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Ryan Hahn Biology 302 AA GFP Transformation and Selective Activation in in Esche  Escherichia richia Coli Coli Introduction

As medical research advances, scientists have found ways to treat a range of diseases that affect humans including diabetes. Using plasmid transformations containing the recombinant DNA for human insulin in  Escherichia Coli, scientists are able to manufacture insulin on a large scale for patients 1. The

manufacturing manufacturing of insulin in Escherichia Coli is cost-effective, manageable, productive, and efficient. However, production has drawbacks including improper protein folding, an absence of post-translation modifications, and plasmid loss 1. In the  Escherichia Coli, the use of GFP can be used to demonstrate successful plasmid transformation confirming the presence of the insulin gene in bacterial colonies and has applications for quantifying protein production under the same gene promoter 4.

GFP or Green Fluorescent Protein is a protein that emits wavelengths in the green area of the visible emission spectrum. GFP has two main applications for monitoring gene expression: fusion tagging and transcription reporting2. The latter application uses GFP as a reporter gene that is expressed under a common gene promoter found in Escherichia coli demonstrating that the transformed gene is expressed in correlation with GFP expression 3.

In this experiment, we wanted to identify which sugar media(s) induce GFP production in  Escherichia coli after a plasmid transformation containing GFP and bla, a gene encoding the beta-lactamase protein

that digests ampicillin antibiotic 4. Differential GFP production is confirmed using SDS-PAGE and quantified by spectrophotometric analysis of each sample.

Results

Phenotypic Presentation of Escherichia coli under UV Light

 Escherichia coli Observed under under UV Light Following Transformation, Growth Growth in Different Sugar Medias

Ryan Hahn Biology 302 AA Figure 1: The top three photos are Escherichia coli colonies grown in LB/ampicillin media with fructose, lactose, and arabinose respectively. The remaining photos are colonies with LB/ampicillin media, LB/ampicillin/arabinose media with professional cells, and LB/ampicillin media with professional plasmid. Colonies that glow under UV lighting demonstrate GFP expression. The arabinose colonies glow the brightest, demonstrating higher levels of expression for GFP than any other sugar media.

The presence of Escherichia coli colonies grown in an antibiotic media demonstrates a successful selection and transformation of the GFP+bla plasmid. Fluorescence intensity under UV indicates GFP expression. Comparing phenotypic presentation based on observations, arabinose sugar media induces GFP transcription at higher levels than fructose or lactose (Figure 1). Observational data alone does not conclude that the fluorescence is derived from GFP or the levels of expression for GFP, requiring SDSPAGE to confirm the expression of GFP in conjunction and the use of spectrometry to measure the levels of GFP production.

GFP Screening in E. coli

Potential GFP Expression Confirmed via SDS-PAGE in Different Escherichia coli Treatments

Figure 2: Following cell lysing using SDS solution, the following protein bands were detected on an SDS-PAGE gel. The arabinose and professional cells solutions all share sharp bands above the 25kDa marker, indicating high levels of GFP expression. The remaining solutions have faint bands suggesting GFP had low expressions in their cells or non-GFP proteins of the same size were present at low levels.

Following cell lysing and protein denaturing, denaturