risks and benefits?

13. Ask students whether they think all industry groups or environmental advocacy groups would hold the same perspective as these respective groups. What might be similar? What might be different? 14. To reinforce students’ ability to detect bias, assign the following activity for homework. Ask each student to find two articles (using newspapers, magazines, and Internet) that represent opposing views on a particular genetically engineered crop. They should summarize the main points of each article and list the type of source and any types of bias that may be evident. Alternatively, they can bring in a single article from a recent newspaper that discusses some type of biotechnology. Ask them to read the article and to summarize the viewpoint of the author. Ask them to consider whether the way the information was presented led to bias (e.g., were only positive consequences discussed? What were the sources of information presented in the article?).

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STUDENT P AGE Genetically Engineered Organisms – Perspective A General Information – Genetically engineered (GE) plants and animals have the potential to be one of the greatest discoveries in the history of farming. Improvements in crops so far relate to improved insect and disease resistance and weed control. Those improvements using bioengineering or GE technology lead to reduced cost of food production. Future GE food products may have health benefits. Scientific Impact – Genetic engineering is a technique that has been used to produce food products that are approved by the Food and Drug Administration (FDA). Genetic engineering has brought new opportunities to farmers for pest control and in the future will provide consumers with nutrient-enhanced foods. GE plants and animals have the potential to be the single greatest discovery in the history of agriculture. We have just seen the tip of the iceberg of future potential. Human Impact – The health benefits from genetic engineering can be enormous. A special type of rice called Golden Rice has already been created and has higher levels of vitamin A. This rice could be very helpful because vitamin A deficiency (VAD) is devastating in third-world countries. VAD causes irreversible blindness in more than 500,000 children and is also responsible for more than one million deaths annually. Because rice is the staple food in the diets of millions of people in the third world, Golden Rice has the potential for improving millions of lives a year by reducing the cases of VAD. The FDA has approved GE food for human consumption, and Americans have been consuming GE foods for years. Although every food product may pose risks, there has never been a documented case of a person getting sick from GE food. Financial Impact – Genetically engineered plants have reduced the cost of food production, which means lower food prices, and that result can help feed the world. In America, lower food prices help decrease the number of hungry people and also let consumers save a little more money on food. Worldwide, the number of hungry people has been declining, but increased crop production using GE technology can also help further reduce world hunger. Environmental Impact – GE technology has produced new methods of insect control that can reduce chemical insecticide application by 50 percent or more. This change means less environmental damage. GE weed control is providing new methods to control weeds, which are a special problem in no-till farming. Genetic engineering of plants has the potential to be one of the most environmentally helpful discoveries ever.

Adapted from A. Tegene, W. Huffman, M. Rousu, and J. F. Shogren, “The Effects of Information on Consumer Demand for Biotech Foods,” Technical Bulletin No. 1903, U.S. Department of Agriculture, April 2003, www.ers.usda.gov/publications/tb1903/.

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STUDENT P AGE Genetically Engineered Organisms – Perspective B General Information – Genetic engineering is one of the most dangerous things being done to your food sources today. There are many reasons that genetically engineered (GE) foods should be banned, mainly because unknown adverse effects could be catastrophic! Inadequate safety testing of GE plants, animals, and food products has occurred, so humans are the ones testing whether or not GE foods are safe. Consumers should not have to test new food products to ensure that they are safe. Scientific Impact – The process of genetic engineering takes genes from one organism and puts them into another. This process is very risky. The biggest potential hazard of genetically engineered foods is the unknown. This process is a relatively new technique, and no one can guarantee that consumers will not be harmed. Recently, many governments in Europe assured consumers that there would be no harm to consumers over mad cow disease, but, unfortunately, their claims were wrong. We do not want consumers to be harmed by GE food. Human Impact – Genetically engineered foods could pose major health problems. The potential exists for allergens to be transferred to a GE food product that no one would suspect. For example, if genes from a peanut were transferred into a tomato, and someone who is allergic to peanuts eats this new tomato, that individual could display a peanut allergy. Another problem with genetically engineered foods is a moral issue. The foods are taking genes from one living organism and transplanting them into another. Many people think it is morally wrong to mess around with life forms on such a fundamental level. Financial Impact – GE foods are being pushed onto consumers by big businesses, which care only about their own profits and ignore possible negative side effects. Those groups are actually patenting different life forms that they genetically engineer and have plans to sell them in the future. Studies have also shown that GE crops may get lower yields than conventional crops. Environmental Impact – Genetically engineered foods could pose major environmental hazards. Sparse testing of GE plants for environmental effects has occurred. One potential hazard could be the effect of GE crops on wildlife. One study showed that one type of GE plant killed monarch butterflies. Another potential environmental hazard could come from pests that begin to resist GE plants that were engineered to reduce chemical pesticide application. The harmful insects and other pests that get exposed to such crops could quickly develop tolerance and wipe out many of the potential advantages of GE pest resistance.

Adapted from A. Tegene, W. Huffman, M. Rousu, and J. F. Shogren, “The Effects of Information on Consumer Demand for Biotech Foods,” Technical Bulletin No. 1903, U.S. Department of Agriculture, April 2003, www.ers.usda.gov/publications/tb1903/.

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STUDENT P AGE Genetically Engineered Organisms – Perspective C General Information – Bioengineering is a type of genetic engineering where genes are transferred across plants or animals, a process that would not otherwise occur (in common usage, genetic engineering means bioengineering). With bioengineered pest resistance in plants, the process is somewhat similar to the process of how a flu shot works in the human body. Flu shots work by injecting a virus into the body to help make a human body more resistant to the flu. Bioengineered plant-pest resistance causes a plant to enhance its own pest resistance. Scientific Impact – The Food and Drug Administration (FDA) standards for genetically engineered (GE) food products (chips, cereals, potatoes, etc.) are based on the principle that they have essentially the same ingredients, although they have been modified slightly from the original plant materials. Oils made from bioengineered oil crops have been refined, and this process removed essentially all the GE proteins, making them like non-GE oils. So even if GE crops were deemed to be harmful for human consumption, it is doubtful that vegetable oils derived from GE crops would cause harm. Human Impact – Although many genetically engineered foods are in the process of being put on your grocer’s shelf, there are currently no foods available in the United States where genetic engineering has increased nutrient content. All foods present a small risk of an allergic reaction to some people. No FDA-approved GE food poses any known unique human health risks. Financial Impact – Genetically engineered seeds and other organisms are produced by businesses that seek profits. For farmers to switch to GE crops, they must see benefits from the switch. However, genetic engineering technology may lead to changes in the organization of the agribusiness industry and farming. The introduction of GE foods has the potential to decrease the prices to consumers for groceries. Environmental Impact – The effects of genetic engineering on the environment are largely unknown. Bioengineered insect resistance has reduced farmers’ applications of environmentally hazardous insecticides. More studies are occurring to help assess the effect of bioengineered plants and organisms on the environment. A couple of studies reported harm to monarch butterflies from GE crops, but other scientists were not able to recreate the results. The possibility of insects growing resistant to GE crops is a legitimate concern.

Adapted from A. Tegene, W. Huffman, M. Rousu, and J. F. Shogren, “The Effects of Information on Consumer Demand for Biotech Foods,” Technical Bulletin No. 1903, U.S. Department of Agriculture, April 2003, www.ers.usda.gov/publications/tb1903/.

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STUDENT P AGE Detecting Bias Tips for Effectively Evaluating Information • Question each argument. Imagine you are involved in a debate, and think about how you would present the story from a different side. • If the sources of facts are listed, go back to the original source and interpret it for yourself. Does your interpretation agree with that of the author? • Read other articles on the same topic. Seek out sources that disagree, and evaluate then their arguments. • Realize that authors often unintentionally produce biased material. Critically evaluate everything you read, regardless of the source. Even scientists, doctors, teachers, and politicians can produce biased material.

Questions that Can Help You Detect Bias Where is the source from? The location of the source can often give you information about its potential for bias. It is important to remember that even sources that claim to be neutral (such as newspapers or news programs) can often be biased in the way they choose to present information. Types of sources include the following: • Newspaper—In theory, the news articles are supposed to be neutral and to present both sides of the story. But factors such as choice of language, headline, and placement can all influence a reader. • Magazine—Magazines often depend on selling ads for revenue, so their articles can be influenced by companies that choose to advertise in their pages. • Peer-reviewed journal—Peer-reviewed journals contain articles that have been assessed and accepted by other experts in the field. Although those types of articles are generally thought to be factual and neutral, they can still contain forms of bias. • Internet—All the types of sources listed earlier, in addition to others (blogs, websites, etc.), can be found on the Internet. Although the Internet can be an excellent source of information, it is extremely important that you evaluate the information for yourself because anybody can post information, whether or not it is factual. Who is the author? If you know who the author is, you can potentially assess his or her level of expertise or knowledge. You don’t have to know authors personally; you can “know” them through their job title, by becoming familiar with other examples of their work, or through their public reputation. Consider the following: • Does the article you are using list the author, or is it anonymous? • Which would you trust more: an article with an author’s name associated with it or one that lists the author as “anonymous”? • Under what circumstances would you accept information from an anonymous source? • What about information from encyclopedias? • Is there a group or society that is linked to the article? • Is there an author for each entry? • Would you accept an argument or a conclusion from an author simply because of his or her qualifications? Are facts or opinions being stated? Facts can be proved true or false with data, whereas opinions cannot. Consider the following: • Does the author attempt to use opinions as facts? • Are opinions backed up with facts that can be independently evaluated?

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STUDENT P AGE Detecting Bias (continued) Are all sides being addressed? Ask yourself if the story being presented is balanced. Consider the following: • Are all sides of the story being presented? Note that there can be more than two sides to an issue (not everything is black or white; there are often various shades of gray). • Why would only some sides be presented? • Is there evidence that the author is slanting the article (picking and choosing certain facts that support his or her argument)? Use of language. Evaluate the author’s choice of language. Choice of words can influence how we feel about an event. Consider the following: • Does the author use words that are charged with emotion? • Ask yourself how the author’s choice of words has influenced how you feel about the story. Are the arguments logical? Break down each argument presented, and ensure that the logic makes sense. • Is the author using circular reasoning (supporting a premise with another premise)? • Are there facts and, importantly, sources for those facts presented? • Authors use citations as a way of sharing with you where they learned the information they are presenting. Although citations are not necessarily appropriate in every type of writing, most academics will use them. Newspapers generally cite their sources of information; ideally, reporters will check out the validity of information gained through their sources. Should all writing and reporting be free of bias? Can you think of situations where presenting a biased argument is appropriate? Do you think it is possible to be completely free of bias, either as an author or as a reader?

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Part D. Case Studies of Genetically Engineered Crops Students will analyze the risks and benefits of a crop that has been developed in one of three focus areas of agricultural engineering, including nutritional enhancement, pesticide resistance, and resistance to environmental stressors.

getting ready ƒ Make enough copies of the following student pages (one for each group): – Student Page: Types of Agricultural Engineering – Student Page: Genetically Engineered Plants ƒ Make a copy of Student Page: Risks, Benefits, and Management Strategies of Genetically Engineered Plants for each student.

doing the activity 1. Initiate a discussion with the class by asking students if any of them have ever eaten a genetically engineered organism. Record the percentage of those who say yes and the percentage of those who say no (you may have a group of students who say they don’t know; if so, record that information). Compare this statistic with the national average that 75 percent of crops have been genetically engineered.1 Ask the students whether they are surprised by such numbers. 2. Ask the students how they would know whether the food they are eating has been genetically engineered. This question should lead into a discussion of food labeling, ways to test for genetically engineered foods, and the definition of organic. Current U.S. regulations require labeling only if a GEO is found to be significantly different from its traditional counterpart. Currently in the United States, no foods have been genetically altered that require special labeling. There are relatively simple genetic tests (such as polymerase chain reaction and gel electroActivity 1: Biotechnology and You © American Forest Foundation

phoresis) that can test whether a food product was derived from a genetically engineered source. Genetically engineered foods cannot be sold under the organic label; however, just because a food has not been genetically engineered does not mean it is necessarily organic.In addition to not including genetically engineered organisms, organic foods are grown without the use of pesticides, antibiotics, synthetic hormones, or irradiation. Policies governing the labeling of genetically engineered organism vary among countries. More information on this can be found in Box 1.2 in the Introduction. 3. Break the students into small groups, and ask each group to research and to create a list of plants that have been genetically engineered, along with a list of the trait or traits introduced to each plant. They can use a variety of resources, including the Internet. Ask them to arrange the list into similar categories according to the type of modification. Have each group assign both a recorder and a reporter. 4. After giving the students time to conduct their research, have the reporter from each group write its categories on the board. Discuss the similarities and differences among the groups. 5. Pass out Student Page: Types of Agricultural Engineering. Have the students compare their categories with the ones listed, as well as the specific types of genetically engineered plants. Discuss any discrepancies among the lists. 6. Pass out the Student Page: Genetically Engineered Plants. Ask the students how their lists of specific plants that have been genetically engineered compare with those listed on the student page. 7. Break the class into three groups (or more if the students have decided there are more categories than those represented on Student Page: Types of Agricultural Engineering). Have each group make a list of what it thinks are the benefits and risks associated with each category of genetically engineered crops. 33

8. Bring the class back together as a group, and have each group take turns listing one of the benefits by writing it on the board—until all potential benefits have been listed. Repeat this activity with the risks. 9. Once the class has listed the risks associated with genetically engineered plants, begin a discussion of risk management. Explain that risk management is the process of identifying, evaluating, selecting, and implementing actions to reduce risk to human health and to ecosystems.2 (See PLT’s Focus on Risk secondary module for more information.) 10. Divide students into small groups again, and have them come up with a risk management strategy for the risks associated with each category of genetically engineered crops. Again, have each group read out each of its answers until all responses are represented on the chalkboard. 11. Pass out Student Page: Risks, Benefits, and Management Strategies of Genetically Engineered Plants. Ask the students to read it and compare it with the list that the class came up with. Discuss any discrepancies.

13. You can end this activity by asking the students to consider if the reasons for genetically modifying an organism affect whether or not they support the use of transgenics. For example, in this activity they were considering different ways to modify agricultural plants. But what about using genetic engineering to produce pharmaceuticals (such as insulin and vaccines)? Ask them to consider the case of the GloFish mentioned in the introduction (use of genetic engineering for “re-creation” (i.e., the creation of pets).

endnotes 1. “U.S. vs. EU: An Examination of the Trade Issues Surrounding Genetically Modified Food,” Pew Initiative on Food and Biotechnology, December 2005, www.pewtrusts.org/uploadedFiles/ wwwpewtrustsorg/Reports/Food_and_ Biotechnology/Biotech_USEU1205.pdf. 2. “Framework for Environmental Health Risk Management,” Final report, Vol. 1, Presidential/ Congressional Commission on Risk Assessment and Risk Management, 1997, www.riskworld. com/nreports/1997/risk-rpt/pdf/EPAJAN.PDF.

12. As they use the information and ideas learned in class, have each group come up with a position statement on whether it supports the genetically engineered organisms in its category. Not all members of the group necessarily have to agree with the position statement, but everyone should participate in its presentation.

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STUDENT P AGE Types of Agricultural Engineering Nutritional Enhancement: Genetic engineering can increase the nutritional qualities of plants. Nutritional enhancements can include modifying plants to produce higher yields, to contain novel nutrients such as vitamins, or to decrease the prevalence of nonnutritious components such as trans fats. Genetic engineering introduces novel genes that were not originally present in an organism.1 Herbicide Resistance: Weeds can have significant adverse effects on crop production. Methods of weed control generally fall into three categories: (a) mechanical (tilling or hoeing), (b) cultural (rotating which crops are grown in certain fields), and (c) chemical (through the application of herbicides). Farmers often rely on a combination of all three types of control. Plants that are genetically engineered to be resistant to common herbicides can survive the application of herbicides that are used to control weeds.1 Resistance to Environmental Stressors: A variety of environmental conditions can stress plants and adversely affect plant growth and crop yield. Potential stressors include pests, salinity, and drought. Various plants have been genetically engineered to be immune to or more tolerant of such types of stresses. The genetic modifications can either increase tolerance for varying environmental conditions (allowing plants to survive higher or lower temperatures than usual) or allow plants to visually indicate potential stressors that farmers can then act on (e.g., producing a color change when water availability is critically low).1,2,3 Endnotes: 1. L. A. Castle, G. Wu, and D. McElroy, “Agricultural Input Traits; Past, Present, and Future,” Current Opinions in Biotechnology 17 (2006): 105–12. 2. T. Umezawa, M. Fujita, Y. Fujita, K. Yamaguchi-Shinozaki, and K. Shinozaki, “Engineering Drought Tolerance in Plants: Discovering and Tailoring Genes to Unlock the Future,” Current Opinions in Biotechnology 17 (2006):113–22. 3. S. L. Singla-Pareek, M. K. Reddy, and S. K. Sopory, “Genetic Engineering of the Glyoxalase Pathway in Tobacco Leads to Enhanced Salinity Tolerance,” Proceedings of the National Academy of Sciences 100, no. 25 (2003): 14672–77.

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STUDENT P AGE Genetically Engineered Plants (Examples of genetically engineered plants that have been marketed or are in late-stage development) Plant Tomato Rice Canola Turfgrass Banana Sunflower Potato Flax Corn Carnation Cotton Dairy Papaya Peanut Soybean Alfalfa Apple

Modification Delayed softening,1 increased salt tolerance,1 disease resistance1 Contains vitamin A1 Herbicide resistance,2 contains no trans fats2 Herbicide resistance1 Vaccine delivery (cholera, hepatitis B, and diarrhea),1 increased shelf life,2 pest resistance2 Herbicide resistance,1 mold resistance1 Pest resistance2 Herbicide resistance2 Pest resistance,2 drought resisitence,2 increased energy availability,2 increased nutrients,2 amalyase production (assists in production of ethanol)2 Produces a purple carnation2 Pest resistance,2 herbicide resistance2 Enzyme that curdles milk to make cheese,2 protein that increase milk production2 Virus resistance2 Increased oleic acid content2 Herbicide resistance,2 increased oleic acid content3 Herbicide resistance2 Pest resistance2

Endnotes: 1. “Transgenic Crops: An Introduction and Resource Guide,” Department of Soil and Crop Sciences, Colorado State University, 2004, http://cls.casa.colostate.edu/TransgenicCrops/future.html. 2. “Agriculture Biotechnology Reference Guide,” National Corn Growers Association and U.S. Grains Council, www.ncga.com/files/guide.pdf. 3. O. V. Singh, S. Ghai, D. Paul, and R. K. Jain, “Genetically Modified Crops; Success, Safety Assessment, and Public Concern,” Applied Microbiology and Biotechnology 71 (2006): 598–607.

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STUDENT P AGE Risks, Benefits, and Management Strategies of Genetically Engineered Plants1 Benefits of Genetically Engineered Plants • Increased yield: Genetic engineering can make plants grow faster or produce bigger fruit. • Decreased use of pesticides: Genetically engineering plants to resist certain pests can eliminate the need to use toxic pesticides to protect the crop. • Decrease in energy needed to produce crops: Genetic engineering that results in fewer pesticide applications will result in less energy use by farmers (fewer trips with the tractors, thereby saving fuel). • Increased nutritional quality: Genetic engineering can introduce vitamins and increase protein levels. • Ability to grow in severe or extreme environmental conditions: Genetic engineering can enable some crops to grow in areas that otherwise may not be able to support a crop; extreme conditions can involve high or low temperatures, flooding, or drought. • Increased shelf life: Genetic engineering may result in crops that are easier to store and ship. • Use for human health: Genetically engineered organisms can be used to improve human health such as in the case of edible vaccines. Risks of Genetically Engineered Plants • Introduction of allergenic or harmful proteins into food: Concerns have arisen about increasing the presence of known allergens in novel plant species (such as nut allergies) and about the possible toxicity of transgenic proteins not normally consumed by humans. • Detrimental effect on nontarget species and the environment: Transgenic crops that have insecticidal properties may affect nontarget organisms (e.g., butterflies that naturally feed on the plants). • Potential for increased invasiveness and weediness of genetically engineered crop plants: Genetic engineering of an organism that already possesses invasive traits could exaggerate such traits, potentially causing widespread environmental damage. • Pest resistance: The possibility of pests’ developing resistance to transgenic proteins has generated concern. • Effect on biodiversity: Transgenic crops could hybridize with native plant species, thus decreasing overall biodiversity; cross fertilization through wind-borne pollen or seeds could allow transgenic plants to grow in fields considered free of genetically engineered organisms. Risk Management Strategies • Using male sterile plant lines (no pollen produced) prevents crop cross-contamination. • Making seed viability dependent on the application of a chemical that is not normally found in the environment can prevent seed dispersal. • Using nonfood crops such as tobacco can limit the possibility of genes entering the food chain. • Using greenhouses can contain transgenic crops and their pollen and seeds. • Using phenotypic markers (such as fluorescence) could allow for visual monitoring of transgenes. • Labeling genetically engineered foods will enable consumer choice. • Instituting regulatory requirements will ensure that certain safety considerations are met. • Harvesting crops before they are reproductively mature will limit pollen or seed escape. Endnotes: 1. O.V. Singh, S. Ghai, D. Paul, and R. K. Jain, “Genetically Modified Crops: Success, Safety Assessment, and Public Concern,” Applied Microbiology and Biotechnology 71 (2006): 598–607. Activity 1: Biotechnology and You © American Forest Foundation

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Part E: Issues of Containment The purpose of this activity is to introduce some of the concerns about the regulation and control of transgenic plants. It is a real-life example of how regulation and containment efforts that were put in place to prevent the unintended escape of a transgene failed. By the end of this activity, students will have a greater understanding of the complexities and potential consequences of transgenic organisms. They will understand that although the potential benefits of transgenic organisms can be great, the risks can be difficult to predict and control. For more information on how risk is communicated, please read Activity 5 in PLT’s Focus on Risk module.

getting ready ƒ Duplicate Student Page: Transgenes Escape to Taco Bell for each student. ƒ Make four copies of Student Page: Timeline of Events Linked to the Escape of the StarLink™ Transgene. ƒ Make one transparency of the Timeline of Events Linked to the Escape of the StarLink™ Transgene Student Page.

3. Ask each student to read the story on Student Page: Transgenes Escape to Taco Bell. Once all students have finished reading, initiate a class discussion to answer any questions they may have and to ensure that they understand the following definitions and concepts: ƒ Transgenes and transgenic plants ƒ Methods of containing transgenic plants

doing the activity 1. Begin this activity by asking the students to consider the words containment and escape and to come up with definitions for both. Ask them to suggest things or items that need to be contained and that may escape. Ask students to consider the words in a biological sense. Can they suggest examples of plants and animals that humans have tried to contain but failed? Lead the discussion so that the students eventually consider the idea of gene escape. For example, if a transgenic plant is not contained and ends up producing pollen that fertilizes a nontransgenic plant, the transgene is considered to have escaped.

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2. Write the following two phrases on the board: Physical Containment and Biological Containment. Lead the group in a discussion of the differences between the two, and ask the students to come with definitions and examples of each. Write each new example on the board. For physical containment, they might suggest growing plants in a greenhouse, separating male and female plants, or growing only plants of a single sex. For examples of biological containment, they might suggest using only sterile plants or using plants that have been genetically altered to reproduce only in the presence of some external mechanism. Wrap up the discussion by asking the class to consider the following questions: Are physical and biological containment methods mutually exclusive? Will one type of method be best for all types of plants? If not, what are some plant characteristics that might influence containment methods?

ƒ Consequences of not containing transgenic plants ƒ The role of the Food and Drug Administration in regulating transgenic organisms 4. Divide the class into four groups. Each group will be assigned one of the following four categories: (a) political, (b) economical, (c) ecological, and (d) human health. Assign a recorder and a reporter in each group. Pass out a copy of Student Page: Timeline of Events Linked to the Escape of the StarLink™ Transgene. Ask each group to fill in its category (political, economical, ecological, and human health) at the top of the last column.

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5. Ask each group to read over the timeline of events associated with the StarLink incident and indicate with a check in the column labeled “Category” which of the events described fall under the category assigned to their group. 6. Have the recorder from each group fill in the group’s information on the master overhead using a symbol assigned to each particular category. The groups can each choose a symbol, or symbols can be created and assigned by the teacher. Make sure each group writes a key (e.g., $ = economical) at the bottom of the transparency. Display the results to allow the students to see that each event can have consequences in a variety of categories.

9. As a follow-up activity, ask each student to write a brief essay that addresses what could be done to minimize the chances of this issue occurring in another transgenic organism and whether he or she feels more limitations should be placed on transgenic organisms.

Enrichment Ask your students to use the Internet to find popular press articles of this incident (they can start by typing the terms “StarLink” and “Taco Bell” into the Internet search engine). Ask them to print an article, read it, and look for signs of bias (they may need to consult Student Page: Detecting Bias). Have them write a one-page paper that summarizes the type of bias (if any) that they find.

7. Have the reporters from each group stand in front of the class and give a brief summary explaining why the events so indicated fall into that group’s category. 8. After all groups have reported on their categories, initiate a discussion with the students about the variety of consequences associated with the StarLink transgene escape. Ask the students to consider how human error and unforeseen consequences should figure into policy decisions. What can be learned from this particular case?

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STUDENT P AGE Transgenes Escape to Taco Bell® In September 2000, a genetically modified variety of corn, known as StarLink™, was detected in Taco Bell® taco shells. StarLink corn contains a gene known as Cry9C, which codes for a protein that makes plants resistant to certain insects. Because of a concern that this particular gene might induce allergic reactions in humans, the corn was approved for use as feed for nonhuman animals only.1 Once the StarLink variety was detected in the taco shells, a massive recall was launched to ensure that all foods containing traces of the StarLink corn intended for human consumption were destroyed. However, despite massive efforts to destroy food products containing StarLink corn and to destroy StarLink seed stocks, the StarLink transgene was still detectable in U.S. corn supplies up to 3 years later.2 This story illustrates several very important points. First, efforts to ensure that only livestock ate StarLink corn failed. As a result of the StarLink transgene escape, the U.S. Environmental Protection Agency no longer approves transgenic plants unless they are intended for both human and animal consumption. Second, despite the massive effort, complete removal of the escaped transgene once detected was impossible. Although the prevalence of StarLink corn in the U.S. corn market has decreased dramatically as a result of massive containment efforts, it is still detectable years after having been completely banned. Endnotes: 1. Luca Bucchini and Lynn R. Goldman, “Starlink Corn: A Risk Analysis,” Environmental Health Perspectives 110, no. 1 (January 2002): 5–13. 2. “StarLink Test Results,” Grain Inspection, Packers andStockyards Administration, U.S. Department of Agriculture, 2003.

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STUDENT P AGE Timeline of Events Linked to the Escape of the StarLink™ Transgene1 Year Month Event Category 2000 Sept. • StarLink™ corn found in taco shells. • Kraft Foods announces recall of taco shells that have been found to contain StarLink corn. • Aventis (the biotechnology company that produces StarLink corn) announces it will purchase entire crop of StarLink corn from this year to prevent any further use of the corn in food products. Oct. • Aventis chooses to cancel the U.S. registration of StarLink corn. This move means that StarLink corn can no longer be planted for any agricultural purpose. • Aventis asks the Environmental Protection Agency (EPA) to temporarily allow the use of StarLink corn for human consumption because it has already appeared in many food products (EPA considered and later rejected this request). Nov. • The U.S. Department of Agriculture (USDA) announces plans to test corn shipments bound for Japan for the presence of StarLink grain. The move was taken to reassure consumers in Japan, the largest importer of U.S. corn, that the StarLink gene had not escaped. • Aventis confirms reports that the StarLink protein (Cry9C) was present in corn hybrids having no known connection to StarLink varieties. USDA officials begin working with the companies involved to investigate the mix-up. Dec. • An EPA-appointed Scientific Advisory Panel concludes that the Cry9C protein has a medium likelihood of causing allergic reactions in humans.* • Because the Cry9C protein has been found in several varieties of non-StarLink hybrid corn, USDA recommends that U.S. seed companies test all their corn seed lots for the presence of the Cry9C protein. 2001 Jan. • Farmers whose nontransgenic crops were found to contain the Cry9C protein will be compensated for losses they may incur, under an agreement signed by Aventis and the attorneys general for 17 corn-producing states. Feb. • The president, general counsel, and vice president for market development of the U.S. crop sciences division of Aventis Crop Science are fired. A spokesperson for Aventis said it was fair to link the firings to the StarLink fiasco. Mar. • Tainted seed is found in inventories slated to be sold to corn farmers this spring. There is concern that U.S. corn exports will suffer again this year if the crop contains traces of the Cry9C protein. • USDA announces that it will buy Cry9C-tainted corn seed from small seed companies that are not affiliated with Aventis and were not licensed to sell StarLink corn last year. Major seed companies and companies licensed to sell • An Aventis executive estimates the amount of corn contaminated with the Cry9C protein to be 430 million bushels, far more than the amount of contaminated corn seed that will be bought by the U.S. government to prevent its being planted this spring.

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STUDENT P AGE Timeline of Events Linked to the Escape of the StarLink™ Transgene1 (continued) Year 2001

Month Event Category June • The Centers for Disease Control releases its finding that the transgenic protein in StarLink corn was probably not the cause of the apparent allergic reactions that have been attributed to it by people who suffered symptoms shortly after eating corn products. July • Stores remove from their shelves a brand of tortilla chip made from white corn because traces of StarLink corn were found in it. Makers of tortilla chips have been switching to white corn as a precaution because the Bt Cry9c transgene was incorporated only into a yellow corn variety. Avoidance of yellow corn was believed to eliminate the presence of the StarLink protein, which has been found widely—at low levels—in stores of corn destined for human consumption. • The panel of scientists who have been advising EPA on the safety of StarLink corn declines to recommend lifting the ban on human consumption of the corn, saying it is not yet satisfied that the transgenic product is safe. 2002 Dec. • Japan’s Agriculture Ministry reports that it has found traces of StarLink corn in a shipment of U.S. corn that docked at Nagoya Harbor. USDA officials say they believe the last stocks of StarLink corn were destroyed last year. 2003 Sept. • The genetically engineered (GE) gene from StarLink corn, along with the GE genes from other types of GE corn, is reported to have entered the native corn populations of Mexico. The commercial cultivation of any kind of GE corn is prohibited in Mexico because of concerns about gene flow to Mexico’s indigenous corn varieties, but GE corn kernels can be imported for use as food. 2007 Oct. • EPA proposes to cease testing yellow corn for the presence of the StarLink variety after concluding that “potential exposure of the U.S. population to the Cry9C protein in StarLink corn in the current U.S. food supply is extremely low, and continued testing of corn grain by grain handlers and millers for the presence of Cry9C provides no additional human health protection.”2 *There is no evidence that the StarLink variety of corn has caused harm to any individuals. Endnotes: 1. “Transgenic Crops: An Introduction and Resource Guide,” Department of Soil and Crop Sciences, Colorado State University, 2004, http://cls.casa.colostate.edu/TransgenicCrops/starlink_news.html. 2. “Monitoring for StarLink™ Corn to End,” U.S. Environmental Protection Agency, April 2008, www.epa.gov/ pesticides/biopesticides/pips/starlink_corn_monitoring.htm.

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project learning tree Exploring Environmental Issues: BioTechnology © American Forest Foundation

Part F. Genetically Engineered Organisms and Clothing In this activity, students will identify the main traits that are genetically engineered in cotton plants. They will learn how non–genetically modified cotton can be identified and will explore the reasons some clothing manufacturers choose to use organic cotton. 1. Your students will explore the use of genetically engineered cotton in today’s society. Begin by challenging them to identify products that they use in their everyday life and that come from cotton or cottonseed oil or both. On a large piece of paper or the chalkboard, list all the products they identify. 2. Ask your students to arrange the items into categories. The two main categories likely to appear are food and clothing. 3. Using the Internet or other resources, have your students determine what the major modifications are in genetically engineered cotton. They should come up with herbicide resistance and pest resistance. 4. Divide the class into small groups. Challenge each group to identify a clothing manufacturer that chooses to use cotton that has not been genetically engineered. Ask the students to make a magazine advertisement that is for the company and that expresses the reasons for the company’s choosing to use this type of cotton in its products.

ƒ One way to determine whether cotton is genetically engineered is to determine if it has been certified organic. Certified organic cotton must come from plants that have not been genetically engineered. ƒ Some big-name clothing manufacturers such as Patagonia sell all organic cotton, whereas others such as Nike advertise that they use a certain percentage of organic cotton. Students will generally find that many brands that use organic cotton tend to be smaller, less global names. ƒ The Organic Trade Association’s website (www.ota.com) contains links and information on organic cotton. 5. Display the advertisements around the classroom, and allow all students to read each one. Initiate a class discussion that touches on the common themes. You may use the following questions to help guide a discussion: ƒ What is the most common reason companies express for using organic cotton? ƒ Are the opinions expressed by the companies supported by facts? ƒ Are there any flaws or gaps in the logic of the companies? ƒ What would the economic effect be if all cotton sold were produced from non–genetically modified organisms? ƒ Did the information from your classmate’s advertisement change your view on genetically engineered cotton? Why or why not?

If students are having trouble finding a clothing manufacturer that does not use genetically engineered cotton, the following tips may help them:

Activity 1: Biotechnology and You © American Forest Foundation

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notes

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project learning tree Exploring Environmental Issues: BioTechnology © American Forest Foundation

Activity 2: Bioremediation

In this activity, students will learn how living organisms have been used traditionally to process materials or to produce certain products or results. Students will also explore how modern bioengineering techniques have been used to mimic natural processes for the cleanup of sewage, oil spills, and other environmental toxins.

Subjects: Biology, AP Biology, Ecology, Environmental Science, AP Environmental Science, Microbiology, Politics, Social Studies Concepts: 1.4, 1.5, 1.6, 2.1, 3.3, 4.4 Skills: Collaborating, Comparing, Defining Problems, Formulating Questions, Identifying Attributes, Observing, Predicting, Reasoning, Representing, Researching, Synthesizing and Creating Materials: For each class: A color printer, One map of your county, One Edvotek oil-consuming bacteria demonstration kit One large Erlenmeyer flask to prepare the demonstration, Bleach (optional), An incubator (optional) For each group: Order the following supplies (enough for each lab station): • One strain of bacteria for each lab group (order the following from Carolina Biological Supply Company or WARDs Natural Science): Escherichia coli, Rhodospirillum rubrum, Pseudomonas fluorescens, Enterococcus faecalis, Serratia marcescens D1, Branhamella catarrhalis (Branhamella is not available at WARDs); at a minimum, use the first four strains listed if you cannot order and use all six • Four different types of prepoured nutrient media plates (order one each of the following prepoured plates per lab group): Nutrient agar, Brain heart infusion agar, Tryptic soy agar, MacConkey agar) • Parafilm® or tape and plastic wrap • 30 disposable inoculation loops • One small Erlenmeyer flask For each student: Copies of the student pages, Computers with Internet access, and Microsoft Office Publisher. Time Considerations: Preparing the Activity Part A: 30 minutes Part B: 60 minutes Part C: 15 minutes Doing the Activity Part A: Two 50-minute periods Part B: Two 50-minute periods, 3–5 days apart Part C: One 50-minute period

Activity 2: Bioremediation © American Forest Foundation

Objectives: ƒ Students will become familiar with the historical events leading to federal regulations and assistance in waste reduction. ƒ Students will research toxic substances that affect their community. ƒ Students will identify current methods used to clean up hazardous waste. ƒ Students will be introduced to methods to use artificially selected or transgenic species to clean up various types of waste under a range of environmental conditions. ƒ Students will learn about the current methods used to clean up sewage and to extend this concept of bioremediation to other types of waste. Assessment Opportunities: ƒ Use Student Page: Presentation Evaluation Rubric to grade the group presentations. ƒ You may choose to have the students complete the Group Evaluation Form in Appendix XX to evaluate how well they worked together to produce their Superfund site presentation. ƒ Use Student Page: Growing Bacterial Strains on Various Media—Lab Experiment and Student Page: Oil-Consuming Bacteria—Lab Experiment to identify strengths and weaknesses in your students’ understanding of the scientific process.

ƒ Use Student Page: Municipal Wastewater Treatment Facility Field Trip to assess your students’ understanding of how organisms are currently being used in bioremediation. ƒ Use Student Page: Oil-Consuming Bacteria—Lab Experiment to assess your students’ understanding of how Activity A and Activity B relate to each other. Tell the students you would like to produce a bacterial strain for sale to companies that will be cleaning up a contaminated site, but you must know the quantity of toxin that the bacterial strain can break down over a given time. What simple scientific process(es) could be performed to find out this information? – Ask the students to work in small groups to create their own experiment quantifying the amount of toxin (metal, chemical, or oil based) that a particular bacterial strain consumes in a given time. Remind them to write a procedure that tests this question and to include only one variable and one control. Ask them to use Student Page: Oil-Consuming Bacteria—Lab Experiment to record their final ideas. – Ask one representative from each group to share its best experiment idea with the class using the information recorded on Student Page: Oil-Consuming Bacteria Lab Experiment as a guide.

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background One man’s trash is another man’s treasure. That adage could not be truer than in the example of bioremediation. Plants, bacteria, and even fungi can benefit from metabolizing the concentrated nutrients that are present in some types of waste generated by human activities. Bioremediation is the breakdown of certain contaminants by living organisms in an effort to restore an ecosystem to its natural condition. Often, as in the case of most surface waters, this type of remediation is unplanned and occurs naturally as native organisms eat the excess sewage, runoff, industrial by-products, or farm wastes as part of the biogeochemical nutrient cycling process. Plants can absorb metal contaminants such as arsenic, cadmium, and lead, or they may be used simply to hold the contaminants in place so chemicals that are toxic do not migrate so fast into groundwater or surface water. Microorganisms, fungi, and invertebrates that are naturally occurring in soil or water can be encouraged to reproduce at higher rates in order to consume toxins that have leaked into an area. For centuries, humans have intentionally influenced the natural processes of nutrient cycling as a means of cleaning up concentrated contaminants, such as planting sugar beets to remove salt from agricultural fields or adding worms or other macroinvertebrates to compost piles, which will speed the decomposition of agricultural waste. Methods of Bioremediation Plants are easy to grow and monitor, and they are able to draw out toxins from soil or water as long as the chemicals are within the range of their root system. In addition, plants reduce soil erosion and can trap particles in the soil to slow the migration of contaminants. Often, fungi that are root symbionts catalyze the uptake of certain materials or may even absorb contaminants directly.1 In places where contaminated substances have permeated deep into the soil beyond the reach of surface plants, naturally occurring soil bacteria have been found to break down pollutants by metabolizing the toxic molecules into inert molecules.

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Two primary methods exist by which organisms clean up wastes in a region.2 In the first process, termed hyperaccumulation, a plant absorbs a toxin through its roots and concentrates the substance in its tissues. That uptake and sequestering are most common for plants that are absorbing metals such as arsenic, lead, nickel, copper, cadmium, mercury, or even radioactive substances such as strontium or uranium. For example, sunflowers hyperaccumulate strontium and have been used at Chernobyl to help contain radioactive dust. Plants cannot use large quantities of metals for metabolism, so why do they absorb those toxic substances from the soil? It is thought that toxin-tolerant plants have adapted to absorb those substances as an evolutionary advantage to outcompete plants that are unable to grow in contaminated soils.3 After the metals have been absorbed into the plant tissue, the plants can be removed and incinerated to further concentrate the metal for disposal or reuse. The use of plants to control, contain, or hyperaccumulate a contaminated site is a form of phytoremediation. While the plants are growing, there is a risk that herbivores will ingest plants that have hyperaccumulated a metal, thus reintroducing the metal into the ecosystem through the food chain, so scientists must monitor and control a site that is being restored using this method. The second process by which toxins are removed from an ecosystem by living organisms is through metabolism. By using the toxins as a nutrient source, some bacteria, plants, and fungi can alter the toxic molecule into an inert molecule that is safe to release into the environment. An example of this process is in the cleanup of gasoline or oil spills in the ocean or on land. Specialized, naturally occurring bacteria that live in soil, freshwater, and the oceans consume hydrocarbon chains for cell respiration. The resulting products are those typical of metabolism: carbon dioxide gas and water. Bacteria can also metabolize chlorinated hydrocarbons and a wide range of organic solvents commonly used in industry and agriculture.4 In this case, the organisms chemically alter the pollutants rather than simply contain them, so there is no need to recover the organisms after the pollutants have been consumed.

project learning tree Exploring Environmental Issues: BioTechnology © American Forest Foundation

Traditional Biotechnology Still Applicable Today Bioremediation has been a familiar method of disposing of or reducing the quantity of concentrated waste since humans have lived in sedentary groups. Early sewage systems and compost piles give evidence of rudimentary waste treatment by early civilizations. These practices rely on native bacteria and soil organisms to break down waste through metabolism so the nutrients in the waste can be returned to the surface water or soil in a form that is either less harmful or not harmful. Modern techniques of sewage treatment and landfill composting promote the same processes that have been used for centuries. Current techniques encourage bacteria, algae, snails, worms, and other invertebrates to ingest nutrient-rich sewage or landfill waste by aerating the wastewater or soil while providing moisture or substrates for organisms to cling to while they eat and reproduce. In addition to using native organisms, artificial selection of plants to restore agricultural fields and the selection of bacterial strains to consume a particular toxin such as cyanide or sulfur are common cleanup procedures. Scientists also encourage populations of native soil bacteria and microorganisms to flourish in order to clean up highly contaminated sites such as the tailing piles from mining operations, brownfields, or industrial waste burial sites. Using fertilizers or chemicals such as chelating agents, electron acceptors, or electron donors, scientists can help local bacteria metabolize the waste at those sites to minimize the spread of contamination into groundwater wells and surface water.5 Modern Biotechnology Used to Deal with Modern Problems Scientists have been experimenting with encouraging the growth of certain native bacteria and other soil or water microorganisms in situ by fertilizing regions that have been contaminated by oil spills or other types of petroleum-based pollutants. The addition of nitrate or sulfate fertilizers or electron acceptors (such as phosphorus, oxygen, or carbon) can remove the factors that would normally limit the growth and reproduction of certain oilconsuming species. If nutrient or electron or both acceptors are made available through the use of Activity 2: Bioremediation © American Forest Foundation

injection wells, the microorganism population can be stimulated to bloom and consume the contaminant while this food source is abundant. This form of bioremediation is termed biostimulation. The latest techniques in bioremediation involve the use of genetically modified or transgenic species to target the cleanup needs of a particular substance in a specific region. The introduction of naturally occurring strains of microorganisms or genetically engineered microorganisms to assist in chemical cleanups is called bioaugmentation, which is often used in conjunction with biostimulation. By isolating and copying metabolic genes from bacteria that produce enzymes that can break down certain chemicals, scientists can insert the gene of interest into another species that can more adequately address the demands of quantity or drainage in an area. The species that receives the genes for a particular enzyme may be another bacterium, or any other organism that is suitable for growth in the contaminated region, such as an annual or perennial plant. An example of bioaugmentation is the insertion of a mercury-absorption gene that is found in bacteria into a fast-growing tree species that has a deep root system such as willow, cottonwood, or poplar trees.6 The mercury is absorbed from soil or water in a highly toxic form; then mercury vapor, which is significantly less toxic, is released from leaf surface areas. In the past, bacteria have been used to perform this cleanup process, but the vapor release rate is slow because of the low surface area and the slow rate of diffusion of the concentrated vapor in soil. Trees have helped speed up the rate of mercury vaporization, keeping the more toxic contaminants from spreading farther into the soil and groundwater. In the past century, applications of bioremediation have expanded with advances in microbiological techniques and the use of genetic recombination.7 Although we have increased our consumption and waste production exponentially, and although—as a society—we are realizing the extent of the damage we have already inflicted on terrestrial and aquatic ecosystems, there is hope in the form of living organisms that the environment may be returned to its original conditions. 47

endnotes 1. R. S. Boyd and S. N. Martens, “The Significance of Metal Hyperaccumulation for Biotic iIteractions,” Chemoecology 8 (1998): 1–7. 2. E. Philon-Smits and J. Freeman, “Environmental Cleanup Using Plants: Biotechnological Advances and Ecological Considerations,” Frontiers in Ecology and the Environment 4, no. 4 (2006): 203–10. 3. M. N. V. Prasad, “Emerging Phytotechnologies for Remediation of Heavy Metal Polluted and Contaminated Soil and Water,” Hyderabad, India, December 29, 2006, http://wgbis.ces. iisc.ernet.in/energy/lake2006/programme/ programme/proceedings/lc2.htm. 4. “Advances in Remediation of Contaminated Water and Soil Systems II,” session held at the 2006 Western Pacific Geophysics Meeting, Beijing, China, July 24–27, www.agu.org/ meetings/wp06/wp06-sessions/wp06_H24B. html. 5. S. A. Thomas, “Mushrooms: Higher Macrofungi to Clean Up the Environment,” Battelle Environmental Issues, Fall 2000. 6. D. Glass, “Current Trends in Phytoremediation,” International Journal of Phytoremediation 1, no. 1 (1999): 1–8. 7. D. R. Lovley, “Cleaning Up with Genomics: Applying Molecular Biology to Bioremediation,” Nature Reviews Microbiology 1, no. 1 (October 2003): 35–44.

Part A: Superfund Sites

doing the activity 1. Ask a few questions to stimulate and connect the students to the concept of environmental toxins and the removal or cleanup of these hazards. ƒ Do you know anyone who has become ill from exposure to chemicals in his or her environment? ƒ Do you live in a place that is safe from toxins and hazardous waste? Is your drinking water free of chemicals that could harm you or make you ill later in life? ƒ Relatively, how safe do you think you are from encountering radioactive waste, hazardous waste, or toxic chemicals? How do you know that you are relatively safe? ƒ How are toxic substances cleaned from contaminated soil or water? ƒ What government regulations provide protection from exposure to hazardous waste, and how effectively are those regulations monitored and enforced? ƒ Where do you think the nearest small amount of radioactive, chemical, or hazardous toxic substance is?

getting ready ƒ Copy Student Page: CERCLA Case Study: Love Canal, Part I and Student Page: Researching Superfund Sites for each student and Student Page: “Presentation Evaluation Rubric for each group. ƒ Reserve the computer lab for use by your class so that every student or every pair of students has Internet access.

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ƒ Run through the procedure below to find the nearest Superfund sites in your region so you can direct your students to the correct locations to offer them the appropriate number of sites when they perform the activity.

2. Give the students Student Page: CERCLA Case Study of Love Canal, Part I, and ask them to read about the situation that led to exposure of leaking toxins in that neighborhood. 3. Begin a Socratic discussion to help the students explore their own opinions on issues of toxicity and public risk (see Box 2.1 for discussion tips). Here are some questions you might use to initiate and encourage discussion:

project learning tree Exploring Environmental Issues: BioTechnology © American Forest Foundation

ƒ If you lived in the Love Canal neighborhood, what would it take for you to get involved in opposing and correcting that situation? Would one of your neighbor’s children being harmed be enough, or would you get involved only if your own child was affected? ƒ Who is responsible for paying for the cleanup of a contaminated area such as Love Canal?

4. Ask the students to go to the Environmental Protection Agency’s (EPA’s) website at www. epa.gov/superfund/20years/index.htm. Click on “Chapter 2: The Birth of Superfund.” Have the students read the first three pages of Chapter 2 that describe the events that led to the creation of the Comprehensive Environmental Response, Compensation and Liability Act (CERCLA) in 1980.

ƒ What should be done if the people or company responsible for the contamination cannot be found or no longer exist?

5. Distribute Student Page: Researching Superfund Sites so the students can record a brief definition and history of this act.

ƒ Do you think something should be required of industries that are producing substances that might pollute the soil, surface water, or groundwater? And if so, what?

6. Ask the students to search for Superfund sites in their county by going to www.scorecard. org and typing their zip code or their school’s zip code into the box provided. The zip code entered will bring up the Pollution Report Card for that county or city (you may also use a nearby county, a county of interest, the nearest large city, or an area where your students may potentially relocate).

ƒ How “clean” should a site be after it has undergone remediation? Should any contamination be allowed to remain? ƒ Often, the process of cleaning up a site requires the removal of massive amounts of soil, or freshwater may be needed to flush out contaminated groundwater. What should be done with the contaminated water, soil, or chemicals after they have been collected and contained?

7. After taking some time to explore the information presented on the Pollution Report Card for the county they selected, ask the students to record what they have found in questions 2–6 on Student Page: Researching Superfund Sites.

Box 2.1: Tips for Guiding a Socratic Discussion A Socratic discussion is a style of teaching whereby the teacher uses questions to draw out what the students already know about a given topic. In such discussions, the students take a central position in self-education and peer education, while the teacher takes the role of facilitator, asking pertinent questions and encouraging participation. The information that the students offer one another helps them review and tutor their peers. The responses and the discussion also allow the teacher to look for misconceptions and the depth of the students’ understanding. With incremental questions, a teacher can encourage students to build on their existing knowledge and to make conjectures into areas where they are less confident. Peer questioning, playing devil’s advocate, and pressing the students to back up their ideas with facts or examples will help reinforce critical thinking and logic.

Activity 2: Bioremediation © American Forest Foundation

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8. Ask the students to click on the bullet “See how your county stacks up” under the heading “Worst Toxic Waste Sites (Superfund Sites).” The students can then scroll down to the blue heading “Data Summary” and can use the information that follows to answer questions 7–10 on their student page.

13. After each group has given its peers an idea of the problem and proposed cleanup, ask the groups one or two of the following questions to assess their understanding of the problem they have described:

9. By clicking on the bullet “See what Superfund sites are in your community” on the previous “Pollution Report Card” page under “Worst Toxic Waste Sites (Superfund Sites),” students will find a list of all the Superfund sites in their county.

ƒ Why are those particular methods being used to clean up the site?

10. Ask each group to choose a Superfund site in its selected county. When each group has chosen a particular Superfund site, explain that the group will need to plan a presentation about that site using information gathered from www.scorecard.org/ and the form at www.epa. gov/superfund/sites/query/basic.htm. Students can use their student page to guide them through each of these websites to find the information needed for their presentation. 11. After the students have filled in the questions on the student page, ask them to prepare a summary of their Superfund site to present to the rest of the class. They can (a) use the county map to give the location of the Superfund site, (b) describe the events leading to the presence of toxins on that site, (c) describe the contaminants, and (d) go over the proposed cleanup. Presentation topics can be split up within the group if each group has a different Superfund site or between groups if more than one group is researching a single site.

ƒ Which chemical is cause for the most concern?

ƒ What was the time frame for the cleanup of that area? ƒ What could that site be used for after it has been cleaned up (park, gas station, school, etc.)? ƒ Which of the sites presented would you be most comfortable living next to after remediation has been completed? ƒ Which chemicals are the most persistent or the most difficult to clean up? ƒ Is soil or water contamination more difficult to clean up? ƒ Which is more expensive to clean up, the first 90 percent of the contamination or the last 10 percent of the contamination? 14. To conclude this activity, present the case study in Box 2.2 of a Superfund site that was cleaned up using bioremediation methods. Use that case study to generate curiosity for part B, which explores how bioremediation could be used more extensively in conjunction with hazardous waste cleanup.

12. Use Student Page: Presentation Evaluation Rubric to grade the group presentations.

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project learning tree Exploring Environmental Issues: BioTechnology © American Forest Foundation

Box 2.2: Bioremediation Case Study At the Hastings Ground Water Contamination Superfund Site in Nebraska, one of the areas was contaminated with benzene, toluene, xylene, and styrene from a gas manufacturing plant. During the evaluation of this site, inspectors saw that naturally occurring soil bacteria were already reducing the contamination by using the toxic chemicals as a nutrient source. One of the treatments called for in the decontamination plan for this site was to add an oxygen-releasing chemical to the soil so the local soil bacteria could multiply and clean up the chemicals more rapidly. Currently, this site has had a reduction in the original contaminants and is now in the final phase of cleanup.

enrichments ƒ Ask students to research how the laws of a country promote or deter the production of hazardous waste. Ask them to debate how placing a high cost on the disposal of hazardous waste would limit or increase the production of hazardous waste or illegal disposal or both. Ask how disincentives (monetary penalties, increased paperwork, surprise inspections, poor reputation with the general public, etc.) or incentives (easyto-schedule pickups by a public service agency, company advertising that brags about reduction and compliance, payment by government for clean production, etc.) could be used to limit hazardous waste production and the containment and cleanup of waste. ƒ Require the students to explore specific cases where a country other than the United States has reduced the amount of hazardous waste produced or released into the environment. Ask them to explain how the private and public sectors have influenced the reduction of production or inappropriate disposal or both.

waste cleanup vs. the consumer should have to pay for hazardous waste cleanup. Ask the students to defend their positions using actual costs and a proposal of what they think would be a fair way to deal with the issue.

sources for more information “Hastings Groundwater Contamination (Second Street Subsite)” fact sheet, 2007, www.epa.gov/superfund/accomp/factsheets04/ secondst.htm. www.epa.gov/region7/cleanup/npl_files/ ned980862668.pdf “A Citizen’s Guide to Bioremediation,” EPA 542F-01-001, Office of Solid Waste and Emergency Response, U.S. Environmental Protection Agency, April 2001, www.epa.gov/tio/download/citizens/ bioremediation.pdf.

ƒ Ask the students to debate the role of the consumer in the production and cleanup of hazardous waste. Propose two sides to the issue: the consumer should not have to pay for hazardous

Activity 2: Bioremediation © American Forest Foundation

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STUDENT P AGE CERCLA Case Study of Love Canal, Part I The article below was written by Eckhardt Beck, who was administrator of the U.S. Environmental Protection Agency’s Region 2 from 1977 to 1979. This article describes his account of the Love Canal situation until 1979, when the tragedy was still emerging. It ends with his plea to create a law that will hold polluters accountable for their actions. The Love Canal Tragedy by Eckardt C. Beck, EPA Journal, January 1979 Quite simply, Love Canal is one of the most appalling environmental tragedies in American history. But that’s not the most disturbing fact. What is worse is that it cannot be regarded as an isolated event. It could happen again— anywhere in this country—unless we move expeditiously to prevent it. It is a cruel irony that Love Canal was originally meant to be a dream community. That vision belonged to the man for whom the three-block tract of land on the eastern edge of Niagara Falls, New York, was named: William T. Love. He felt that by digging a short canal between the upper and lower Niagara Rivers, power could be generated cheaply to fuel the industry and homes of his would-be model city. But despite considerable backing, Love’s project was unable to endure the one-two punch of fluctuations in the economy and Nikola Tesla’s discovery of how to economically transmit electricity over great distances by means of an alternating current. By 1910, the dream was shattered. All that was left to commemorate Love’s hope was a partial ditch where construction of the canal had begun. In the 1920s, the seeds of a genuine nightmare were planted. The canal was turned into a municipal and industrial chemical dumpsite. Landfills can, of course, be an environmentally acceptable method of hazardous waste disposal, assuming they are properly sited, managed, and regulated. Love Canal will always remain a perfect historical example of how not to run such an operation. In 1953, the Hooker Chemical Company, then the owners and operators of the property, covered the canal with earth and sold it to the city for one dollar. It was a bad buy. In the late ’50s, about 100 homes and a school were built at the site. Perhaps it wasn’t William T. Love’s model city, but it was a solid, working-class community. For a while. On the first day of August 1978, the lead paragraph of a front-page story in the New York Times read: NIAGARA FALLS, N.Y.—Twenty-five years after the Hooker Chemical Company stopped using the Love Canal here as an industrial dump, 82 different compounds, 11 of them suspected carcinogens, have been percolating upward through the soil, their drum containers rotting and leaching their contents into the backyards and basements of 100 homes and a public school built on the banks of the canal. In an article prepared for the February 1978 EPA Journal, I wrote, regarding chemical dumpsites in general, that “even though some of these landfills have been closed down, they may stand like ticking time bombs.” Just months later, Love Canal exploded. The explosion was triggered by a record amount of rainfall. Shortly thereafter, the leaching began.

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project learning tree Exploring Environmental Issues: BioTechnology © American Forest Foundation

STUDENT P AGE CERCLA Case Study of Love Canal, Part I (continued) I visited the canal area at that time. Corroding waste-disposal drums could be seen breaking up through the grounds of backyards. Trees and gardens were turning black and dying. One entire swimming pool had popped up from its foundation, afloat now on a small sea of chemicals. Puddles of noxious substances were pointed out to me by the residents. Some of these puddles were in their yards, some were in their basements, and others yet were on the school grounds. Everywhere the air had a faint, choking smell. Children returned from play with burns on their hands and faces. And then there were the birth defects. The New York State Health Department is continuing an investigation into a disturbingly high rate of miscarriages, along with five birth-defect cases detected thus far in the area. I recall talking with the father of one the children with birth defects. “I heard someone from the press saying that there were only five cases of birth defects here,” he told me. “When you go back to your people at EPA, please don’t use the phrase ‘only five cases.’ People must realize that this is a tiny community. Five birth defect cases here is terrifying.” A large percentage of people in Love Canal are also being closely observed because of detected high whiteblood-cell counts, a possible precursor of leukemia. When the citizens of Love Canal were finally evacuated from their homes and their neighborhood, pregnant women and infants were deliberately among the first to be taken out. “We knew they put chemicals into the canal and filled it over,” said one woman, a long-time resident of the Canal area, “but we had no idea the chemicals would invade our homes. We’re worried sick about the grandchildren and their children.” Two of this woman’s four grandchildren have birth defects. The children were born and raised in the Love Canal community. A granddaughter was born deaf with a cleft palate, an extra row of teeth, and slight retardation. A grandson was born with an eye defect. Of the chemicals that compose the brew seeping through the ground and into homes at Love Canal, one of the most prevalent is benzene—a known human carcinogen, and one detected in high concentrations. But the residents characterize things more simply. “I’ve got this slop everywhere,” said another man who lives at Love Canal. His daughter also suffers from a congenital defect. On August 7, New York Governor Hugh Carey announced to the residents of the Canal that the state government would purchase the homes affected by chemicals. On that same day, President Jimmy Carter approved emergency financial aid for the Love Canal area (the first emergency funds ever to be approved for something other than a “natural” disaster), and the U.S. Senate approved a “sense of Congress” amendment saying that federal aid should be forthcoming to relieve the serious environmental disaster that had occurred. By the month’s end, 98 families had already been evacuated. Another 46 had found temporary housing. Soon after, all families would be gone from the most contaminated areas—a total of 221 families have moved or agreed to be moved. State figures show more than 200 purchase offers for homes have been made, totaling nearly $7 million. A plan is being set in motion now to implement technical procedures designed to meet the seemingly impossible job of detoxifying the Canal area. The plan calls for a trench system to drain chemicals from the Canal. It is a difficult procedure, and we are keeping our fingers crossed that it will yield some degree of success. I have been very pleased with the high degree of cooperation in this case among local, state, and federal governments, and with the swiftness by which the Congress and the president have acted to make funds available. But this is not really where the story ends. Quite the contrary.

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STUDENT P AGE CERCLA Case Study of Love Canal, Part I (continued) We suspect that there are hundreds of such chemical dumpsites across this nation. Unlike Love Canal, few are situated so close to human settlements. But without a doubt, many of these old dumpsites are time bombs with burning fuses—their contents slowly leaching out. And the next victim could be a water supply, or a sensitive wetland. The presence of various types of toxic substances in our environment has become increasingly widespread—a fact that President Carter has called “one of the grimmest discoveries of the modern era.” Chemical sales in the United States now exceed a mind-boggling $112 billion per year, with as many as 70,000 chemical substances in commerce. Love Canal can now be added to a growing list of environmental disasters involving toxics, ranging from industrial workers stricken by nervous disorders and cancers to the discovery of toxic materials in the milk of nursing mothers. Through the national environmental program it administers, the Environmental Protection Agency is attempting to draw a chain of congressional acts around the toxics problem. The Clean Air and Water Acts, the Safe Drinking Water Act, the Pesticide Act, the Resource Conservation and Recovery Act, and the Toxic Substances Control Act—each is an essential link. Under the Resource Conservation and Recovery Act, EPA is making grants available to states to help them establish programs to ensure the safe handling and disposal of hazardous wastes. As guidance for such programs, we are working to make sure that state inventories of industrial waste disposal sites include full assessments of any potential dangers created by these sites. Also, EPA recently proposed a system to ensure that the more than 35 million tons of hazardous wastes produced in the United States each year, including most chemical wastes, are disposed of safely. Hazardous wastes will be controlled from point of generation to their ultimate disposal, and dangerous practices now resulting in serious threats to health and environment will not be allowed. Although we are taking these aggressive strides to make sure that hazardous waste is safely managed, there remains the question of liability regarding accidents occurring from wastes disposed of previously. This question covers a missing link. But no doubt the question will be addressed effectively in the future. Regarding the missing link of liability, if health-related dangers are detected, what are we as a people willing to spend to correct the situation? How much risk are we willing to accept? Who’s going to pick up the tab? One of the chief problems we are up against is that ownership of these sites frequently shifts over the years, making liability difficult to determine in cases of an accident. And no secure mechanisms are in effect for determining such liability. It is within our power to exercise intelligent and effective controls that are designed to significantly cut such environmental risks. A tragedy, unfortunately, has now called upon us to decide on the overall level of commitment we desire for defusing future Love Canals. And it is not forgotten that no one has paid more dearly already than the residents of Love Canal.

Source: Adapted from www.epa.gov/history/topics/lovecanal/01.htm.

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STUDENT P AGE Researching Superfund Sites So far, approximately 1,300 Superfund sites have been placed on the National Priorities List. Sites on the National Priorities List are those places that represent an immediate and severe threat to human health. Although those sites are scheduled for total cleanup, the degree to which they are improved varies and takes an average of 12 years. Cleanup of a site must use the most cost-effective methods, which have taken approximately $30 million per site. Use the following questions and the websites listed to learn more about Superfund sites in the United States. Understanding What Has Passed Go to the Environmental Protection Agency’s (EPA’s) website at www.epa.gov/superfund/20years/index.htm, and click on “Chapter 2: The Birth of Superfund.” Read pages 1, 2, and 3 of that chapter. (Note that the numbers used herein match the numbers in the teacher’s guide.) In the space below, briefly describe what the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA) is and why it was created. The Road That Lies Ahead For the next part of the activity, you will be researching the pollutants that affect the air, soil, and water of a particular county using www.scorecard.org. After entering the zip code of your selected county, scroll down to the section titled “Toxics” to answer the following questions. How is your county ranked among other counties in the United States? What are the names of the top polluting companies in your county? What are the top polluting chemicals that are released in your county? What is the risk of exposure to lead in the houses in your county?

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STUDENT P AGE Researching Superfund Sites (continued) Click on the bullet “See how your county stacks up against all others in the U.S.” under the heading “Toxic Chemicals Released by Factories, Power Plants, and Other Industrial Companies.” Then scroll down to the blue heading “TRI Data Summary” to find information to answer questions 6–10. How many National Priority List or “Superfund” sites are in your county? How many sites are in the preliminary stages (they would be listed as remedial assessment or emergency removal of contaminants)? How many sites are currently being prepared for decontamination (they would be listed as study under way, remedy selected, or design under way)? How many sites are currently being decontaminated (construction under way)? How many sites have completed cleanup? Return to the “Pollution Report Card” page. Under the heading “Worst Toxic Waste Sites (Superfund Sites)” in the “Toxics” section, click on the bullet “See what Superfund sites are in your community.” Select a Superfund site to research with your lab group. You will need to gather as much information as possible so that you can explain how this site became contaminated, can describe its contaminants, and can explain what the EPA expects to do to clean up this site. What is the source of the contamination? What type of industry has contaminated the site? How could the contamination have been avoided? What are the primary chemicals that have been released at this site? Read the “Threats and Contaminants” section for your Superfund site, and answer the following questions. What are the greatest threats these chemicals pose to the environment?

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STUDENT P AGE Researching Superfund Sites (continued) How has the area been affected by the contamination (groundwater, surface water, air, soil, or other)? Include a description of any information that is directly below this chart. What are the “Pre-Cleanup Rankings” for this site? Click on one of the chemicals, and read the information on the page that pops up. Scroll down, and click on the “Links” section after you have read this page; next click on “EPA Health Effects Notebook for Hazardous Air Pollutants” or “EPA Office of Groundwater and Drinking Water Contamination Fact Sheet.” Read this page, and then write a summary of where this chemical comes from and how it affects human health. Note: Links for different locations may have other summaries. Preparation for Your Presentation Go to www.epa.gov/superfund/sites/query/basic.htm to look up your county and state (press “Submit Query” after county and state have been entered). If there are any blue checkmarks on the chart that appears, you can click on those checkmarks to get information about that site. Use the Internet to search for more information on your Superfund site or the contaminants in preparation for your presentation. You will probably need to conduct a search to find the most current information concerning cleanup progress. You must address these categories in your presentation: ƒ History of your Superfund site: Describe what industry used this site and what it did to contaminate the site. ƒ Pre-cleanup state: Describe how bad the contamination is rated for this site, as well as the areas contaminated (air, soil, water). ƒ Contaminants: Name and describe each chemical that is contaminating this site. Describe what the chemical does to human health and environmental health. ƒ Cleanup: Describe how the EPA plans to decontaminate this site. Describe what has been accomplished so far and what still needs to be completed. Describe what the level of contamination will be after the cleanup has been completed.

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STUDENT P AGE Presentation Evaluation Rubric Use the following rubric to grade each group presentation.

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Score

Qualities Apparent in the Presentation

5

All members of the group participate in dispensing information. All group members demonstrate that they are informed about all aspects of this topic by answering questions and by assisting in explanations in any area ,either spontaneously or when called on.

4

All members of the group participate in dispensing information. Some group members demonstrate that they are informed on aspects of this topic other than the topic they presented by answering questions and by assisting in explanations in those other areas.

3

Each group member dispenses at least one fact or area of information. Each group member demonstrates knowledge of the particular area that the group is presenting, but they do not seem well versed in the areas of knowledge of their teammates.

2

One person in the group does not participate and does not demonstrate knowledge of the subject being presented.

1

More than one person in the group does not participate and does not demonstrate knowledge of the subject being presented.

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Part B: Bacteria Lab Experiment getting ready Copy Student Page: Growing Bacterial Strains on Various Media–Lab Experiment for each student. If you plan to have your students reinforce their skills using the scientific process, you may ask them to complete Student Page: Oil-Consuming Bacteria—Lab Experiment after they have completed Student Page: Growing Bacterial Strains on Various Media—Lab Experiment. For the class ƒ One Edvotek oil-consuming bacteria kit to use as a demonstration ƒ One large Erlenmeyer flask to prepare the demonstration ƒ Bleach ƒ Incubator (optional) For each group Order the following supplies to allow sufficient materials at each lab station: ƒ One bacterial strain (order the following from Carolina Biological Supply Company or WARDs Natural Science: Escherichia coli, Rhodospirillum rubrum, Pseudomonas fluorescens, Enterococcus faecalis, Serratia marcescens D1, and Branhamella catarrhalis (Branhamella is not available at WARDs)—at a minimum, use the first four strains listed if you cannot order and use all six ƒ Four different types of prepoured nutrient media plates (order one each of the following prepoured plates per lab group): nutrient agar, brain heart infusion agar, tryptic soy agar, MacConkey agar) ƒ Parafilm or tape ƒ Plastic wrap ƒ 30 disposable inoculation loops ƒ One small Erlenmeyer flask

The students should use sterile techniques to spread the bacteria on the petri dishes. Petri dishes should be purchased prepoured or prepared using sterile techniques and then stored upside down in the refrigerator until 1 hour before use. All culture tubes should remain closed and refrigerated except for the few seconds needed to remove the cap to take a sample. All samples should be spread with a disposable inoculation loop or a metal inoculation loop that has been sterilized with alcohol and flamed. All culture tubes, petri dishes, and other instruments used in this lab should be (a) autoclaved at 121° C for 15–60 minutes or doused with hydrochloric acid or bleach, (b) left overnight, and (c) then disposed of properly after use. Students can participate in the setup of this experiment to learn microbiological lab skills, and the teacher can discuss the cleanup procedure so students understand the risks of working with cultured organisms.

doing the activity 1. Pique student interest by asking some questions about how bacteria live and flourish. Use the questions to review the basic biology of bacteria, as well as to arouse curiosity. ƒ How do bacteria affect your life? They can be pathogens for diseases such as strep throat and pneumonia; they are the biotechnology behind the production of foods such as yogurt and cheese. ƒ How do bacteria reproduce? Asexually through binary fission; sexually through conjugation, transformation, and transduction. ƒ How quickly can bacteria reproduce? Some strains undergo cell division every 20–30 minutes; as bacteria reproduce, they form masses of clone cells called colonies.

ƒ Gloves

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ƒ What do bacteria eat? Some bacteria are photosynthetic autotrophs or chemoautotrophs and are, therefore, able to make their own carbon compounds for food using ultraviolet radiation or other chemical energy such as hydrogen sulfide or ammonium. Many bacteria are heterotrophs that consume carbon chains produced by autotrophs (glucose, dextrose, lactose, etc.) or by eating other organisms. Most bacteria need oxygen for cell respiration, and other elements along with micronutrients such as magnesium, potassium, calcium, and so forth to maintain healthy cellular growth and reproduction. ƒ What would happen if the bacteria were given some nutrients to live on that they could digest easily and a warm moist place to grow? They would grow quickly, dividing until there was no open space on the given substrate—it would be considered a “lawn” if the substrate was a petri dish containing agar. ƒ What would happen if bacteria were given some nutrient to live on that was difficult for that particular organism to break down? The bacteria would grow more slowly or not at all if they were unable to obtain the proper nutrients from their environment. 2. Give your students an introduction to the ways in which bacteria have been used in biotechnology, from traditional uses to those techniques that have become common in the past few decades. Use the Background section of this activity as a guide. 3. Let the students know that they will be gathering data about this question: Do bacterial strains have the same success growing on media that offer different types of nutrients? Ask them if they can think of a few ways in which this question could be useful in biotechnology applications. There are numerous connections between bacterial growth nutrients and biotechnology. Here are a few ideas that you might hear: 60

(a) bacteria that grow well on high levels of nitrogen or phosphorus can be used to digest sewage, animal manure, agricultural runoff, or industrial waste; (b) bacteria that grow on petroleum products can be used to clean up refinery spills, oil-transportation spills, and leaking underground containers; (c) bacteria that grow on certain toxins can be used to metabolize those substances so they are reduced to an inert form; and (d) bacteria that can metabolize hydrogen sulfide as an energy source can clean up mining waste and reduce the acidity of the soil and water in these areas. 4. Ask the students what type of experimental procedure could be used to answer the earlier question. Encourage their ideas by accepting all suggestions while helping them mold the concepts into techniques that would work. Use questions to guide them to an experimental procedure that has consistency between sample groups, that tests one variable at a time, and that has a control. 5. Hand out Student Page: Growing Bacterial Strains on Various Media—Lab Experiment to each student, and have them reinforce the scientific process of experimentation by filling out the page on the basis of the procedure that follows next or one that you and your students have created. 6. Demonstrate the sterile technique that the students will use to spread the bacterial cultures on each media plate. You may want to refer to Student Page: Using Sterile Technique to Inoculate Bacterial Plates to review the procedure yourself and as an information sheet for your students to follow. Ask each student to sketch the separate steps of the process on Student Page: Growing Bacterial Strains on Various Media—Lab Experiment before they begin the procedure. 7. Provide each lab group with a different bacterial strain to spread on four different nutrient media petri dishes. Each bacterial strain grows best on a particular type of agar, depending

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on the nutrients available in the agar. First, the nutrient agar is a standard agar that can be used to grow most bacteria because it contains a range of common nutrients in a form that is accessible to most bacteria. Second, the brain heart infusion agar is a medium that is begun with a base of nutrient agar or tryptic soy agar that has some additional nutrients taken from mammalian blood at a concentration of 5–10 percent. That agar encourages the growth of bacteria that are pickier or more delicate in their tastes. Third, the tryptic soy agar has the same nutrients as the nutrient agar but contains fewer nutrients than the brain heart infusion agar; it is intermediate to those two agar media.

Fourth, the MacConkey agar is a specialized agar that allows scientists to select against Gram-positive bacteria. Gram-negative bacteria will grow on this agar, whereas Gram-positive bacteria are inhibited by the bile salts and crystal violet that are added to this medium. A pH indicator that is added to the MacConkey agar will allow for the differentiation of lactose-fermenting and non-lactose-fermenting bacteria. Gram-negative bacteria that ferment lactose for consumption will turn pink, whereas Gramnegative bacteria that cannot ferment lactose will grow well but the colonies will remain translucent.

8. Ask the students to label their media plates with (a) the type of medium, (b) the bacterial strain that will be spread onto that plate, (c) their lab group number, and (d) the date. 9. Let the students know that all strains will grow on at least one type of media plate. This plate will be the positive control for each bacterial strain and the positive control for the media. To help the students consider why they are performing this activity, ask them the following questions: ƒ Do you think that all the different types of bacterial strains will grow on each type of media? No.

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ƒ How much diversity do you think there is in bacteria compared with the biodiversity you see in the animal kingdom or the plant kingdom? Bacteria have a wide range of forms, nutritional needs, and habitat preferences. There are also some generalist strains and some specialist strains just as you see in the animal and plant kingdoms. ƒ Why are positive controls are necessary? We need to be certain that the bacteria have not died in shipping and that they are alive and able to reproduce. 10. Ask the lab groups to designate someone in each group to be the copilot who directs the student who is doing the sterile technique using the steps that the group has sketched out. The others in the lab group should watch the student spreading the plates to ensure that all plates are spread using the same technique. Each student in the lab group should take a turn copiloting the technique and a turn performing the technique until all four plates have been spread with bacteria and each lab member has had an opportunity to play each role. Although this is an excellent opportunity to teach your students the lab skills they will use in microbiology, the bacteria that have been chosen are fairly easy to grow and will not be greatly inhibited by the quality of your students’ ability to spread the plates evenly.

You may see some differences in results between the lab groups or from the chart that is included in this procedure if the students do not inoculate the media plates very well. You can use these discrepancies to allow the students to discuss their technique and the factors that may have affected bacterial growth. Do stress that the students should not allow the bacteria to touch anything other than the media plate and that all inoculating loops, sealed culture tubes, and other materials must be place directly in a trash container that will be doused in bleach before disposal. Students should wear gloves and wash their hands thoroughly after they have finished the procedure. 61

11. After the plates have been completed, have the students wrap the edge of the plates in Parafilm (or tape and plastic wrap), and then place all the plates (except the Serratia marcescens D1) upside down in the incubator overnight at 37° C. It is very important that all media plates are completely sealed and are not opened from this point forward. If you do not have an incubator, all media plates grow well at room temperature, but some strains may need 2–6 days to develop visible growth. The Rhodospirillum rubrum needs to be placed under a light or where it can get some sunlight without getting too hot. As it grows, it will become deep red and turn to deep purple if allowed to grow for 2–4 weeks at room temperature. The plates must be tightly sealed with Parafilm (or tape and plastic wrap) in order for the color to develop. Serratia marcescens D1 will grow deep-red colonies (without light), which will turn deep purple with time; however, it must be grown at room temperature for these results (at 37° C, colonies will appear white).

Because some of the bacterial strains are pathogens that can inhabit the human body, from this point on the bacterial plates should not be opened again by any student for any reason. 12. Introduce the concept of oil-consuming bacteria by describing the molecular structure of the hydrocarbon chains that make up all petroleum products. The simple hydrocarbons depicted next can be drawn on the board or constructed as three-dimensional models with an organic chemistry set. Remind the students that the carbon–carbon bond has a fairly high amount of harnessed energy, and therefore energy is released when the bond is broken. This release of energy is what allows carbon molecules to be a source of fuel for organisms and machinery. You may want to share the background information in the instructions for the Edvotek bioremediation using oil-eating bacteria. H H C H H

H H H C C H H H

H H H H C C C H H H H

Methane

Ethane

Propane

(H = Hydrogen, C = Carbon, — indicates a chemical bond between atoms) 13. Perform the oil-consuming bacteria demonstration according to the first step of directions included in the kit: Parcel out a portion of the solution into a flask for each lab group. Each lab group should add 1.3 grams of oil-eating bacteria powder to a clean 250-milliliter flask containing 125 ml of tap water. The flasks can be covered loosely with a piece of foil and placed on a stir plate to aerate the contents. If the cultures are allowed to grow overnight, the students should see a milky suspension in the flasks that indicates the presence of a healthy culture. Students can then add different food or mineral oils to each flask, while one group maintains a control flask with no additives. Bacterial plates after 2 days’ growth at room temperature 62

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14. Ask your students to record their observations under the results section of Student Page: Growing Bacterial Strains on Various Media— Lab Experiment as they watch the reaction develop. The bacteria will need to grow for 7–14 days in order to have enough time to consume a visible quantity of oil present in the flask and to produce a visible by-product of cell respiration. Students can place the flasks in a quiet area of the classroom on a stir plate or a shaking platform to aerate the cultures and can take a few moments each day to make observations. Depending on the ambient temperature of your lab, your bacteria media plates may need 2–7 days to grow to their optimal observation stage.

Preparation of oil-consuming bacterial cultures

Oil-consuming bacterial cultures with the addition of various types of oil

15. When you feel that the bacteria have grown to an adequate stage, draw a data chart on the board similar to the chart on Student Page: Growing Bacterial Strains on Various Media— Lab Experiment, and ask the class what results were recorded in each box. The expected results are noted in parentheses in the Table 2.1.

Table 2.1 Record of Bacterial Growth for Various Strains and Media Lab Nutrient Brain Heart Tryptic Soy Bacterial Strain Group Agar Agar Agar Escherichia coli (+) (+) (+)

MacConkey Agar (+) fuschia

Rhodospirillum rubrum—seal plates tightly to get good results

(+) clear

Will grow but can be difficult

(+) deep red

(–)

Pseudomonas fluorescens

(+)

(+)

(+)

Enterococcus faecalis

(+)

(+)

(+)

(+) clear, green tint (–)

Branhamella catarrhalis

(–)

Serratia marcescens D1

(+) red

Will grow but can be difficult (+)red

Will grow but can be difficult (+)red

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(–) (+)red

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16. Begin a discussion about the results on Student Page: Growing Bacterial Strains on Various Media—Lab Experiment by asking the students to draw some inductive and deductive conclusions. Use the last few questions to include their observations of the oil-consuming bacteria. ƒ If a particular bacterial strain did not grow on a certain medium, how do we know the reason is the lack of appropriate nutrients instead of a flaw in the bacteria (e.g., maybe the strain died during shipping)? Because there should be at least one positive control for each strain—each strain should have grown on at least one agar medium.) ƒ If the bacteria did not grow on the medium, how do we know that the medium was not the problem (contamination, temperature, etc.)? Each medium also had a positive control— each medium should have at least one strain that grew on it well. ƒ Which type of agar contained the best variety of nutrients? Nutrient agar will likely have grown the most types of bacterial strains because this type of agar is not selective and contains a wide variety of nutrients in forms that are easy for most bacteria to use. ƒ What types of abiotic and biotic factors do bacteria need to grow? For example, oxygen, a steady temperature (usually warm), moisture/humidity, light, a source of carbon, phosphorus, nitrogen, and micronutrients, etc. ƒ How do we determine the exact combination of abiotic and biotic factors needed by different strains of bacteria? We can use a scientific experimental process to find the ideal conditions for any particular strain.

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ƒ If we had an area that was contaminated with PCBs (polychlorinated biphenyls—a toxic chemical used as heating and cooling fluids and in the production of plastic coating of electrical wires) and the bacteria that naturally occurred in that area were slowly breaking down those chemicals, how could scientists support the local population to allow the bacteria to multiple and flourish? They could try to grow the bacteria on different media to see which nutrients the bacteria needed and then add those nutrients to the contaminated soil; this process is called biostimulation. ƒ Would it be best to use the local bacteria of an area to clean up waste products or to add bacteria cultures to the soil or water that have been grown in a laboratory setting for a specific purpose (this is termed bioaugmentation)? Answers will vary; some lab strains will be outcompeted by the local organisms. If the local organisms can consume the waste, it would be best to help them flourish instead of introducing a nonnative species or having to grow and give nutrient support to a specialized strain. ƒ What might be some of the problems with introducing a population of bacteria to soil or water where they do not currently exist? There are many problems that may occur from the introduction of a nonnative species: (a) the population may grow out of control; (b) it may not have a natural predator or limiting factor; (c) it may outcompete other organisms for resources; or (d) it may spread to other regions nearby or far away to disrupt other ecosystems, etc.) 17. After the oil-consuming bacteria have been observed for 1 or more weeks, ask your students to send one person from each lab group to the board to share the observations they are making on the oil-consuming bacteria. This study skills technique will allow students to

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confirm their own observations and to notice things that other groups might have missed, while pressing them to be more astute than the other lab groups in order to make unique contributions. 18. Discuss what the students are observing to verify that they understand the main points of the demonstration. You might use the following questions to look for comprehension and misconceptions: ƒ Why are the bacteria breaking down the oil? They are using proteins to break down the carbon chains for use in cell respiration to create ATP—the carbon chains are a source of energy just as the carbohydrates consumed by humans are carbon chains used for energy. ƒ Have you seen a difference in the amount of oil in the container over time or the clarity of the solution over time? The solution should become clearer, and the amount of oil should be reduced as it is consumed. ƒ Name a few oil-based products that bacteria similar to these could break down. Oil from drill rigs and oil spills from shipping and transport, plastic containers, fleece, polypropylene, and other carbon polymer clothing, oil-based solvents, oil-based paints, etc. 19. Press your students to draw some inductive and deductive conclusions about the possible uses of this strain of oil-consuming bacteria. Here are some Socratic discussion questions you might use to stimulate and encourage critical thinking and scientific reasoning:

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ƒ Do you think these bacteria eat only petroleum products, or do you think they could survive or thrive on other types of nutrients? Most bacteria can survive on many different nutrients; the students should be able to reach this conclusion based on Student Page: Growing Bacteria on Various Media—Lab Experiment. ƒ What happens to the petroleum product after the bacteria have consumed it? It is broken down into carbon dioxide, water, and energy in the same way that our carbonbased food is metabolized. ƒ Many Superfund sites have metal wastes such as mercury, cadmium, and lead. Do you think it might be possible for bacteria to break down metals? Yes, it is possible. ƒ What do we do with the bacteria colonies that have consumed, and so contain a persistent substance like a metal? These colonies can be incinerated to recover the metals in a concentrated form. 20. If you have time, you may want to have your students fill out Student Page: Oil-Consuming Bacteria—Lab Experiment to reinforce the skills of designing a scientific experiment. Although the students may not know all the techniques necessary to conduct proper microbial experiments, they can suggest thoughtful ideas that are supported by logic and consistent use of the scientific process.

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STUDENT P AGE Using Sterile Technique to Inoculate Bacterial Plates A sterile, or aseptic, technique is used to prevent microbial organisms from contaminating any surface other than the specific location where they are being transferred or encouraged to grow. To keep bacteria from growing where they are not wanted (surfaces such as your hands, desk, or media plates where they were not intended), the bacteria—and anything that touches the bacteria— must not touch any additional surfaces. To grow a particular bacterial strain on a specified media plate, you will use a disposable or flame-resistant inoculating loop (these instructions will cover the use of a disposable plastic inoculating loop). Read all the steps below, and study the pictures carefully. Perform a “dry run” of this technique with a partner while he or she watches and points out improvements. When you are ready, follow the steps below, paying very careful attention not to touch any surfaces with the inoculating loop, the bacterial tube, or the cap of the bacterial tube. If you are to keep the media plates, culture tubes, and inoculating loops free of any additional unwanted organisms, it is important that they remain closed at all times, except when the bacteria are being transferred. The photos in this series are taken with ungloved hands so the students can get a clear view of the position of the fingers. All students should wear gloves and should wash their hands and fingernails with soap and water after the procedure. 1. Arrange the media plates, bacteria source, inoculating loops, and trash receptacle within easy reach. Do not open the inoculating loops, culture tubes, or media plates. Label the media plates with (a) the date, (b) the bacterial strain, and (c) the medium name before beginning. 2. The inoculating loops should remain in the sterile packaging. Touch them only at the center of the handle with gloved hands. Never touch either end to anything other than the bacterial culture tube or a media plate. Read the rest of this procedure so you understand what you will do with the inoculating loop.

One end of the inoculating loop will be used to dip into the bacteria growing in the culture tube. This end will be used to make a zigzag pattern (six to eight times back and forth) on half of the media plate. The inoculating loop can then be turned over so the other end can be used to slash across the zigzag one time and to make a second zigzag pattern across the remaining half of the media plate.

Second zigzag streak using the opposite end of the inoculating loop

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First zigzag streak of the inoculating loop

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STUDENT P AGE Using Sterile Technique to Inoculate Bacterial Plates (continued)

This technique will smear the bacteria from the culture out across the medium in a thin film during the first streak, and then the second streak will spread a small number of bacteria away from the others to allow the growth of individual colonies.

3. The bacterial culture tubes should be opened only briefly by lifting the cap just enough to slide the inoculating loop inside without touching the loop to the cap. The cap should never be set down in the process, and the tube should be sealed immediately after the loop is removed. You may do this procedure using two people, or you can practice the technique of unscrewing the top and holding the cap and tube in one hand while you work with the inoculating loop.

4. The media inside the plates should never be touched by anything except the inoculating loop as it adds the bacteria. The covers to the media plates should always remain closed and should be lifted only enough to allow the inoculating loop to spread the bacteria. When the lid is lifted, it should remain facing down and should hover over the bottom of the plate to minimize the chance of contaminating fungal or bacterial particles entering. The lid should never be turned over or set on the table and should be closed immediate after inoculation.

5. After a single plate has been inoculated, the inoculating loop should go directly into a trash receptacle. A new loop will be needed to inoculate the next media plate using the same technique. After all the plates have been inoculated, the plates should be taped shut using Parafilm® and plastic wrap or tape. The Rhodospirillum rubrum bacterial plates will need to be taped so they are airtight in order to encourage the bacteria to grow anaerobically. None of the media plates should be opened for any reason after this point in the procedure.

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STUDENT P AGE Growing Bacterial Strains on Various Media—Lab Experiment Introduction Provide the information requested below. What is the question that you are asking in this experiment? Give two possible answers to the question above (these are your hypotheses). 1. 2. The null hypothesis for this experiment is this:, Different strains of bacteria will grow equally well on various types of nutrient media. After we have seen the results of this experiment, we will either accept or reject the null hypothesis. At this point (before you have seen any experimental results), do you think the null hypothesis will be accepted or rejected? Using three examples, explain how this lab experiment could help a scientist use bacteria in biotechnology. Methods Fill in the information requested below. Materials—List the materials you need to perform this experiment. Procedure—Make a quick sketch of each step in this procedure: Setting up:

Spreading the bacteria on the plates:

Cleaning up:

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STUDENT P AGE Growing Bacterial Strains on Various Media—Lab Experiment (continued) Results Fill in the data chart with a plus (+) symbol if the bacteria grew or a minus (–) symbol if the bacteria did not grow. Record observations about your results in the space below. Include color, thickness of colonies, and any other details. Data chart:

Name of Bacterial Strain Escherichia coli Rhodospirillum rubrum Pseudomonas fluorescens Enterococcus faecalis Branhamella catarrhalis Serratia marcescens D1

Lab Group

Nutrient Agar

Brain Heart Tryptic Soy Agar Agar

MacConkey Agar

Observations on the plated media: Observation on the oil-consuming bacteria: Conclusion Answer the following reflection questions. Explain why some bacterial strains grew on certain nutrient media but did not grow on other nutrient media. Which strains of bacteria were able to grow on the most types of media? Which bacterial strains appeared to have more specific nutrition or environment needs?

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STUDENT P AGE Growing Bacterial Strains on Various Media—Lab Experiment (continued) Explain how positive controls were used in this experiment. Revisit your hypotheses in the Introduction section. Explain which predictions were true, and back up your explanations with data from the experiment. Explain which parts of your hypotheses were not quite accurate, and back up your explanations with data from the experiment. Now that you have seen the results of this experiment, would you accept or reject the null hypothesis? What types of errors occurred or could have occurred to alter the results? If you were to perform another experiment using the same materials, what question would you test? What does the variation in growth of these bacteria suggest about the growth habits of bacteria that occur naturally in soil or water of an ecosystem? If you were growing a bacterial strain in the lab on one particular type of nutrient agar and you found that those bacteria could consume plastic grocery bags as a carbon energy source, what are some ways that you could encourage those bacteria to help clean up a landfill?

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STUDENT P AGE Oil-Consuming Bacteria—Lab Experiment Introduction Oil-consuming bacteria have a number of interesting applications such as detoxifying landfill waste, cleaning up oil spills, or decontaminating groundwater. To better sell these specialized organisms to companies that need them, you would need to quantify how much of a particular substance the bacteria can consume over a given time. Design an experiment that addresses the following question: How much oil does this bacterial strain consume in a given time? Methods List the materials you will need for this experiment: Write out the steps of your lab procedure next, and then draw the steps of the procedure on a large piece of poster board to present the experiment to the class. Procedure: 1. 2. 3.



4. 5. 6. 7. 8. 9.

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STUDENT P AGE Oil-Consuming Bacteria—Lab Experiment (continued) What are the variables in this experiment? What are the controls? Results Draw a data table that would be used to collect the information you expect to get from your procedure.

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Part C: Wastewater Field Trip getting ready ƒ Order transportation and obtain permission for students to tour the local municipal wastewater treatment facility. ƒ Copy Student Page: Municipal Wastewater Treatment Facility Field Trip for each student.

doing the activity 1. Gather students on the bus for a tour of the local wastewater treatment facility. Encourage the students to bring cameras or camcorders to record the steps of the process (if permitted by facility). If you are unable to take your class on a field trip, you may choose to do a virtual field trip by showing a video that describes the process of wastewater treatment (Carolina Biological Supply Company offers a video called Affluent Effluent, #49-1220V, and there are many short videos on www.youtube.com). 2. On the bus, pass out Student Page: Municipal Wastewater Treatment Facility Field Trip, and ask the students to read the background information and the reflection questions before they arrive at the facility.

Activity 2: Bioremediation © American Forest Foundation

3. While the students are touring the facility, remind them to fill out the answers to questions on the student page, and consider any additional questions they may have. 4. After they have toured the facility, ask your students to sketch a map of the facility before they leave. Require them to finish any remaining reflection questions on the bus on the return to school so they can turn in this assessment when they arrive.

enrichments ƒ Perform some of the lab experiments that your students suggested in the Conclusion section of Student Page: Growing Bacterial Strains on Various Media—Lab Experiment or some of the experiments created for Student Page: OilConsuming Bacteria—Lab Experiment (see part B). ƒ Career Connection: Visit a microbiology research lab at a university or in private industry to introduce students to the equipment, the research questions, and the daily work lives of people in this field of science. Ask a microbiologist to come to your class as a guest speaker to talk about current research and job opportunities in the field.

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STUDENT P AGE Municipal Wastewater Treatment Facility Field Trip 1. How many people and businesses are served by this plant? 2. What is the maximum capacity of this plant? 3. Where does the water for this plant come from? 4. Where does the effluent water go after it has been treated? 5. Describe the primary treatment of wastewater at this plant. What are some other types of primary treatment that would be possible? 6. Describe the secondary treatment of wastewater at this plant. What are some other types of secondary treatment that would be possible? 7. Describe the tertiary treatment of wastewater at this plant. What are some other types of tertiary treatment that would be possible? 8. What are the resulting products of the treatment process? 9. What is the sludge made of? 10. How much sludge is produced each day? 11. What is done with the sludge? What else could be done with the sludge? 12. What are suspended solids?

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STUDENT P AGE Municipal Wastewater Treatment Facility Field Trip (continued) 13. How are suspended solids removed? 14. How do the scientists at this facility quantify the amount of suspended solids that have been removed? 15. What are dissolved solids? 16. How are dissolved solids removed? 17. How do the scientists at this facility quantify the amount of dissolved solids that have been removed? 18. Which are more difficult to remove, suspended or dissolved solids? 19. What living organisms are used at this facility for bioremediation? 20. What does this facility do to encourage these organisms to grow and flourish?

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STUDENT P AGE Municipal Wastewater Treatment Facility Field Trip (continued) 21. What types of substances are in the wastewater that cannot be removed by this treatment facility? 22. If this facility were located in an agricultural region where large quantities of fertilizers were applied to the land, how could the wastewater facility be adapted to remove excess nitrogen and phosphorus nutrients using bioremediation? a. Describe some ways that treatment process could benefit the environment. b. Considering the definition of biotechnology to be “any technological application that uses biological systems, living organisms, or derivatives thereof to make or modify products or processes for specific use,” how does this plant use biotechnology in the treatment of sewage? c. Describe something interesting that you learned on this field trip that you did not expect to learn. d. Write one additional question that you asked on this tour:

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Activity 3: Biotechnology and Human Health Biotechnology plays a role in human health. Examples include using biotechnology to diagnose disease, prevent disease through the use of vaccines, and treat disease (e.g., genetic therapy). In this activity, students simulate the spread of disease, learn about different types of vaccines and the controversies surrounding them, and explore how genetically engineered plants can be used to produce a new class of edible vaccines. Subjects: Biology, AP Biology, Environmental Science, AP Environmental Science, Health Sciences, Social Studies Concepts: 1.4, 2.3, 2.8, 3.5, 5.6 Skills: Discussing, Identifying Relationships and Patterns, Reasoning, Researching Materials: baking soda, test tubes or small cups, pH indicator (phenolphthalein), copies of student pages, computer, PowerPoint software, projector Time Considerations: Preparing the Activity Part A: 15 minutes Part B: 15 minutes Part C: 30 minutes Part D: 30 minutes Doing the Activity Part A: Two 50-minute periods Part B: Two 50-minute periods Part C: Two 50-minute periods Park D: Two 50-minute periods

Objectives: Students will (a) model the transmission of an infectious disease; (b) research the types of vaccines they have received; and (c) explore the development, use, risks, and benefits of different types of transgenic plants. Assessment Opportunities: ƒ To assess the students’ understanding of how diseases spread, have the students draw a logistic curve and label (a) what the carrying capacity is, (b) where accelerated growth is represented, and (c) where decelerated growth is represented. Ask your students to predict what the graph would look like if there were 10 interactions and if there were 20 interactions. Ask them to explain the shape of the curve. Plasmids are small, circular, extra-chromosomal pieces of DNA that are typically

double-stranded and are found in bacteria. Because plasmids replicate autonomously and do not undergo recombination, they can pass on the inserted gene without interruption. – Use the rubric provided on page XX to assess student presentations in Part C. You may also want to use the Group Evaluation Form in Appendix XX titled “Successful Cooperative Learning” to help assess individual contributions to group work. – Use the rubric provided on page XX to assess student presentations in Part D. You can also allow the students an opportunity to assess the group presentations by having them also use the rubric provided.

background Everyone has heard the word vaccine. But how many people have really thought about what it means? Why do we get the vaccines that we do? And what exactly are we being vaccinated against? A vaccine is a foreign agent (antigen) that is injected into the body. Vaccines are developed through the use of biotechnology. The body’s immune system responds to this foreign agent, thereby triggering an immune response. The idea behind a vaccine is that the level of exposure is too low to cause serious disease, but it is high enough to trigger the immune system. Essentially, vaccines introduce your body to a disease agent, thus preparing it to fight off the agent more effectively in the future. Why don’t vaccines themselves cause the disease? The answer depends on the type of vaccine used. There are several different types of vaccines, including whole organism (dead or attenuated), partial organism (consisting of a subunit of the disease-causing agent), and recombinant vaccines (made from DNA).1 The first type (whole organism) contains whole disease-causing microorganisms that have been killed by scientists (through the use of heat or chemicals). Those types of vaccines pose no risk of infecting the patient with the actual disease, because the injected vaccine contains only dead microorganisms.

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However, just the presence of those microorganisms—even though they are no longer living—can initiate the desired immune response, thus preparing the body against future infection. The second type mentioned earlier is an attenuated vaccine. Attenuated vaccines contain a living sample of the disease-causing microbe; however, they have been treated to make them less virulent (that is, they are no longer as dangerous). Those types of vaccines work well to provoke a strong immune response so that the patient is capable of fighting off the disease in the future. Attenuated viruses can, however, cause some people—particularly those with compromised immune systems (the very young; the elderly; those with HIV, diabetes, and other diseases)—to become sick. Therefore, attenuated viruses are most often recommended for people who are healthy and have a strong immune system. A third type of vaccine is the subunit vaccine. Those vaccines carry only a part of the diseasecausing microorganism (typically a surface protein or capsid) that the body will react to. Because those vaccines do not carry the entire microorganism, there is no chance of contracting the actual disease. Recombinant vaccines are the most recent form of vaccinations. They work by using a type of vector (viral, bacterial, plasmid) to deliver a piece of DNA (a gene) that codes for (directs the synthesis of) the protein from a disease-causing organism. Those types of vaccines are currently being researched for use against diseases such as HIV and cancer. Transgenic plants can be used for both the production and delivery of recombinant DNA vaccines.2 Edible vaccines, that is vaccines produced and delivered in a plant-based product, are currently being investigated for diseases such as measles, cholera, foot-and-mouth disease, and hepatitis B and C.3 A variety of risks and benefits are associated with edible vaccines. Risks include contamination of the food supply, transfer of genes to nontarget organisms, and development of oral tolerance.3 Benefits include reduced production and delivery costs, ability to remain stable at room tem-

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perature, elimination of safety concerns associated with needles, and increased compliance.3 The use of vaccines to prevent disease is not without controversy. Many people are concerned that the dramatic increase in immunologic and neurological disorders in recent years is linked to the widespread practice of immunization. For more information about the concerns of vaccinations causing illness, go to www.cdc.gov/ vaccines/vac-gen/safety/. In addition to preventing disease, biotechnology is also used to detect disease. One type of biotechnology test is called the enzyme-linked immunosorbant assay, or ELISA. The ELISA can detect the presence of an antigen or an antibody in a sample, thus making it useful for detecting various diseases (for example, HIV and West Nile virus), as well as food allergens (for example, nuts and dairy). Pregnancy tests are another use for an ELISA. The basic steps of an ELISA test for HIV are as follows: First, antibodies for HIV are mixed with a serum sample taken from a patient. Those antibodies will bind with any HIV antigens present. Second, researchers add an enzyme that will result in a color change if the antibodies have combined with the antigens. In this case, a color change indicates a positive response (the patient has been exposed to HIV). The absence of a color change indicates that no HIV antigens were detected. The ELISA is only one type of biotechnology that is used to detect disease; there are many others, including the polymerase chain reaction and gel electrophoresis. Biotechnology can also be used to treat diseases. Scientists are researching ways to manipulate and modify people’s genes to cure genetic illness. This type of work is referred to as gene therapy. The main difficulties associated with gene therapy include the short-lived nature of genetic therapy, the risk of a negative immune response with insertion of foreign DNA, the issues associated with viral vectors used to deliver the DNA, and the fact that many genetic disorders are multigenic (that is, they are not caused by a single gene).4

project learning tree Exploring Environmental Issues: BioTechnology © American Forest Foundation

In addition to the scientific problems, many ethical issues are associated with gene therapy. For example, are all disabilities necessarily disorders that require a cure? Should somatic cell gene therapy (manipulations that cannot be passed on to offspring) be held to a different standard than is germ line cell manipulation (changes that can be passed on to offspring)?4 Although no Food and Drug Administration–approved gene therapies are available in the United States, scientists are continuing to research gene therapy to cure genetically caused diseases. While the potential for genetic therapy to improve human health exists, care must be taken to give the ethical issues as much consideration as the scientific ones. Other emerging areas of biotechnology research focusing on human health include the use of stem cells and cloning to regenerate tissue and organs. Both those issues are at the center of much political and ethical debate. A solid understanding of the science behind those issues is crucial when forming your opinion about such issues.

endnotes 1. “Understanding Vaccines,” National Institute of Allergy and Infectious Diseases, 2008, www3. niaid.nih.gov/healthscience/healthtopics/ vaccine/PDF/undvacc.pdf. 2. “Human Genome Project Information,” U.S. Department of Energy Office of Science, 2009, www.ornl.gov/sci/techresources/Human_ Genome/medicine/genetherapy.shtml. 3. P. H. Lambert and P. E. Laurent, “Intradermal Vaccine Delivery: Will New Delivery Systems Transform Vaccine Administration?” Vaccine 26 (2008): 3197–208. 4. Diane E. Webster, Merlin C. Thomas, Richard A. Strugnell, Ian B. Dry, and Steve L. Wesselingh, “Appetising Solutions: An Edible Vaccine for Measles,” Medical Journal of Australia 176, no. 9 (2002): 434–37. 5. Carol O. Tacket, Marcela F. Pasetti, Robert Edelman, John A. Howard, and Stephen Streatfield, “Immunogenicity of Recombinant LT-B Delivered Orally to Humans in Transgenic Corn, Vaccine 22 (2004): 4385–89.

Activity 3: Biotechnology and Human Health © American Forest Foundation

Part A. Spread and Detection of Infectious Diseases In this activity, students will model the spread of an infectious disease. They will observe how biotechnology can be used to diagnose disease.

getting ready Before class, prepare a small solution of a contaminant (a basic solution). It will serve as the source of the infection. You can mix water with baking soda (1 tablespoon in 200 milliliters [ml] of water will allow detection of up to six exchanges) to obtain a basic solution. Alternatively, you can use 0.01 M of sodium hydroxide (NaOH) (dissolve 0.4 gram of NaOH in 100 ml of distilled water; this mixture will allow detection of at least eight exchanges). Obtain one test tube (or small cup or container) for each student in the class, plus two extra containers to serve as a positive control and a negative control. Fill two test tubes halfway (2 ml) with the contaminant (basic) solution. Set aside one for the positive controls, and use the remaining one as the source for the class activity. The tube that contains the “contaminant” or basic solution should be marked in an inconspicuous manner, so that only you are aware of who starts the activity with the contaminated sample. Fill the remaining tubes halfway (2 ml) with distilled water. Label one as the positive control. Obtain 120 ml of phenolphthalein (pH indicator). You will add approximately 25 microliters (μl) of phenolphthalein (about 1 drop) to each sample to test for contamination. Make enough copies of Student Page: Fluid Exchange Record Sheet for each student.

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doing the activity 1. Begin this activity by asking your students to raise their hands if they have ever had a viral or bacterial infection. Ask them how they think they became exposed to that particular illness. Did other members of their family develop the same illness? Ask them what factors can affect the transmission of a contagious disease. For example, the density of the population, the age and health of the population, the virulence of the disease. Tell your students that in this activity they will be modeling the spread of an infectious disease. You can use any infectious disease (such as the common cold, the flu, HIV). 2. Give each student a copy of Student Page: Fluid Exchange Record Sheet and a container that is filled halfway with liquid. One student should be given the “contaminated” (basic) water solution. You should record this information, but not let the students know who has it. Explain that one person in the room has been infected by a contagious disease, but you do not know who it is. Tell them you are going to mimic the spread of disease by mixing the contents of each student’s test tube. You will then conduct an assay to determine which fluids have been infected. In this activity, the assay is simply a pH-induced color change. One common assay that is used to detect disease in reality is an enzyme-linked immunosobant assay. For more information on ELISAs, see the Background section for this activity. 3. Have each student in the class exchange liquid with three different people, making sure to record the identity and order of contacts on the student page. When they exchange liquids, tell the students to combine two test tubes of liquid into a single test tube. This step will cause the liquids to mix. Then have them redistribute half the liquid into the other test tube. Students should end up with the same volume of liquid as when they started. Ask them if they can detect whether or not they have been infected. 80

4. Explain to the students that you have a solution that will change color in the presence of the disease. At this point, you can add the indicator (phenolphthalein) to the positive control so that the students can see the reaction. You can also add it to the negative control so that they see what happens when the “contaminant”isn’t present. Ask students to come to the “clinic” (front of room) and add several drops of the indicator solution to each test tube or set out the indicator solution and have the students add it themselves. Tell the students that a color change indicates a “positive” response (they have the contaminant in their samples). Have them fill out the results on their student page. At this point, you can introduce the topic of an ELISA test, which is an optional enrichment activity (see the Background section for more information on ELISAs). 5. Collect all the used test tubes (or cups). You may now decide to have the students repeat the experiment, varying the number of exchanges (for example, they can compare how the results change if they exchange fluid with only one other person). Please note that if you choose to do this experiment, you will need several sets of test tubes that are set up for each new round. 6. If you had students repeat the experiment varying the number of exchanges, you can ask them to graph their results. The independent variable is the number of exchanges on the x-axis, and the dependent variable is the number of infections on the y-axis. ƒ Ask your students to predict the shape of the curve on the graph as the number of exchanges increases. Have one student volunteer to draw his or her graph on the board. Ask the students to explain the shape of the graph. A logistic growth curve is an S-shaped curve, characterized by an exponential middle phase and a leveling off at the end. In the students’ experiment of disease transmission,

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Number of infections

the number of infections will eventually level off (reach carrying capacity) because only a finite number of students in the class can become infected.

Carrying capacity Exponential growth phase Lag phase

Number of exchanges 7. Use the following questions to assess the students’ understanding of the disease transmission activity and its connection to biotechnology: ƒ Ask the students how the real-life spread of diseases might differ from the way the “disease” spread in the activity they just completed. Possible answers could include that some people (older, younger, immunocompromised) might be more susceptible than others (i.e., exposure may not lead to infection); or airborne contamination; preventative measures (washing hands, covering mouths, etc.). ƒ What type of biotechnology was used in this activity? An assay (in this case, a change in pH detected by an indicator solution) was used to detect the presence of a particular “pathogen” (in this case, a basic substance). ƒ Ask the students to investigate the types of assays that are available “over the counter” to detect disease (or other physiological conditions). Activity 3: Biotechnology and Human Health © American Forest Foundation

Possible answers include pregnancy tests, glucose meters that test for blood sugar levels, kits that test for HIV, and kits that can detect the presence of specific drugs in urine. 8. As a final exercise, ask the students to try to work out the identity of the original carrier for that experiment using the combined information. Confirm their conclusions with the actual identity of the students who began with the contaminated liquid in each experiment. They may be able to narrow it down to only one or two students. ƒ Ask the students if they were surprised by the number of students “infected” after just a few exchanges.

enrichments ƒ Learn about Career Connection—Genetic Counseling Advances in medical biotechnology have necessitated the creation of a new type of job, a genetic counselor. Genetic counselors help people decide whether or not to conduct genetic testing, as well as help people interpret and work though the results of such tests. Genetic counselors must have a strong science background in order to understand and interpret the results of genetic testing. In addition, they must have excellent communication skills and must enjoy working with people. Decisions surrounding genetic testing often involve complicated personal and ethical issues, and it is the job of a genetic counselor not only to understand the science but also to understand how to help people through the emotional side of testing. More information about the field of genetic counseling can be found on the website of the National Society of Genetic Counselors at www.nsgc.org/. ƒ Perform an ELISA activity with your students. Information about how to purchase classroom kits that contain all the necessary materials to perform an ELISA activity can be found in Appendix XX.

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STUDENT P AGE Fluid Exchange Record Sheet Student Name: ___________________________________________________ In this activity, you are going to model how diseases can spread. You will be given a container full of a liquid. One person in the class will have a liquid that is already “infected.” You will combine your fluid with three of your classmates and then will use a biotechnology technique called an assay to determine if your sample has been infected. The assay involves the addition of an indicator solution that will cause your sample to change color if it is infected. 1. Examine the liquid in your container. Briefly explain what it looks like by describing the color. Then indicate the volume (for example, is your container half full, three-quarters full, etc.). 2. Pick a classmate and combine the liquid from your two containers into one. This action will cause the liquid to mix. If the infection is present in one liquid, it will spread to the other liquid. After mixing the two liquids, pour half back into the other container. You should each end up with the same volume of liquid as when you started. Record the name of the person you exchanged fluids with below, as well as the color of your liquid after the exchange. Person you exchanged fluids with: ____________________________________ Color of your liquid after exchange:_ __________________________________ 3. Repeat the fluid exchange with a different classmate, and record the information below. Person you exchanged fluids with: ____________________________________ Color of your liquid after exchange:_ __________________________________ 4. Repeat the fluid exchange for a third and final time. Make sure to pick someone you have not already exchanged fluids with. Record the information below. Person you exchanged fluids with: ____________________________________ Color of your liquid after exchange:_ __________________________________ 5. You are now ready to conduct an assay to determine if your sample is infected. First, you will want to test the assay to ensure that it works by using a positive and negative control. Your teacher has set up a single positive and negative control for the class. Record the results of the controls below, and write a brief description below each entry of what the results mean. (For example, what does a color change indicate? What does it mean if there was no color change?). Positive Control (circle one):

Solution changed color

Solution did NOT change color

What does this result indicate?

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STUDENT P AGE Fluid Exchange Record Sheet (continued) Negative Control (circle one):

Solution changed color

Solution did NOT change color

What does this result indicate? 6. Now, it is time to test your sample. Follow your teacher’s directions for adding the indicator solution to your samples. Record the results here: Experimental Sample (circle one):

Solution changed color

Solution did NOT change color

What does this result indicate? 7. Your teacher will ask how many students are infected. Write the total below. 8. How do you think this number might vary if you increased or decreased the number of exchanges? Explain here: 9. The following information was obtained from a class of 25 students who conducted the same experiment but varied the number of exchanges. Graph the data, making sure to label the axes. Number of Exchanges 1 2 3 4 5 6 7 8 9 10

Number of Infections 2 4 8 14 23 25 24 23 25 25

Dependent Variable

Independent Variable

Describe the shape of the curve that you drew. Why does it never exceed 25 infections?

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Part B.1. Vaccinations

ƒ Which of these are examples of biotechnology?

Students will research and learn about common vaccines and will discuss some of the benefits and risks of vaccines.

getting ready Make enough copies of Student Page: Types of Vaccines for each student. You may choose to make a single copy on a transparency and to show it to the entire class using an overhead projector. Internet access or library access will be necessary for students to conduct research. Alternatively, you can assign research as homework.

doing the activity 1. Generate a discussion about vaccines by asking your students to describe what they know about vaccines. Ask them to list some of the vaccinations they have received. Make a list of those vaccines on the board. 2. Next, ask your students if they know what each vaccine is designed to prevent. Add this information to the list on the board. 3. Next, ask your students if they know how vaccines work. Ask them if they know the differences among live, attenuated, and recombinant vaccines. Use the Activity 3 PowerPoint to introduce them to the basics of vaccines. This information will provide an opportunity for students to learn how the process of biotechnology is used in the area of disease prevention. 4. Allow the students time to read Student Page: Types of Vaccines. Generate a discussion using the following questions. Although students may not have an advanced understanding of some of the complexities associated with vaccines, these questions will generate thoughts about some of the basic difference among the vaccine types.

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All are examples of biotechnology. Biotechnology is the use of living organisms to produce useful products. ƒ What would be a potential advantage of introducing only a part of a disease-causing organism? It would not be able to actually cause a disease. ƒ What is a potential disadvantage of introducing only a part of a disease-causing organism? It may not be as effective at inducing an immune response as a whole organism. ƒ Do you think each method is equally safe? Why or why not? Safety can be measured as the risk of acquiring the disease. The safest methods involve introducing either dead microorganisms or subunits of the microorganisms. They are used because there is then zero risk of becoming infected. However, safety must also be weighed against effectiveness. For example, attenuated vaccines are often more effective than dead vaccines in stimulating a defensive response. In cases where individuals are healthy, they may be able to effectively fight off an attenuated version of the disease. However, the same vaccine may not be tolerated as well by individuals who are less able to produce a strong defense, such as the very young, elderly, or immunocompromised individual. ƒ Ask the students if they have heard of any controversy surrounding vaccines. Controversies associated with vaccines center on potential side effects (such as neurological and immunological disorders), as well as on individual rights (many vaccines are legally required).

project learning tree Exploring Environmental Issues: BioTechnology © American Forest Foundation

ƒ What are the challenges associated with making vaccines for diseases such as the flu or HIV? Viruses such as the flu and HIV rapidly mutate. Therefore, the challenge is developing a wide variety of vaccines that can respond to the various strains. 5. Return the students’ attention to the list generated in step 1. Ask them if they think the list is complete. The list generated by your students will likely be quite short and will miss a lot of information. Give students an opportunity to research on their own what types of vaccines they have received. They can do this by searching the Internet for information, consulting the school’s admittance policy, contacting their doctor, and so forth. 6. Once your students have had an opportunity to gather more information on the topic, bring them back together as a group, and fill out more information on the chart that was started in step 1. You may add a column to indicate whether this vaccine is optional or required. 7. Next, ask your students to pick a vaccine to research and to use the information they find so they can make an informational poster. You may choose to have students work in pairs for this activity, but they can also work individually. Tell your students that you would like for them to address the following questions through their research:

Activity 3: Biotechnology and Human Health © American Forest Foundation

ƒ What is the vaccine designed to prevent? ƒ How is the vaccine made? ƒ How does the vaccine work? ƒ Is the vaccine live, attenuated, or recombinant? ƒ Is the vaccine required for all people? If not, explain. ƒ At what age is the vaccine given? Are boosters required? ƒ What are the benefits of the vaccine? ƒ What are the risks associated with the vaccine? ƒ What are two debate topics associated with your vaccine? 8. Hang the posters around the room, and allow the students to walk around and to read each poster. Finally, ask each student to give a brief presentation (less than 5 minutes) to the class about the vaccine he or she chose to study, highlighting what it does, how it works, and any controversies surrounding it. Leave a minute for questions from the class. If multiple students have chosen the same vaccine, you may want them to give a group presentation.

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STUDENT P AGE Types of Vaccines1 Whole Organism

Whole organism is the first type of vaccine invented. The idea is to expose a healthy individual to a mild form of a disease, allowing the body to generate antibodies. The production of antibodies will allow the body to mount a strong defense if it is exposed to the same disease in the future. With this type of vaccine, the entire disease-causing organism is introduced into the body of a healthy individual. However, it is modified so that it is not as virulent (disease causing) as the original. This modification is often made by using heat or chemicals. If the modification process actually kills the organism, it is considered a dead vaccine (this is how the original polio vaccine was created). If the organism has been weakened, but not completely killed, it is referred to as an attenuated virus (such as the common measles, mumps, and rubella vaccine,).

Partial Organism

A partial organism vaccine is referred to as a subunit vaccine. This type of vaccine contains only a part of the disease-causing organism. For example, certain proteins are found on the surface of organisms; it is those proteins that antibodies recognize. If only the surface proteins are introduced into a healthy individual, the body is essentially “‘tricked” into making antibodies, even though the actual disease-causing organism is not present.

Recombinant Vaccine

Recombinant vaccines are similar to subunit vaccines in that they “trick” the body into thinking it is being invaded by a specific microorganism. The body then responds by producing antibodies against that organism. However, no actual parts of the disease-causing organism are injected. Instead, genes that code for the proteins made by disease-causing organisms are injected. The genes then produce the proteins, triggering the body’s immune response. The vaccine for hepatitis B is an example of a recombinant vaccine. Endnotes: 1. “Understanding Vaccines,” National Institute of Allergy and Infectious Diseases, 2008, www3.niaid.nih.gov/ healthscience/healthtopics/vaccine/PDF/undvacc.pdf.

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Part B.2. Online Enrichment Students will learn about a recently introduced vaccine for human papillomavirus (HPV).

getting ready Review the “Suggestions for Conducting a Debate”—Option 2: Informal Debate (Focus on Risk module’s Appendix 5). Make enough copies of Student Page: Gardasil® for each student.

doing the activity 1. Ask your students to list some of the vaccinations they have received. Next to each type of vaccine, list the type of disease or illness it is designed to prevent. 2. Ask your students if they have heard of any controversies surrounding any of the vaccines. List their examples on the board or introduce the few below if they have not come up with those examples on their own. Examples include (a) MMR (mumps, measles, and rubella) shots and autism;1 (b) pertussis vaccine (whooping cough) and brain damage;2 and (c) neurological disorders associated with Menactra® meningococcal conjugate vaccine.3 3. With your students, investigate one or more of the controversies using the websites listed. Identify the perspectives presented in the controversy and the data supporting the perspectives. Ask students if enough information is presented for them to make a decision on whether they would have the vaccination. If not, what further information would they want?

Activity 3: Biotechnology and Human Health © American Forest Foundation

4. Pass out copies of Student Page: Gardasil® to your students, and ask them to read it. Ask them if they think any controversies are associated with this vaccine. List any ideas they come up with. Pick one or two of the most common ideas. Split the class into two groups so they may have an informal debate on the idea(s), with one team being pro-vaccine and the other team being anti-vaccine. Sample debate topics include the following: ƒ Will vaccinating young girls encourage sexual activity? ƒ Why is there a need to vaccinate girls at an age when they are unlikely to be sexually active? ƒ Should parents have the option of not vaccinating their child against this virus for nonmedical reasons? ƒ Should a vaccine be developed for males?

endnotes 1. “Measles, Mumps, and Rubella (MMR) Vaccine and Autism Fact Sheet,” Centers for Disease Control and Prevention, July 5, 2007, www. cdc.gov/vaccinesafety/concerns/mmr_autism_ factsheet.htm. 2. Alan R. Hinman, “The Pertussis Vaccine Controversy,” Public Health Reports 99, no. 3 (May–June 1984): 255–59, www. pubmedcentral.nih.gov/picrender.fcgi?artid=14 24579&blobtype=pdf. 3. “GBS and Menactra Meningococcal Vaccine,” Centers for Disease Control and Prevention, February 26, 2008. www.cdc.gov/vaccinesafety/ concerns/gbsfactsheet.htm.

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STUDENT P AGE Gardasil® Background

Gardasil® is the first vaccine developed to prevent cervical cancer and genital warts caused by a human papillomavirus (HPV). In June 2006, Gardasil® was licensed by the Food and Drug Administration (FDA). The Centers for Disease Control and Prevention and the FDA monitor the safety and effectiveness of the vaccine. What is a human papillomavirus? An HPV is a common virus that is passed on through sexual contact. Most of the time, an HPV has no symptoms so people do not know they have it. There are many different strains or types of HPVs. Some types can cause cervical cancer in women and can also cause other kinds of cancer in both men and women. Other types of HPVs can cause genital warts in both males and females. In most people, HPVs go away on their own and do not cause health problems. Experts do not know why HPVs go away in some cases, but not in others. How common are HPVs? HPVs are the most common sexually transmitted infections in the United States, with about 20 million people currently infected. Women have an 80 percent chance of getting an HPV by the time they are 50. Every year in the United States, about 6.2 million people get a new HPV infection. HPVs are most common in young people who are in their late teens and early twenties. How common is cervical cancer? The American Cancer Society estimates that in 2007 more than 11,000 women in the United States will be diagnosed with cervical cancer and approximately 3,600 will die from it. What is the HPV vaccine? The vaccine Gardasil® is the first vaccine developed to prevent cervical cancer and genital warts from an HPV. It works by protecting against the four types of HPVs that most commonly cause these ailments. The vaccine is given as an injection into muscle in three doses. The vaccine is licensed by the FDA for girls and women ages 9 through 26. How is the HPV vaccine made? Gardasil® is a recombinant protein that is produced by a strain of yeast that has been genetically engineered to produce virus-like particles (VLPs). The VLPs are similar enough to the actual human papillomavirus (which causes cervical cancer and genital warts) to cause your body to produce antibodies against it. The antibodies are then available to fight off the HPV if you are exposed to it at a later time. Because the VLPs are not an actual virus, they cannot cause any of the problems caused by an HPV, such as cervical cancer and genital warts. Who should get the HPV vaccine? Doctors recommend this vaccine for 11- and 12-year-old girls. The vaccine can also be given to girls and women ages 13 through 26 who did not get the vaccine when they were younger or who did not complete the vaccination series. Ideally, girls and women should get this vaccine before their first sexual contact when they could be exposed to the HPV. This timing is because the vaccine prevents disease in girls and women who have not previously acquired one or more types of HPVs prevented by the vaccine. It does not work as well for those who were exposed to the virus before getting the vaccine. Is the HPV vaccine effective? This vaccine targets the types of HPVs that most commonly cause cervical cancer and genital warts. This vaccine is highly effective in preventing those types of HPVs in young women who have not been previously exposed to them. The vaccine will not treat existing HPV infections or existing diseases or conditions caused by HPVs. The vaccine will also not protect against diseases and infections caused by other HPV types that are not included in the vaccine.

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STUDENT P AGE Gardasil® (continued) Is the HPV vaccine safe? The vaccine has been licensed as safe. Before it was approved by the FDA, the vaccine was studied in thousands of females ages 9 through 26 in the United States and around the world. The most common side effect is soreness at the injection site. Are there other ways, besides the vaccine, to prevent HPV? The surest way to prevent genital HPVs is to avoid sexual contact. For individuals who are sexually active, condoms may lower their chances of getting an HPV, if used always and used correctly. Condoms may lower a person’s chances of developing genital warts and cervical cancer. But an HPV can infect areas that are not covered by a condom—so condoms may not fully protect against HPVs. Will girls and women be protected against HPVs and related diseases even if they don’t get all three doses of the vaccine? The HPV vaccine is recommended as a three-dose vaccine. It is not yet known how much protection girls and women would receive if they get only one or two doses of the vaccine. For this reason, it is very important that they get all three doses of the vaccine. Why is the vaccine indicated only for girls and women ages 9 through 26? The vaccine has been widely tested in females ages 9 through 26. But research on how well the vaccine works in older women has just recently begun. The FDA may license the vaccine for those women when research shows that it is safe and effective for them. What about vaccinating boys? We do not yet know if the vaccine is effective in boys or men. Studies are being conducted to find out if the vaccine is effective in males. When more information is available, this vaccine may be licensed and recommended for boys and men as well. Does the vaccine protect against other sexually transmitted diseases or pregnancy? No. Girls and women who have had all doses of this vaccine can still get other sexually transmitted diseases or get pregnant if they are sexually active.

Adapted from “HPV Vaccine—Questions & Answers for the Publicabout the Safety and Effectiveness of the Human Papillomavirus (HPV) Vaccine,” Centers for Disease Control and Prevention, July 17, 2008, www.cdc.gov/vaccines/vpd-vac/hpv/hpv-vacsafe-effic.htm.

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Part C. Edible Vaccines

3. Pass out Student Page: Benefits of Edible Plant Vaccines. Have students compare their list with the one on the student page. Discuss any discrepancies.

In this activity, students will design their own edible vaccine. The purpose of this activity is to encourage students to organize the information they are given and to create a presentation that effectively synthesizes and delivers that information.

4. Divide the class into small groups. Have each group come up with some of the risks associated with edible plant–based vaccines. Ask each group to assign a reporter and recorder.

getting ready

5. Have the recorder from each group list the risks. Then ask the reporter to write each unique suggestion on the board.

Make enough copies of the following student pages for each student: ƒ Student Page: Benefits of Edible Plant Vaccines ƒ Student Page: Risks of Edible Plant Vaccines ƒ Student Page: Creation of an Edible Plant Vaccine ƒ Student Page: Rubric for Assessing Students’ Poster and Oral Presentation

doing the activity 1. Ask your students to identify the most common way to administer a vaccine (needle injection). Ask them if there are any other delivery methods (nasal, topical, edible). Review the Activity 3 PowerPoint to explain the different ways of administering vaccines. 2. Introduce the concept of an edible plant– based vaccine. Ask students to come up with a list of the benefits of edible vaccines. If you have not completed Activity 1: Biotechnology and You, review with the students the meaning of transgenic. Transgenic plants are created by inserting novel genes into an organism for a specific trait. Review the information presented in the Background section of Activity 1 for a more in-depth explanation of transgenic organisms.

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6. Pass out Student Page: Risks of Edible Plant Vaccines. Discuss any discrepancies between the groups’ lists and the student page. 7. To allow students to use the information they have just discussed so they more fully explore some of the risks and benefits of edible plant vaccines, have each group design an edible plant vaccine. Tell the groups to prepare an oral presentation (using a poster as a visual aid) that is of their plant and that addresses the questions on the student page. You can pass out copies of the Student Page: Rubric for Assessing Transgenic Plants to assist them in preparing their presentation and poster. Have the students consult Student Page: Creation of an Edible Plant Vaccine for information and a list of considerations. 8. After giving the groups time to prepare their poster and presentation, have them present their edible plant vaccine to the rest of the class. Have each student assess the presentations from the other groups while using Student Page: Rubric for Assessing Students’ Poster and Oral Presentation.

project learning tree Exploring Environmental Issues: BioTechnology © American Forest Foundation

9. Use the following questions to assess your students’ understanding of edible plant vaccines: ƒ Was the plant you created a transgenic organism? Why or why not? ƒ Yes, it is a transgenic organism because it was made by inserting a gene from another organism into its genome. ƒ Where in the world would edible vaccines be most useful? Why? The benefits of edible vaccines would be most strongly felt in developing countries where storage conditions, transportation systems, and trained medical personnel are in short supply. ƒ Can you list some of the main differences in the risks and benefits of edible vaccines vs. the more common injectable vaccines? You can refer to the student pages for risks and benefits. Briefly, benefits of edible vaccines include increased heat tolerance, reduced production costs, reduced need for trained medical personnel, and decreased risk of inadvertent needlesticks. Risks include difficulty in precisely quantifying the amount of vaccine delivered, the development of oral tolerance, and the escape of transgenes into nontarget organisms. Benefits of injectable vaccines include the ability to deliver precise amounts of antigen and no concern about transgenes’ escaping into a nontarget food supply. Risks include potential for needlesticks, less compliance among patients because of needle aversion, and more expensive production and transport.

Activity 3: Biotechnology and Human Health © American Forest Foundation

ƒ In this activity, we discussed using plants to produce edible vaccines. But this is not the only way plants are being genetically modified to produce vaccines. Nonedible plants (such as tobacco) can be used to produce vaccines that are then purified from the plant and administered through a separate delivery system (such as an injection). Can your students think about the main differences between plant-based vaccines and edible vaccines? As the teacher, you should encourage students to consider some of the benefits and risks associated with each. Edible vaccines are meant to be eaten directly from the plant in which they are produced. This process eliminates a potentially costly step of purifying the protein and then preparing it for injection. Additionally, edible vaccines tend to be more tolerant to heat, making them more stable during transport. Conversely, using nonedible plants as a means of producing vaccines has the benefit of decreasing the risk of having the transgenes escape into the food supply, because they can be made in plants that are not ingested by humans. Additionally, the use of nonedible plants such as tobacco for production of vaccines can offer economic benefits to communities that are dependent on agriculture.

enrichments ƒ What health issues that relate to biotechnology are currently in the news? Ask your students to bring in a newspaper or magazine article that relates to this question.

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STUDENT P AGE Benefits of Edible Plant Vaccines1,2 • Edible means of administration Because the vaccines’ delivery is painless (there is no needle!), people may be more willing to receive the vaccine, especially children. • Reduced need for medical personnel and sterile injection conditions Because needles are not used, there is no risk of an accidental needlestick (and possible transmission of disease) to the people giving the vaccines. Additionally, an edible vaccine will not necessarily require trained medical personnel to deliver. This fact is an important consideration in developing countries where doctors are not always available. • Reduced costs to mass-produce and transport Plants can be grown cheaply and in large quantities, and they can be shipped easily. • Heat stability Vaccines made from plants are more stable and are less affected by high temperatures, reducing the need for refrigeration during transport and onsite storage. This fact is important in rural areas where electricity is not readily available to refrigerate samples. • Subunit vaccine Vaccines produced from plants contain only a part (or a subunit) of the disease-causing organism. For this reason, there is no chance of actually getting the disease. That finding is not necessarily true of vaccines that are made from the whole organism (referred to as live or attenuated vaccines). Endnotes: 1. Diane E. Webster, Merlin C. Thomas, Richard A. Strugnell, Ian B. Dry, and Steve L. Wesselingh, “Appetising Solution: An Edible Vaccine for Measles,” Medical Journal of Australia 176, no. 9 (2002): 434–37. 2. Hugh S. Mason, Heribert Warzecha, Tsafrir Mor, and Charles J. Arntzen, “Edible Plant Vaccines: Applications for Prophylactic and Therapeutic Molecular Medicine,” Trends in Molecular Medicine 8, no. 7 (2002): 324–29.

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STUDENT P AGE Risks of Edible Plant Vaccines1,2 • Oral tolerance Repeated exposure to oral (taken by mouth) antigens may result in oral tolerance. Oral tolerance is when your body no longer responds to antigens that are ingested repeatedly. In the case of oral vaccines, this repetition would lead to people’s becoming immune to the vaccine and, therefore, being susceptible to the disease. • Accumulation of enough antigens The plant must be able to produce antigens (vaccine) in sufficient quantity. Ideally, you would want a normal serving size of the food to contain the correct dosage. For example, it would be unrealistic to expect a person to eat 30 bananas in one sitting to obtain the correct dosage of a vaccine. • Public perception of genetically modified organisms People may not consider an edible vaccine made from a transgenic plant a viable option if they are opposed to consuming genetically engineered organisms. • Transfers of genes to nontarget organisms There are concerns that plants genetically engineered for use as a vaccine may interbreed with crops that are not intended for vaccine usage. Furthermore, herbivores (animals that eat plants, such as cows) and other organisms that eat the plant material (sucking insects, soil microbes) may then be exposed to the antigen. • Dosage The amount of antigen in each plant could vary, making it difficult to determine the correct dosage for each patient. Endnotes: 1. Diane E. Webster, Merlin C. Thomas, Richard A. Strugnell, Ian B. Dry, and Steve L. Wesselingh, “Appetising Solution: An Edible Vaccine for Measles,” Medical Journal of Australia 176, no. 9 (2002): 434–37. 2. Hugh S. Mason, Heribert Warzecha, Tsafrir Mor, and Charles J. Arntzen, “Edible Plant Vaccines: Applications for Prophylactic and Therapeutic Molecular Medicine,” Trends in Molecular Medicine 8, no. 7 (2002): 324–29.

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STUDENT P AGE Creation of an Edible Plant Vaccine (Questions and considerations to address when making your oral presentation and using your poster as a visual aid) • What is the name of your transgenic plant? • Describe the transgenic organism, and describe where the vaccine proteins are produced (stem, fruit, leaves). Explain why you chose this particular type of plant. You can refer to the list of plants and some of their advantages and disadvantages in Table 1 when choosing the type of plant to use for your vaccine. • What is the purpose of your edible plant vaccine (or transgenic plant)? What type of disease does it protect against? • What are the benefits of this transgenic organism? • What are the risks of this transgenic organism? • In what country do you plan to market this plant-based vaccine? Why?

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STUDENT P AGE Creation of an Edible Plant Vaccine (continued) Table 1. Examples of Plants That Can Be Used to Produce Edible Vaccines, plus Advantages and Disadvantages for Each Species1 (Remember that vaccines produced from plants are proteins.) Plant Tobacco

Potato

Tomato

Advantages Transformation process (insertion of new genes into plant) is efficient. Plant produces large quantities of proteins. Plant can be eaten raw. Plant can be grown as a clone (no fertilization with other plants, which decreases risk of mixing with other plants). Transformation process (insertion of novel genes into plant) is efficient. Fruit is edible raw.

Raw potatoes taste bad, and cooking could destroy proteins. Plant has relatively low fruit protein (vaccine) content.

Plant is grown widely in developing countries where vaccines are badly needed. Plant can be eaten raw by infants and adults.

Lots of space is required for growth.

Legumes or Production technology is widely established. cereals High protein (vaccine) content exists in seeds. Protein (vaccine) remains stable in seeds during storage.

Alfalfa

Potential exists for outcrossing (breeding with other non–genetically modified plants) in field. Plant has a relatively low quantity of proteins (vaccine).

Acidic fruit may damage certain vaccines and may be unhealthy for some people (such as babies). Transformation process (insertion of novel genes into plant) is inefficient.

Greenhouse production is well established. Banana

Disadvantages Plant is toxic when eaten.

Plant is expensive to grow in greenhouse. Transformation process (insertion of novel genes into plant) is inefficient. Heating or cooking would destroy vaccine protein.

Potential exists for outcrossing (mixing with plants not intended to contain vaccine) in field for some species. Transformation process (insertion of novel genes Potential exists for outcrossing in field. into plant) is efficient. Plant produces deep roots that are hard to get High protein content exists in seeds. rid of after plant is harvested. Leaves are edible uncooked.

Endnotes: 1. Hugh S. Mason, Heribert Warzecha, Tsafrir Mor, and Charles J. Arntzen, “Edible Plant Vaccines: Applications for Prophylactic and Therapeutic Molecular Medicine,” Trends in Molecular Medicine 8, no. 7 (2002): 324–29.

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STUDENT P AGE Rubric for Assessing Students’ Poster and Oral Presentation Criteria for Evaluations • Transgenic plant is represented with graphics or pictures. • Reasons for choice of plant are clearly addressed both orally and visually on poster. • Graphics or pictures are labeled clearly and accurately. • Students consistently refer to and use poster as an effective visual aid during presentation. • Both risks and benefits of their transgenic plant are clearly addressed, both on poster and during oral delivery (presentation is not biased). • Transgenic plant is represented with graphics or pictures. • Reasons for choice of plant are clearly addressed, either orally or visually on poster. • Graphics or pictures are labeled but may not be clear and accurate. • Students refer to and use poster as a visual aid during poster presentation, but could use it more effectively. • Risks and benefits of their transgenic plant are addressed, either on poster or during oral delivery (presentation is not biased). • Transgenic plant is represented with graphics or pictures. • Reasons for choice of plant are addressed, either orally or visually on poster. • Students occasionally refer to and use poster as a visual aid during presentation. • Presentation is biased, addressing only risks or only benefits of transgenic plant in poster or oral delivery or both. • Transgenic plant is not clearly represented with graphics or pictures. • Reasons for choice of plant are not clearly addressed, either orally or visually on poster. • Graphics or pictures are not labeled accurately. • Students fail to refer to and use poster as a visual aid during presentation. • Presentation of risks and benefits of their transgenic plant is biased, or risks and benefits are not addressed at all.

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Score

4 3 2 1

project learning tree Exploring Environmental Issues: BioTechnology © American Forest Foundation

Part D. Transgenic Plants: Risk and Benefit Assessment Students will identify different types of agricultural engineering. They will then be challenged to present the risks and benefits associated with a particular type of agricultural engineering in a PowerPoint presentation.

getting ready Duplicate enough copies of Student Page: All Transgenic Plants Are Not Created Equal and Student Page: Rubric for Presentation for each student in the class.

doing the activity 1. Ask your students to suggest the different ways in which humans use plants. They will likely come up with some of the following categories: food (crops), shelter (using trees as shade), ornamental (using plants for decoration), environmental (using plants for erosion control), and medical (producing medicine). Initiate a discussion about how views on genetic engineering of plants might differ for the different categories. For example, ask your students if they would be more concerned about genetic engineering of a food crop or genetic engineering of an ornamental plant. 2. Pass out copies of Student Page: All Transgenic Plants Are Not Created Equal to all members of the class, and ask them to read it individually.

Activity 3: Biotechnology and Human Health © American Forest Foundation

3. Once everyone has finished reading, ask members of the class to volunteer the major points. Write those points on the board as they are listed. Continue to elicit responses until the students are convinced they have covered the major points. 4. Divide the class into small groups of no more than four students. Assign each group one of the following topics (some topics may be assigned to more than one group, depending on class size). ƒ Risks and benefits of agronomic transgenic plants ƒ Risk and benefits of pharmaceutical transgenic plants ƒ Risks and benefits of using food crops versus nonfood crops as transgenic organisms 5. Tell the members of each group that they will be preparing a 5-minute PowerPoint presentation about their assigned topic. Each group should be sure to include definitions of terminology used, as well as examples to illustrate the risks and benefits that are listed. Provide each student with a copy of the rubric that will be used to assess the presentations. Research for this project can be conducted during class using available resources (Internet, library) or can be assigned as homework. 6. Allow each group to give its presentation to the class while using the available rubric to assess each presentation. You may choose to have each member of the class assess each presentation using the same rubric.

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7. Once each group has given its presentation to the class, initiate a class discussion to consider the following questions: ƒ How should regulatory organizations (such as the Food and Drug Administration) deal with the different categories of transgenic organisms? ƒ How does human error figure into risk assessment?

endnotes 1. K. Hopkin, “The Risks on the Table,” Scientific American, April 2001, p. 61. 2. Michelle Marvier, “Pharmaceutical Crops Have a Mixed Outlook in California,” California Agriculture 61, no. 2 (2007): 59–66. 3. M. Marvier and R. C. Van Acker, “Can Crop Transgenes Be Kept on a Leash?” Frontiers in Ecology and the Environment 3, no. 2 (2005): 99–106.

ƒ How should the possibility that risks and benefits may not be similar for different groups of people be addressed? For example, the benefits of a particular drug may be realized only by a select group of people with that disease, whereas the risks of unwanted exposure to the transgenic plant may be felt by a much larger group of individuals. ƒ Do people have different criteria for evaluating the safety and usefulness of biotechnology, depending on its intended use (i.e., human health such as vaccines vs. food such as transgenic crops)? Why might this be? 8. Ask each student to write a brief position statement on how he or she feels about transgenic plants. Communicate to your students that there are no right or wrong answers, but they must back up their positions with specific reasons. Explain that this is not a black-and-white issue and that their positions may be quite complex and dependent on a variety of caveats and assumptions.

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STUDENT P AGE All Transgenic Plants Are Not Created Equal Most processed foods in the United States contain some ingredients that were obtained from genetically engineered foods.1 However, not all genetically engineered foods are equal. To date, the majority of genetically engineered plants that have been approved for commercial use contain genetic changes that have to do with herbicide or pesticide resistance. They are classified as agronomic because their main purpose is to make the growing of those crops less costly (farmers save money by losing fewer plants to insect damage or by not having to buy and apply as many herbicides). But future generations of genetically modified crops are being developed to contain pharmaceutically active proteins. A pharmaceutically active protein is simply a drug, such as a vaccine or medicine. Although the biology behind creating those agronomic and pharmaceutical transgenic plants is similar, the consequences of the changes may be very different. For example, the health effects on humans who consume a plant that contains a pharmaceutical transgene can potentially be quite different from the health effects on humans who consume a plant that contains an agronomic transgene. Another issue that must be considered when using plants to make pharmaceutically active proteins is the use of food crops (such as corn, soybeans, rice) versus nonfood crops (such as tobacco).2,3 Food crops offer several advantages over nonfood crops when creating pharmaceutical transgenic plants, such as the ability to eat the plant directly and the fact that proteins are more stable and easier to store for long periods in food plants such as grains. However, the use of nonfood crops has the benefit of potentially limiting unwanted human exposure to the transgenes because they are not consumed directly by humans. Endnotes: 1. K. Hopkin, “The Risks on the Table, Scientific American, April 2001, p. 61. 2. Michelle Marvier,. “Pharmaceutical Crops Have a Mixed Outlook in California,” California Agriculture 61, no. 2 (2007): 59–66. 3. M. Marvier and R. C. Van Acker, “Can Crop Transgenes Be Kept on a Leash?” Frontiers in Ecology and the Environment 3, no. 2 (2005): 99–106.

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STUDENT P AGE Rubric for Presentation Introduction (title and outline)

Content

Does Not Meet Expectations 0 points

Meets Expectations

Exceeds Expectations

12 points

2–4 points

q Title or outline or both do not accurately reflect contents of presentation.

q Title and outline mostly q Title and outline accureflect contents of rately reflect contents presentation. of presentation.

q Title or outline or both are missing. 0 points

q Title and outline are present.

q Title and outline are present.

1–4 points

4–8 points

Score

q Information provided is q Information provided is q Information provided is inaccurate. mostly accurate. accurate.

Visuals

Grammar and spelling

Presentation style (Include a final slide that lists major contributions of each group member)

q Information is not explained clearly.

q Information provided is q Information presented mostly explained. is clearly explained.

q Terminology is not defined. 0 points

q Some terminology is defined. 2–3 points

q All terminology is clearly defined. 3–5 points

q Text is too small to be read.

q Text is mostly effective.

q Text is large enough to be read by entire audience.

q Pictures and graphics are not used.

q Use of pictures and graphics mostly enhance presentation.

0 points

1 points

q There are more than two grammar and spelling errors. 0 points

q There are fewer than two grammar and spelling errors 2–3 points

q There are no grammar or spelling errors.

q Contribution of all group members is unclear.

q Most members of the group contributed to presentation.

q Each member of the group contributed to the presentation.

q Use of pictures and graphics enhances audience’s understanding of the content. 3 points

3–5 points

q Diction and voice level q Diction and voice level q Diction and voice level do not engage audiare mostly effective at engage the audience. ence. engaging audience. Total Score

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project learning tree Exploring Environmental Issues: BioTechnology © American Forest Foundation

Activity 4: Forest Biotechnology In this activity, students will learn how traditional methods of artificial selection and modern methods of bioengineering have been used in an attempt to improve the quality of forests products worldwide. Students will investigate both the risks and benefits of genetically modifying trees. Subjects: Biology, AP Biology, Ecology, Environmental Science, AP Environmental Science, Forestry, Social Studies Concepts: 1.4, 2.3, 3.6, 3.7, 4.5, 5.4, 5.5 Skills: Analyzing, Comparing and Contrasting, Concept Forming, Concluding, Debating, Discussing, Evaluating, Generalizing, Identifying Relationships and Patterns, Interpreting, Observing, Ordering and Arranging, Predicting, Representing, Researching, Synthesizing and Creating Materials: For the class: an overhead projector, copies of a map of the United States on overhead transparency sheets for each group, two different colors of overhead markers For each group: 20 milliliters (ml) of liquid dish soap, 10 grams of table salt, 400 ml distilled water, one 500-ml flask, a fingerprint DNA kit (enough supplies for each group), including three types of sample DNA, restriction endonucleases (or precut DNA), a garose gel, a gel stain, a gel electrophoresis rig and power supply to run the fingerprint results from each lab group or to run a single example gel for use by the entire class, graph paper, one field guide to trees of North America (or trees in your specific region) for each lab group (if you have an area with many ornamental or introduced trees, you may need to create your own field guide) For each student: copies of student pages, computers with Internet and Microsoft Office Publisher, a CD of the Charlie Chestnut slide show from the American Chestnut Foundation, a computer and projector to show the information on the CD to the class (or if you have Internet access, the slide show can be viewed at www. charliechestnut.org), one strawberry, one small Ziploc® (or similar) plastic bag, one 50-ml disposable tube with a cap, one 15-ml disposable tube with a cap, one 15-centimeter x 15-centimeter square of four-ply cheesecloth, 5 ml of ice-cold 95 percent ethanol, one toothpick-diameter wooden stick long enough to reach the bottom of the 15-ml tube, one 1-ml microcentrifuge tube

Objectives: ƒ Students will discover the ways in which botanists and forest scientists have manipulated trees to promote the expression of desired characteristics. ƒ Students will research the detrimental effects of an introduced fungus on the distribution of the American chestnut. ƒ Students will learn how to manipulate DNA in a lab setting while answering ecological questions. ƒ Students will debate the merits of different methods of forest management practices, given today’s traditional and biotechnical options. ƒ Students will perform a species abundance and diversity survey to anticipate changes that may occur if any one species becomes diseased. Assessment Opportunities: ƒ Student Page: Informational Brochure on Tree Improvement Techniques can be used to determine how well each student understands the traditional and modern methods of tree improvement. ƒ The lab results and Student Page: DNA Extraction and Gel Comparison of Cut DNA—Lab Experiment can be used to assess the student’s understanding of DNA manipulation. ƒ Student Page: Peer Review of Informational Brochures and Student Page: Strength of Argument can be used to evaluate the development of each student’s study skills. ƒ Oral and visual presentations of a postdisease map to the members of each student’s group can be used to indicate each student’s comfort and competency with mapping and survey skills.

Time Considerations: Preparing the Activity Part A: 15 minutes Part D: 15 minutes Part B: 15 minutes Part E: 15 minutes Part C: 30 minutes Part F: 30 minutes Doing the Activity: Part A: Two 50-minute periods Part B: One 50-minute period Part C: Three 50-minute periods

Activity 4: Forest Biotechnology © American Forest Foundation

Part D: Two 50-minute periods Part E: Two 50-minute periods, plus homework Part F: Three 50-minute periods

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background About 30 percent of the land surface of Earth is occupied by forests, which play an enormous role in our climate, local habitat, culture, and consumption. Trees are responsible for recycling nutrients such as nitrogen, sulfur, phosphorus, and carbon and decontaminating wastes that have accumulated in the soil, water, and air. Trees play a major role in the water cycle, slowing down surface runoff, drawing water through their roots, and returning moisture to the atmosphere through transpiration. Forests, along with grasslands, shrubs, and other autotrophs, absorb solar radiation to provide the basis of the food chain; regulate abiotic factors such as wind, temperature, and moisture; and offer habitat, nutrients, and renewable supplies of many organisms.

Lumber 30%

Fuel 50%

Global use of wood removed from forests1

Paper 20%

Today, 50 percent of wood worldwide is used as fuel for cooking fires and home heating or for running boilers for electricity or steam. Because of the conversion of tropical forests to agriculture, our planet’s forests are shrinking at an annual rate of 140,000 square kilometers.2 There is an increasing price to pay in energy, effort, and education lost as young children and women travel farther each day to collect firewood for daily cooking and heat. Of the forests that are cleared annually, 90 percent are in the tropical regions where people seek money for immediate use from the sale of this wood and the land that it occupies. Humans have been breeding trees for more than a thousand years to produce organisms that are better suited to specific uses.3 Artificial selection techniques and forest management have provided solutions that 102

reduce human demands on forests worldwide so that smaller amounts of land can be used to provide an abundance of wood products in a manner that is sustainable for centuries to come. Biotechnology in the Historical Management of Forests4 Biotechnology was used as early as 6,000 years ago when people began planting fruit tree orchards and ornamental gardens with select species that displayed desired traits. Agriculture was well on its way by that time, and the biotechnology of artificial selection for specific traits was seen in food crops and animal breeding. Tree reforestation practices began as early as 2,300 years ago in Egypt to reduce erosion and to offset the demand for firewood, but it wasn’t until the 13th century that civilizations began selecting specific high-quality trees to use for reforestation. Tree improvement techniques, which began in earnest worldwide in the 19th century, moved deeper into the practices of biotechnology while using results from experiments in inbreeding and outcrossing, cross-pollination, and the introduction of nonnative tree species. It was determined that trees that combined the gene pools of two separate populations often yielded more desirable progeny. Further experimentation revealed that hybrids (offspring from selected parents of the same species or two related species) could be bred to provide desirable traits such as better lumber, more fruit, or faster growth. Hybrids, both intraspecific and interspecific, became the focus of 20th-century forest improvement techniques. Backcrosses, the offspring of two different parental lineages that are pollinated with other trees from the lineage of a single parent for one or more generations, were used to reinforce the majority of the traits of one parent while maintaining one or more desirable traits from the opposite parent (see part B and the case study of the American chestnut for a more detailed example). Hybridization techniques became the basis of seed orchards using select superior trees, called “plus” trees, to create entire orchards of offspring from various select parents. These seed orchards were then culled to remove the lesser individuals,

project learning tree Exploring Environmental Issues: BioTechnology © American Forest Foundation

leaving entire forests of the choice plus the tree offspring that could interbreed and could provide seeds for reforestation or for initiation of forests in other areas. During the 1950s, the seed orchard method of developing large populations of highly selected trees was introduced to the United States, and scientists across the country began developing seed orchards for native species that could be grown in this country. Early on, it became apparent that limiting the gene pool and planting monocultures increased a forest’s susceptibility to disease. Those forests also had a more simplistic ecosystem that allowed pest species to proliferate without the complex niche structure that would harbor a range of predatory species. In an attempt to remedy the problem of limited gene pools, scientists introduced new plus trees periodically to enlarge the gene pool of the offspring trees and to allow gene flow from a greater outcrossing population. The other problems with single-species forests— such as competition and depletion of certain soil nutrients, the loss of ecosystem diversity and complexity, and a decrease in aesthetic value of a less diverse forest—continue to be issues. Creating seed orchards required grafting the tops of plus trees to existing mature trees for harvesting seeds at ground level and then raising the progeny of those seeds to adulthood until the progeny too produced seeds. This method was time-consuming. By the mid-20th century, scientists began combining seed orchard methods with vegetative reproduction while using root cuttings to quickly obtain several genetically identical trees from a single individual. This process provides genetic clones that can be planted en masse at any location to reforest an area with trees that have already undergone extensive artificial selection techniques. Furthermore, some clonal forests could be planted from cuttings made from any part of the plant by using tissue cultures so scientists could choose the desired state of maturity for the new forest. Choosing the desired state of maturity either allowed trees to jump past a juvenile stage—if needed—so the trees could avoid damage from a pest species or allowed reproductively mature plants to bear fruit immediately. Activity 4: Forest Biotechnology © American Forest Foundation

Biotechnology in Forest Management in the 21st Century Although transgenic species are not used in the vast majority of commercial forestry in North America today, the industry is beginning to see a place for this new technology in coming years. Transgenic species have genes inserted into their developing cells that are designed to resist pest species or herbicide applications; to tolerate drought, salt, or other environmental stresses; and to increase desirable properties by introducing genes that could directly affect wood production or other economically viable qualities.5 The genetically engineered varieties can be used to reduce erosion in specific areas or to serve as windbreaks for agricultural land. Scientists use DNA fingerprints on individual trees to analyze their genome, to trace weak genes, or to target desirable ones. Relationships of ancestry and taxonomy are being resolved so hybrid crosses can become calculated successes. Issues of Costs and Benefits Many believe that sustainable, commercial forestry is being implemented across the landscape today and that it has the ability to protect biodiverse areas, water quality, and wildlife habitat, particularly when compared with other land use alternatives (e.g., residential development and agriculture). Intensive management and use of fast-growing non–genetically engineered trees are techniques that are predominantly used today. Advances in biotechnology (bioengineering) that may become more widely used as we move further into the 21st century have raised some concerns for many scientists. Concerns are rooted in the new risks that such transgenic plants pose. Pest resistance to the genes that were introduced to deter those species may eventually lead to new generations of insects and pathogens that are immune to the novel gene. Because the pollen of plants is highly mobile, the introduced gene will be found in surrounding populations of closely related plants through cross-pollination. In addition, both the transgenic and selected species can increase a tree’s potential for invasive reproduction, thereby allowing it to outcompete native species and to reduce overall biodiversity. 103

The biodiversity problems associated with a monoculture continue to challenge scientists as soils become depleted through intraspecific competition and as ecological complexity is compromised.6 However, if tree plantations were used to meet all the world’s need for forest products, those plantations would make up only 5 percent of the total land mass, freeing millions of acres for preserves and biologically diverse habitats.7 In many parts of the world, including the United States, sustainable commercial forestry is successfully implemented using intensive management and fast-growing, non–genetically engineered trees, thus yielding a forest where trees can be harvested and where biodiversity, water quality, and wildlife habitat are protected. However, in many regions of the world, the natural forest has been removed for human use of the land (e.g., tropical forests that have been harvested, burned, and converted to farmland that is typically productive for only 1–3 years) so there is no longer a sustainable source of timber. One perspective holds that if humans made the choice to introduce fast-growing bioengineered species on marginal and degraded farmland and pastureland that was already accessible by roads, then natural buffers would be in place between wild forests and tree plantations to reduce the chance of having introduced genes escape from their planted host. This setup would also keep new roads from being built into pristine areas, therefore limiting human encroachment and habitat degradation. Another perspective is to perform experimental trials before broad implementation in order to test the risk of crossbreeding and gene escape. Such solutions would still call for monitoring and oversight, but they might reduce the pressure being placed on the old-growth forests and dwindling forests that are being used by the poor to meet their basic needs. With the size of the human population, our personal, economic, and political choices in the coming century will greatly affect the air, soil, and water quality around the world. With advances in biotechnology that apply to forestry and management of public and private land, it will be necessary to have a citizenry that is informed and ready to participate in the decision-making process. 104

endnotes 1. G. Tyler Miller, Living in the Environment (Pacific Grove, CA: Brooks/Cole Thompson Learning, 2002). 2. World Resource Institute, 2000. 3. D. Rowland Burdon and William J. Libby, Genetically Modified Forests: From Stone Age to Modern Biotechnology (Durham, NC: Forest History Society, 2006). 4. Ibid. 5. Ibid. 6. Ibid. 7. Miller, Living in the Environment.

Part A: Tree Improvement Techniques—Are They All the Same? GETTING READY ƒ Copy Student Page: Informational Brochure on Tree Improvement Techniques and Student Page: Peer Review of Informational Brochures for each student. ƒ Reserve the computer lab for two class periods so students have access to a computer with Microsoft Office Publisher, the Internet, and a color printer.

DOING THE ACTIVITY 1. Ask the students a few open-ended questions to point out how the availability of trees for human use has affected their lives: ƒ What resource from trees do you use the most in your life? Answers will vary but may include paper, timber for building structures, particleboard, mulch, cork, chewing gum, maple syrup, cloves, cinnamon, nuts, fruits, chocolate, etc. ƒ Where do the trees you use come from?

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ƒ Are the resources you use from trees renewable? If so, on what time scale? Yes, as long as the management practices maintain soil productivity using methods such as retention of organic matter and prevention of soil erosion, the resource is renewable for many harvest rotations over a period of 5–50 years, depending on the species and size of the tree harvested. 2. Tell students that they will each create an informative brochure about one of the techniques used to improve trees (if you feel your students would be more engaged by creating a skit or a commercial, feel free to modify the activity as needed). Assign one topic to each student from the following list: ƒ Artificial selection ƒ Clonal forestry ƒ Hybrids ƒ Seed orchards ƒ Genetically engineered varieties 3. Give each student a copy of Student Page: Informational Brochure on Tree Improvement Techniques so each knows what information is expected to be covered. 4. Allow time for your students to research their topics and to use Microsoft Office Publisher while making a brochure (under the Publications for Print section of a new document). Mention the importance of referencing the information used. 5. After the brochures have been created, have the students print their brochures in a two-sided color format so the brochures can be folded and presented realistically.

Activity 4: Forest Biotechnology © American Forest Foundation

6. Ask your students to turn in all the brochures using separate stacks for each technique. 7. Give each student a copy of Student Page: Peer Review of Informational Brochures, and ask all the students who researched one topic to sit together to evaluate all the brochures about their topic. 8. Ask the students not to reveal which brochure is theirs until the end of the class period so that all brochures can be graded with anonymity. 9. Pass to members of each group the stack of brochures about the topic they researched, and ask them to grade the brochures according to the instructions on their student page. 10. When they are finished with the evaluations, the students should choose a single brochure that has the highest quality of information within the brochure; they will share that brochure with the other groups. 11. When step 9 is complete, the best brochure from each topic should be passed to the next topic group to be shared among the students who did not research that particular topic. The information in the brochure can be read aloud to the entire group, and then the brochure can be passed around the group to answer the questions from their Student Page: Peer Review of Informational Brochures. (This sheet contains questions that help the students find all the important points regarding that topic.) 12. When members of a group have finished with the brochure about the second topic, they should pass that brochure to the next group and should examine the next topic’s brochure until every group has reviewed all the best brochures for each topic.

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STUDENT P AGE Informational Brochure on Tree Improvement Techniques You will create an attractive and informative brochure to present to your peers and your teacher on one of the following tree improvement techniques: • Artificial selection • Clonal forestry • Hybrids • Seed orchards • Genetically engineered varieties

B

F

Front of page

A

C

D

E

Back of page

1. Place the name of the tree improvement technique that you have researched on flap A of your brochure; people will see this panel first when your brochure is folded. Include any appropriate graphics that will accurately depict this technique. 2. Briefly describe the tree improvement technique on flap B of your brochure; people will see this panel second when your brochure is folded. Make sure your description is clear and accurate. 3. Use the three inside panels (C, D, and E) to describe the steps of this tree improvement technique. Use diagrams or photos plus a written description that is detailed enough for anyone in your class to understand. 4. Include the pros and cons of the tree improvement technique on flap E of your brochure. Make certain your list is complete and accurate. 5. Check the information in your brochure. Make sure you have written accurate, easy-to-understand information in your own words. Check your grammar and spelling, and cite any sources. Check your brochure for attractiveness and clarity. Do not put your name anywhere on your brochure. 6. When you have finished creating your brochure, please print it as a two-sided, trifold, color brochure using the color printer. 7. Please bring your brochure to class on _________________ to share it with your peers.

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STUDENT P AGE Peer Review for Informational Brochures Do not allow the other students to know which brochure is yours so that each brochure can be evaluated according to its merits. Number each brochure clearly on the front flap. Circulate the brochures among your group. Read all parts of each brochure. Rate each brochure using the questions below, and give it a score ranging from 1 to 5, with 5 being the highest. Refer to brochures rated earlier if necessary to give each brochure an accurate score. Do not give any two brochures the same list of scores. Do not give any brochure the same score for all categories. Name of Reviewer____________________________________ Tree Improvement Technique Brochure Number____________________________________ 1 2 3 4 5 How would you rate the accuracy and clarity of the summary found on flap B? How accurate is the explanation of this technique on inside flaps C–E? How easy to understand is the explanation of the tree improvement technique? How accurate and complete is the list of pros for this technique? How accurate and complete is the list of cons for this technique? How accurate are the diagrams and figures in depicting the technique? Rate the visual presentation of this brochure. Rate the editing, citations, grammar, and spelling. Are you confident that this student wrote the information himself or herself? Give this brochure an overall grade that takes all factors into account. After you have examined the brochures from the other tree improvement techniques, complete the following chart. For each of the four techniques that you did not research, explain in your own words the basic process of the technique and the pros and cons of each strategy. Name of Technique

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Basic Process

Pros

Cons

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Part B: Where Are the American Chestnuts?

3. Divide the class into five groups. Explain that each group will create an overhead transparency about the distribution of the American chestnut population at a different point in history.

GETTING READY ƒ Make five copies of Student Page: Description of American Chestnut Distribution. ƒ Make five copies of Student Page: Outline Map of the United States onto overhead transparency sheets. ƒ Make a copy of Student Page: Charlie Chestnut Webquest for each student. ƒ Obtain an overhead projector and a different color overhead marker for each group. ƒ Order a CD of the Charlie Chestnut slide show from the American Chestnut Foundation, and set up a computer and projector to show the information on the CD to the class. ƒ Preview the Charlie Chestnut scrapbook to become familiar with the history of the American chestnut. Or if you have Internet access, the slide show can be viewed at www.charliechestnut.org.

DOING THE ACTIVITY 1. Ask the students if they have ever seen an American chestnut tree or if they have heard the story of the American chestnut. Give them time to share what they know or if they are unaware of how an introduced fungus infected and killed the American chestnuts of the eastern United States, give a very brief explanation of the events. 2. Ask the students why scientists would be interested in a species of any type of organism that (a) has seen a dramatic change in range, (b) has become endangered, or (c) has become extinct. Include a discussion of why the students would or would not be interested in a species that may not be in the region where they live.

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4. Give each group a copy of Student Page: Outline Map of the United States and a copy of Student Page: Description of American Chestnut Distribution (numbers 1–5) to record onto their transparency map. 5. After students have completed the paper map, they will transfer the outline of the American chestnut range of distribution onto an overhead transparency containing a preprinted map of the United States. 6. Members of the group with the oldest data set should color in their distribution range with the lightest color of overhead marker. The marker colors for each group should get progressively darker with more recent data sets so that the distribution range will show up clearly when the transparencies are laid one on the other. 7. When all five groups are finished making their transparencies, introduce and show the progression of the fungal infection that decimated the American chestnut population by laying the oldest transparency on the projector and then covering it with a more recent set of data until the current distribution is laid on the stack. Engage the students in a conversation to discuss the speed of this population’s decline to see if they understand the contributing factors. The following questions are suggestions for initiating a conversation: ƒ How many years did it take to reduce the range of the American chestnut to the point where there were almost no mature trees? About 50 years.

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ƒ The current distribution of mature American chestnut trees is what portion of the original range? There are essentially no American chestnut trees in the wild that are able to grow to maturity in the eastern United States. The fungal blight that has infected this species allows the tree to grow to a certain height, and then it kills the upper portion so the tree never matures but the stump remains. ƒ What role do you think the American chestnut played in the ecosystem when it was the most abundant species in eastern forests? The nuts were an important source of food for many species of birds and mammals; the rotresistant trees were used as homes for many species of birds, insects, and mammals while living and after a tree died, etc. ƒ How do you think people used the American chestnut when it made up 25 percent of the eastern forest? The nuts were a source of both food protein and vitamins for the people who collected them and a source of income through the sale to others; the wood was valuable as lumber; the trees’ shade and beauty were recognized by all and so they were used in parks and left uncut to shade houses and barns. ƒ When people realized that the American chestnut had little to no resistance to the fungal infection, what do you think they did to deal with this problem?

Activity 4: Forest Biotechnology © American Forest Foundation

In some areas, the trees were quarantined; infected trees were cut down so the blight would not spread; and many healthy trees were taken down before they were infected so the wood could be used, further reducing the population of the species. ƒ What do you think should have been done in the early stages of this infection? Answers will vary; explore all ideas. 8. Students should use the scrapbook on the Charlie Chestnut website (www.charliechestnut.org) to learn about the history of the American chestnut and to review the biology and ecology of this species. 9. Discuss the Charlie Chestnut scrapbook information. Review what was done to try to stop the blight and what has been done since the trees were decimated to attempt to preserve this species. Revisit the preliminary discussion by asking the students why the history of the American chestnut matters to people today. Ask them if they can think of an organism in their region that has undergone a change in range or distribution. For any organism that is mentioned in discussion, ask the students what the contributing factors were that affected the population changes in this species. 10. The preview map experience, the slide show, and the ensuing discussion will help students understand the relevance of part C when they compare the genetic similarity of crossbred chestnuts with true-breeding American chestnuts and of part D when they debate the solutions to reductions in biodiversity.

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STUDENT P AGE Group #1 Description of American Chestnut Distribution Data Set for Chestnut Distribution before 1880: The areas of the eastern United States where the American chestnut could be found free of blight in 1880 are listed below. Use these data to create a colored distribution map. Label the map “American Chestnut Distribution before 1880.” Maine—southern quarter of the state New Hampshire—southern quarter of the state Vermont—southern quarter of the state Massachusetts—entire state Connecticut—entire state Rhode Island—entire state New York—southern third of the state Pennsylvania—entire state New Jersey—entire state Delaware—entire state Maryland—northern half of the state Virginia—western quarter of the state West Virginia—entire state Ohio—eastern half of the state Indiana—southeastern fifth of the state Kentucky—eastern half of the state Tennessee—eastern three-quarters of the state North Carolina—western third of the state South Carolina—western quarter of the state Georgia—northern quarter of the state Alabama—northern quarter state Mississippi—northeastern quarter of the state

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STUDENT P AGE Group #2 Description of American Chestnut Distribution Data Set for Chestnut Distribution before 1908: The areas of the eastern United States where the American chestnut could be found free of blight in 1908 are listed below. Use these data to create a colored distribution map. Label the map “American Chestnut Distribution in 1908.” New Hampshire—southern quarter of the state Vermont—southern quarter of the state Massachusetts—entire state New York—southeastern third of the state Pennsylvania—entire state Virginia—western quarter of the state West Virginia—entire state Ohio—eastern half of the state Indiana—southeastern fifth of the state Kentucky—eastern half of the state Tennessee—eastern three-quarters of the state North Carolina—western third of the state South Carolina—western quarter of the state Georgia—northern quarter of the state Alabama—northern quarter state Mississippi—northeastern quarter of the state

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STUDENT P AGE Group #3 Description of American Chestnut Distribution Data Set for Chestnut Distribution in 1911: The areas of the eastern United States where the American chestnut could be found free of blight in 1911 are listed below. Use these data to create a colored distribution map. Label the map “American Chestnut Distribution in 1911.” New York—southern quarter of the state Pennsylvania—northwestern three-quarters of the state Virginia—western quarter of the state West Virginia—northern four-fifths of the state Ohio—central half of the state Indiana—southeastern fifth of the state Kentucky—eastern half of the state Tennessee—eastern three-quarters of the state North Carolina—western third of the state South Carolina—western quarter of the state Georgia—northern quarter of the state Alabama—northern quarter state Mississippi—northeastern quarter of the state

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STUDENT P AGE Group #4 Description of American Chestnut Distribution Data Set for Chestnut Distribution in 1913: The areas of the eastern United States where the American chestnut could be found free of blight in 1913 are listed below. Use these data to create a colored distribution map. Label the map “American Chestnut Distribution in 1913.” Virginia—western quarter of the state West Virginia—northern four-fifths of the state Ohio——eastern half of the state Indiana—southeastern fifth of the state Kentucky—eastern half of the state Tennessee—eastern three-quarters of the state North Carolina—western third of the state South Carolina—western quarter of the state Georgia—northern quarter of the state Alabama—northern quarter state Mississippi—northeastern quarter of the state

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STUDENT P AGE Group #5 Description of American Chestnut Distribution Data Set for Chestnut Distribution in 1930: The areas of the eastern United States where the American chestnut could be found free of blight in 1930 are listed below. Use these data to create a colored distribution map. Label the map “American Chestnut Distribution in 1930.” Indiana—southeastern fifth of the state Kentucky—central quarter of the state Tennessee—central quarter of the state Alabama—northern quarter state Mississippi—northeastern quarter of the state

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STUDENT P AGE Outline Map of the United States

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STUDENT P AGE Charlie Chestnut Webquest Use the information found in the Charlie Chestnut scrapbook to answer the questions below. • Create a timeline that spans from 1799 to today. As you read through the Charlie Chestnut scrapbook, add notations to the timeline that describes significant events in the history of the American chestnut.

• What physical features could you use to identify an American chestnut tree? • How did the American chestnut come to dominate the eastern forests before the blight? • What exactly is a “blight”? • Describe how the blight physically affects a single chestnut tree. • What role did the American chestnut play before 1900? Include a description of this tree’s role in human society, as well as its role in the forest ecosystem. • How did the loss of the American chestnut affect the people of that era? • How do you think the population distribution and diversity of other eastern forest plant and animal species changed during the time the American chestnut was dying out? • Are there any reasons that the scientists, the inhabitants of that region, or the inhabitants of other parts of the United States where the tree may not occur naturally would be concerned about the loss of one particular species? • What specific actions has the American Chestnut Foundation taken to fulfill its purpose? 116

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Part C: DNA Lab Experiment getting ready ƒ Copy Student Page: DNA Extraction and Gel Comparison of Cut DNA—Lab Experiment for each student. ƒ For each student, provide the following: – One strawberry – One small Ziploc® (or similar) plastic bag – One 50-ml disposable tube with a cap – One 15-ml disposable tube with a cap – One 15-cm x 15-cm square of four-ply cheesecloth – A container with 5 ml of ice-cold 95 percent ethanol – One toothpick-diameter wooden stick that is long enough to reach the bottom of the 15-ml tube – One 1-ml microcentrifuge tube ƒ For each class, provide the following: – A small bottle of liquid dish soap – A container with 5 g of table salt – A container with 400 ml of distilled water – One Edvotek Chestnut DNA kit (including three types of sample DNA, agarose gel, a gel stain with enough supplies for six lab groups), and at least one gel electrophoresis rig and power supply. (Each lab group can run its own gel comparison if supplies are available; however, a single gel can be used to run as many as 20 samples at one time.)

DOING THE ACTIVITY 1. If your students have not completed part B, review the history of the American chestnut, its characteristics, and the characteristics of its blight-resistant relative, the Chinese chestnut. 2. Discuss the crossbreeding technique used to hybridize the American chestnut and the Chinese chestnut for resistance to the blight. Explain how scientists have chosen the resistant offspring of the Chinese/American cross and backcrossed those offspring repeatedly with American chestnuts for several generations in order to preserve as many of the original traits of the American chestnut as possible without losing the Chinese blight-resistance genes. 3. Explain to your students how geneticists can visualize the DNA from a particular individual or species to see how closely it resembles the DNA of its relatives. Let them know that they will be performing a lab exercise that will simulate the American chestnut, the Chinese chestnut, and the crossbred offspring. 4. Extract DNA from a plant using the following protocol and Student Page: DNA Extraction and Gel Comparison of Cut DNA—Lab Experiment. The following steps need to be prepared before the students begin their student page instructions: ƒ Thaw the strawberries completely if they are frozen. ƒ Place 95 percent ethanol in the refrigerator or freezer; place it on ice in the classroom for easier access during the lab procedure. ƒ In a 500-ml flask, make a simple DNA extraction buffer using 20 ml of liquid dish soap, 10 g of salt, and 380 ml of distilled water.

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ƒ Each student will need the following supplies: – One small ziplock plastic bag – One strawberry (half if they are big) – One 15-ml disposable graduated tube with a cap – One 50-ml disposable tube with a cap – One 15-cm x 15-cm square of four-ply cheesecloth – A container with 5 ml of ice-cold 95 percent ethanol – A container with 10 ml of DNA extraction buffer – One toothpick-diameter wooden stick that is long enough to reach the bottom of the 15-ml tube – One 1-ml microcentrifuge tube 5. After your students have collected the DNA from their strawberry, relate how DNA can be manipulated using restriction enzymes for comparisons within and between taxonomic groups. Explain how restriction enzymes, or are taken from bacteria and used to cut DNA samples into fragments. Explain how endonucleases yield a fingerprint pattern that can be used to compare the similarities and differences between individuals. Explain that your students will be using DNA that has already been cut with endonucleases to compare the DNA of an American chestnut, a Chinese chestnut, and a hybrid offspring produced from crossing these two parent species.

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6. Using the instructions in the Edvotek No. 114PLT hybrid DNA analysis kit, run out the cut chestnut DNA on a gel. 7. Following the kit instructions, rinse the gel with a stain from the kit to visualize the cut fragments that are now sorted by size within each lane. 8. Compare the DNA “fingerprint” made by the fragments in the lane containing the American chestnut DNA with the two lanes that contain the Chinese chestnut and the hybrid chestnut. The hybrid chestnut will have some fragments of DNA similar to each parent, whereas the two parent species are distinctly different. 9. Use the lab experiment reflection questions to verify that your students understand how the simulation relates to the breeding program devised for the American chestnut. 10. Ask your students how DNA analysis techniques, such as the one they practiced in this activity, can be applied to biotechnology.

The students may have many different ideas. Here are a few they might mention: (a) being able to extract DNA can let scientists read the sequence of base pairs that make up specific genes, (b) comparing one species to another helps scientists identify genes that provide disease resistance that may help fight a spreading pathogen, and (c) comparing DNA sequences allows scientists to find the closer or more distant relatives to use in crossbreeding programs.

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STUDENT P AGE DNA Extraction and Gel Comparison of Cut DNA— Lab Experiment

Introduction

The primary purpose of this lab is to extract DNA from the cells of a plant to help you understand a common step in many microbiology and genetics protocols. To extract the DNA from plant tissue, you must first break open the cell wall and cell membranes using a mechanical action and using a soapy detergent that can break up the lipid membranes. After the cells have been torn apart, the proteins and cellular components must be stripped away from the DNA so they do not denature or degrade the molecule. To remove these components, you will add salt, which will bind to the debris and will cause it to clump or fall to the bottom of a tube. Straining the mixture removes the salty clumps and leaves a supernatant (liquid runoff) of macromolecules. After the cell components have been removed, the DNA can be coaxed away from the macromolecular soup by using an alcohol that attracts the charged portion of the molecule. The procedure of extracting DNA is similar for any cellular tissue from any living organism. After extracting the DNA of an organism, you can thenn cut the DNA with restriction enzymes to create fragments of differing lengths. For the second part of this procedure, you will run out the DNA of three different chestnut trees on a gel matrix to look for similarities and differences between related organisms: (a) the American chestnut, (b) the Chinese chestnut, and (c) a hybrid offspring of those two parent species. The instructions for the hybrid DNA analysis are included in the Edvotek kit that your teacher will give you. Materials • One Ziploc® (or similar) plastic bag • One strawberry (or half if they are big) • One 15-milliliter (ml) disposable graduated tube with a cap • One 50-ml disposable tube with a cap • One 15-centimeter x 15-centimeter square of four-ply cheesecloth • A container with 5 ml of ice-cold 95 percent ethanol • A container with 10 ml of extraction buffer • One toothpick-diameter wooden stick that is long enough to reach the bottom of the 15-ml tube • One 1-ml disposable pipet • One 1-ml microcentrifuge tube (optional) • One rubber band (optional) Procedure 1. Place one strawberry in the Ziploc® plastic bag, and press out the air as you seal the bag. Mash the strawberry until the tissue is smooth and creamy.

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STUDENT P AGE DNA Extraction and Gel Comparison of Cut DNA— Lab Experiment (continued) 2. Add 10 ml of DNA extraction buffer to your plastic bag, reseal it after removing the air, and mix it again for 1–2 minutes. 3. Pour the mixture through four-ply cheesecloth that has been draped over a 50-ml test tube (you may want to ask you neighbor to hold the cheesecloth during this step or may use a rubber band to hold it in place). Do not squeeze the contents of the cheesecloth; you want to collect only the liquid that drips through on its own. 4. Pour off 2 ml of the liquid from the 50-ml tube into a 15-ml tube. 5. Tilt the 15-ml tube at an angle, and slowly pipet 5 ml of ice-cold ethanol onto the top layer of the mixture. Do not mix the layers or shake or swirl the tube. Watch for a transparent, mucus-textured, bubble-filled substance to form at the interface of the two layers. This substance is the DNA from your strawberry. 6. Gently insert the wooden stick into the 15-ml tube until it is below the first layer. Twirl the stick to spool long threads of DNA onto the stick. 7. Gently remove the stick containing the DNA, and scrape your DNA into a 1-ml microcentrifuge tube to keep. Conclusion Answer the following questions after you have finished the DNA extraction lab. • Describe how each of the following cell components was removed. Proteins: Nucleus: Vocuole: Cell wall: Cell membrane: Chloroplast: Cytoplasm:

Proteins Nucleus Vacuole Cell wall Cell membrane Chloroplast Cytoplasm

• How would the DNA extracted from bacteria, fungi, or an animal cell differ from the DNA extracted from a plant cell?

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STUDENT P AGE DNA Extraction and Gel Comparison of Cut DNA— Lab Experiment (continued) Answer the following questions after you have finished the Edvotek No. 114-PLT hybrid DNA analysis lab: • Sketch the gel and the bands found in each lane. Label the lanes for each species.

• Using your gel results, explain the genetic relationships among the three species. • Was the hybrid more genetically similar to the American chestnut or the Chinese chestnut? Support your answer with scientific data. • How does the DNA analysis relate to the physical and chemical traits in the hybrid organism? • Predict what the gel would look like if you were to perform a DNA fingerprint analysis using your DNA and DNA from your biological parents.

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Part D: What’s Your View?

ƒ Do nothing, and allow modern forests with no American chestnuts.

getting ready

3. Ask the students to count off into four teams for a debate on the solution to the American chestnut problem. Assign each team one of the positions discussed earlier that the team will research and defend.

Make enough copies of Student Page: American Chestnut Debate Preparation and Student Page: Strength of Argument for each student for the American chestnut debate.

DOING THE ACTIVITY 1. Write the following question on the board and ask students to write on the board what they know about the question in response: How should scientists preserve the American chestnut? 2. There are essentially four answers to the question above:

5. Allow the students time to research their topics. 6. Initiate a debate using the following parameters:

ƒ Crossbreed the American species with the Chinese chestnut, which is resistant to the blight, and then backcross to reduce as many of the Chinese genes as possible while keeping the blight-resistance traits. ƒ Inoculate fungal cankers on existing American chestnut stumps with a virus that offers the trees blight resistance so they can grow to maturity. ƒ Look for genes that provide the Chinese chestnut with resistance to the blight, and insert those genes into a population of American chestnuts to create a genetically engineered American chestnut.

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4. Use Student Page: American Chestnut Debate Preparation to help the students divide their topics into areas of research so that each member of the team becomes an expert in one particular area of this solution. If groups have more than four members, some topics may need to be shared.

ƒ Each team will take turns initiating a new topic of debate by promoting the strengths of its own position in less than 1 minute. ƒ Opposing teams will take turns attacking those strengths or will use their allotted time of less than 1 minute to mention the merits of the solution they represent. ƒ All members of a single team must take turns speaking so that each member has an opportunity to present his or her findings. ƒ Points will be earned when a new idea that is accurate (pro or con) is mentioned within the given time frame.

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ƒ When the debate begins to get repetitive or when it becomes apparent to the judge or teacher that the participants have exhausted their main ideas, a 5-minute reorganization session will be called to allow teams to review their remaining arguments and to organize themselves for presenting their final points. ƒ Each team will take a turn presenting a 1-minute concluding statement that summarizes the team’s strongest arguments in support of its position. 7. Hand out Student Page: Strength of Argument to each student. Ask the students to answer the questions on the page. 8. Allow students time to reflect on (a) what the effectiveness of the debate was, (b) how their level of understanding increased during this activity, and (c) how the level of preparedness of each student affected the team and the flow of the debate. Discuss their responses to the questions on the student page:

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ƒ Which team made the strongest arguments? ƒ Which person on each team presented its information in the clearest and most persuasive manner? ƒ Disregarding personalities and the team you represented, but taking into consideration only the facts that you now know about this situation, what do you think the best solution is for the case of the American chestnut? 9. Ask the students if they think scientists should debate the strengths and weaknesses of potential solutions to problems in environmental science in the same manner. 10. Ask the students to share their observations with the class and announce their decision as to which team argued its position best.

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STUDENT P AGE American Chesnut Debate Preparation Position

Support for This Position

Arguments against This Position

Crossbreed American species with Chinese species; then backcross to the American species to regain traits while keeping blight resistance genes.

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Inoculate stumps with virus that offers blight resistance so the stumps grow to maturity.

Isolate genes that offer blight resistance; use those genes to make a genetically engineered American species.

Do nothing; allow other species to fill in the habitat and uses of this species, which has been outcompeted.

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STUDENT P AGE Strength of Argument Consider the role that each person played in this debate. Answer the following questions as honestly as you can: • Which team made the strongest arguments? • Which person on each team presented its information in the clearest and most persuasive manner? • Disregarding personalities and the team you represented, but considering only the facts that you now know about this situation, what do you think is the best solution for the case of the American chestnut? Explain why you feel this way.

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Part E: What’s Your View?— Alternative Debate or Discussion Topics

2. Ask your students to read the background information presented on the student page for the topic you have chosen to discuss or debate. 3. After your students have read the student page information, you may choose to discuss the case study as a class, or you might ask the students to divide into teams for or against the production of transgenic crops.

getting ready Copy either Student Page: Chinese Transgenic Poplars Experiment or Student Page: Oregon Bentgrass Gene Escape Story for each student. Each student will also need a copy of Student Page: Strength of Argument if you choose to review the topic in a debate format.

DOING THE ACTIVITY 1. If you would like your students to explore other case studies of transgenic plant use in situations with compelling risks and benefits, you may choose to explore one or both of the following topics: • Chinese transgenic poplars • Oregon bentgrass gene escape

5. Allow the students time to research their topics. 6. Initiate a debate using the following parameters:

Chinese transgenic poplars

ƒ Each team will take turns initiating a new topic of debate by promoting the strengths of its own position in less than 1 minute. ƒ The opposing team will be given an opportunity to attack the strengths presented, or it may use its time of less than 1 minute to mention the merits of the solution it represents.

Source: www.dailygalaxy.com/photos/ uncategorized/2007/10/17/trees_2.jpg

ƒ All members of a single team must take turns speaking so that each member has an opportunity to present his or her findings.

Oregon bentgrass Source: http://calphotos.berkeley.edu/imgs/128x1 92/0000_0000/1205/0437.jpeg

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4. Have each team produce a debate preparation chart similar to Student Page: American Chestnut Debate Preparation to help the students divide their topics into areas of research. Each member of the team should focus on one topic listed on the debate preparation chart so that the team can gather as much information as possible on the arguments for and against that particular issue.

ƒ Points will be earned when a new idea that is accurate (pro or con) is mentioned within the given time frame.

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ƒ When the debate begins to get repetitive or when it becomes apparent to the judge or teacher, a 5-minute reorganization session will be called to allow teams to review their remaining arguments and to organize themselves for presenting their final points. ƒ When all arguments have been exhausted, teams will have 5 minutes to write a conclusion that states their strongest arguments in support of their positions. ƒ Each team will take a turn presenting one concluding statement in less than 1 minute. 7. Hand out Student Page: Strength of Argument to each student. Ask your students to answer the questions on the page. 8. Allow students time to reflect on (a) what the effectiveness of the debate was, (b) how their level of understanding increased during this activity, and (c) how the level of preparedness of each student affected the team and the flow of the debate. Discuss their responses to the questions on the student page:

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ƒ Which team made the strongest arguments? ƒ Which person on each team presented its information in the clearest and most persuasive manner? ƒ Disregarding personalities and the team you represented, but taking into consideration only the facts that you now know about this situation, what do you think the best solution is for the case of the Chinese poplars or the Oregon bentgrass? 9. Ask the students if they think scientists should debate the strengths and weaknesses of potential solutions to problems in environmental science in the same manner. 10. Ask students to share their observations with the class and to announce their decision as to which team argued its position best.

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STUDENT P AGE Chinese Transgenic Poplars Experiment After decades of attempting to reforest the northern regions of China where (a) the top layer of soil is dry, (b) the climate is arid, (c) the winds are continuous, and (d) the pest species are voracious, the Chinese government—with funding from the United Nations Development Programme—introduced a poplar species (Populus nigra) with a Bacillus thuriensis (Bt) gene for insect resistance.1 With the Great Leap Forward in the late 1950s, China wiped out a large portion of its forests, which resulted in disastrous floods in the mid-1990s and an increased demand for imported wood.2 Logging is now banned in the headwaters of major rivers, and the government is aiming to establish a 2,800-mile-long shelterbelt of trees across northwestern China near the Gobi Desert.3 More than 1.4 million genetically engineered poplar saplings were planted between 1997 and 1999 in the northern region of Xinjiang near the border of China and Mongolia covering a 300- to 500-hectare area with the aim of covering more than 44 million hectares by 2012.4 The transgenic species is a fast-growing poplar that establishes deep roots that are capable of tapping into the ample supply of groundwater while reducing soil erosion and providing fuelwood. That reforestation effort is the only widespread use of a transgenic forest tree species planted in the world. Because very little information is available on the results of that reforestation, it is unclear what the effects have been on this arid ecosystem. Because the tree-planting program has been conducted over such a large area with the intention of promoting the maximum amount of forest cover, neither the government nor the scientists who produced the genetically engineered trees have any records of the exact location where those genetically engineered trees have been planted.5 Huoran Wang, the Chinese Academy of Forestry representative in Beijing who is on the United Nations Food and Agriculture Organization (UN FAO) Panel of Experts on Forest Gene Resources told the UN FAO that the “poplar trees are so widely planted in northern China that pollen and seed dispersal cannot be prevented.”6 There is currently no strategy in place to limit, isolate, or avoid vegetative spread or crossbreeding of genetically engineered poplar species with non–genetically engineered species.7 Discussion Questions • What are some of the environmental and economic effects that genetically engineered trees may have on the region where they have been planted? • In what ways will the Bt gene affect the insect population of that region, and what effects might the gene have on the survival of non–genetically engineered trees in the area?

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STUDENT P AGE Chinese Transgenic Poplars Experiment (continued) • If the goal of the reforestation program in China was to reduce soil erosion and to provide a sustainable source of wood, would it be considered a positive or negative development if the genetically engineered poplars prospered enough to merge with and extend the existing forest? • The genetically engineered poplar is a clone species in which all the trees have identical genetic material. How can this trait be a negative or positive feature for this particular situation? Endnotes: 1. Chris Lang, “Genetically Modified Trees: The Ultimate Threat to Forests,” World Rainforest Movement and Friends of the Earth, December 2004, www.wrm.org.uy/subjects/GMTrees/text.pdf. 2. Rebecca Renner, Kris Christen, Catherine M. Cooney, and Paul D. Thacker, “China’s Wild Card on Transgenic Tree Front, Environmental Science and Technology 39, no. 5 (2005): 96A–103A, http://pubs. acs.org/subscribe/journals/esthag-w/2005/jan/tech/kc_chinatree.html. 3. Ibid. 4. Clive Chan, “Supertrees to the Rescue,” Catalyst, Spring 2005, www.carleton.ca/catalyst/2005/s7.html; Renner and others, “China’s Wild Card on Genetic Tree Front”; Yang Zili, Zhou Shouyi, Zhang Weidong, and Yang Zixiang, “Poplar Genetic Resources in North China: the Challenge of Sustainable Forestry,” Forest Genetic Resources No. 27, UN Food and Agricultural Organization, www.fao.org/docrep/008/x4133e/ X4133E02.htm. 5. W. Lida, H. Yifan, and H. Jianjun, Molecular Genetics and Breeding of Forest Trees, ed. S. Kumar and M. Fladung, pp., 2005). 6. Lang, “Genetically Modified Trees.” 7. Ibid.

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STUDENT P AGE Oregon Bentgrass Gene Escape Story In 2003, the Scotts Company obtained permission from the U.S. Department of Agriculture to grow 2 hectares of transgenic creeping bentgrass (Agrostis stolonifera) in a bentgrass containment area in north central Oregon east of the Cascade Mountains. The Willamette Valley region west of the Cascade Mountains supports a $200 million annual export business of turf grass that is used on golf courses throughout the world.1 The transgenic species of bentgrass would contain the CP4 EPSPS gene that would allow the grass to be sprayed with a glyphosate herbicide (commonly called Roundup®) that would kill all plants except the bentgrass.2 Because this grass is used as turf on putting greens, the inclusion of that gene would help golf courses maintain weed-free greens more easily. Bentgrass is a wind-pollinated perennial species of grass that can also grow from stolons (stems that run along the ground horizontally) or from seed. There are 34 species of bentgrass in North America, 14 of which are native to Oregon, and many of the species cross-pollinate. Although concern was expressed about the cross-pollination of the transgenic species with other species of invasive plants or bentgrass in neighboring fields, the transgenic species was not engineered to be sterile because the seeds were meant to be harvested for sale and export. Harvested seeds were transported in sealed containers, and machines that planted or harvested the genetically modified bentgrass were fumigated before leaving the control area.3 After the flowering season in 2003, bentgrass from as far as 21 kilometers outside the containment area was found to have herbicide resistance.4 Because genetically modified organisms are not permitted in Europe or in Japan, the farmers of bentgrass in western Oregon were very concerned about the spread of the herbicide-resistant gene to plants in their own fields.5 Ecologists were concerned about maintaining the genetic integrity of the 26 species of Agrostis that are native to North America. In late 2003, the experimental bentgrass containment area was taken out of production, and a mitigation program to eliminate genetically modified organisms (GMOs) in the region was initiated. In a scientific study conducted in 2006, 62 percent of the 585 creeping bentgrass plants tested in the containment region were GMOs.6 Discussion Questions • What concerns might farmers and scientists have voiced before the genetically engineered bentgrass was planted?

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STUDENT P AGE Oregon Bentgrass Gene Escape Story (continued) • Which of the concerns actually occurred? • What long-term economic and environmental effects might occur as a result of the genetically engineered bentgrass project in Oregon? Endnotes: 1. Doug Tankersley, “Grass Seed Industry Unready for Roundup,” Oregon Magazine, 2003, http:// oregonmag.com/GMGrass.htm 2. J. R. Reichman and Lidia S. Watrud, “Identification of Escaped Transgenic Creeping Bentgrass in Oregon,” ISB News, April 2007, www.isb.vt.edu/articles/apr0701.htm. 3. Eric Baack, “Engineered Crops: Transgenes Go Wild,” Current Biology 16, no. 15 (2006): R583–R584, http://wwwdata.forestry.oregonstate.edu/orb/worddocs/Baack_2006_MolecEcol_Bentgrass.pdf. 4. Reichman and Watrud, “Identification of Escaped Transgenic Creeping Bentgrass in Oregon.” 5. Tankersley, “Grass Seed Industry Unready for Roundup.” 6. M. L. Zapiola, C. K. Campbell, M. D. Butler, and C. A. Mallory-Smith, “Escape and Establishment of Transgenic Glyphosate-Resistant Creeping Bentgrass (Agrostis stolonifera) in Oregon, USA: A 4-Year Study,” Journal of Applied Ecology 45, no. 2 (2007): 486–94,www.blackwell-synergy.com/doi/abs/10.1111/ j.1365-2664.2007.01430.x.

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Part F: Mapping Trees in Our Community

ƒ What percentage of a city should trees cover in order to provide the benefits?

getting ready ƒ Decide what area around your school should be mapped so that each group surveys a large enough region to include 25–50 trees (if species diversity is high or if overall tree density is low, use a smaller number of trees; if species diversity is low or if overall tree density is high, use a larger number of trees). ƒ Using several sheets of graph paper that are taped together, draw a map that encompasses the entire area that will be surveyed by your class (the number of sheets of graph paper that you use should equal the number of groups so the map can be broken apart to allow each group to complete a part of the overall map; see step 3 below). ƒ Copy Student Page: Tree Species and Location Data Sheet for each group. ƒ Obtain graph paper and one field guide to trees of North America (or trees in your specific region) for each group (if you have an area with many ornamental or introduced trees, you may need to create your own field guide). Note: This activity would be much more challenging in the winter when the trees have limited foliage and few, if any, flowers.

DOING THE ACTIVITY 1. Ask the student a few questions to generate interest in the topic they will be studying: ƒ Why do people plant trees in urban areas? What do trees provide in urban areas? To absorb noise pollution, air pollution, and water runoff; to purify air and water; to provide shade; to reduce temperature and reduce wind; to provide habitat and breeding areas for wildlife; etc.

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2. Preview the activity with your students so they understand what they will be doing and why their work needs to be accurate. 3. Review the map of the total area that will be surveyed, and point out some landmarks on the map to orient the students. You can then dissemble the pieces of graph paper that make up the survey area so each group has one piece of graph paper to fill in during the tree identification. 4. Introduce the field guides for trees, and show the students examples of the types of trees found in your region. Give them key characteristics to help them identify trees they are likely to see. If your students have never used field guides, you may want to take a class period to practice using dichotomous keys for the trees in your area so your students feel comfortable using the guides and looking for identifying characteristics before sending them out on the mapping project. You can also collect samples of the trees found in your area and send a tagged sample with each group for easy identification. You may choose to invite a U.S. Forest Service educator or local botanist to help your students identify common trees in your area. You may also need to rely on the knowledge and skills of your students who have grown up in the area or who are familiar with the local plants. 5. Give each group its piece of graph paper and point out the size and scale of the map. Clarify that each group’s objective is to find each tree in its portion of the map, then identify and note the location and the species on its piece of graph paper and on the Student Page: Tree Species and Location Data Sheet. If you have the ability, you may want to use a global positioning system (GPS) to locate the latitude and longitude of the trees in your area so they can be positioned precisely. You may want to agree

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as a class that only trees exceeding a certain height (e.g., knee-high) will be included or that all trees of any size will be included. 6. Allow the groups to go out into the surrounding area to begin their survey. Give them a definite time period to return to class with the day’s results, providing enough time to identify difficult trees when they return to class. Ask them to bring back leaves and fruit or a description of any tree that they were unable to identifying with certainty. 7. Before the period ends for the day, go over the results your students have so far, and help them identify tree samples that they have brought to class. 8. Allow your students to continue the survey until they complete all the maps. Ask the groups to go over their data using markers that are dark colors so the maps will be easy to read after they are photocopied. 9. Photocopy the maps created by each group, and give a copy to each student. 10. Ask the students to combine the group maps so they create one composite map. 11. Ask each student to create a complementary map that reveals how the region would be affected if a disease were to wipe out a particular local species. Give each student a different species, or give several students the same species, depending on the diversity of your area. 12. Ask the students to divide into groups so that each member has created a map that has wiped out a different species. Within the mixed groups, ask your students to take turns sharing their map and describing what the resulting map would look like if that specific species were no longer present.

Activity 4: Forest Biotechnology © American Forest Foundation

enrichments ƒ Visit an agricultural field station where plants are being grown and tested for viability under various treatments. ƒ Career Connection: Take a field trip to a genetics lab where students can observe the equipment that is used in a working research laboratory. Invite them to learn about the research questions that are currently being explored at that lab, and encourage them to consider the career options in this field of science. ƒ Students will be introduced to a case study of the reforestation project in China where fastgrowing, nonnative poplar trees were used to stem erosion problems. Because those trees were challenged by insect infestation, a genetically modified poplar was introduced to overcome the obstacle. ƒ Have the students create a field guide for trees and shrubs in your neighborhood. Each species could be represented on a large index card, and the cards can be hole-punched and placed on a ring clip so they are portable. ƒ Ask the students to research situations where genetically engineered species of trees were used to (a) combat erosion, (b) provide firewood, or (c) solve other cultural or ecological problems (e.g., poplar trees used in China). In the case study, students can be asked to list the positive and negative effects of the biotechnological solution and then to describe whether a traditional method would have had greater success or presented more difficulties.

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STUDENT P AGE Tree Species and Location Data Sheet Species of Tree

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Map Locator Symbol

Species of Tree

Map Locator Symbol

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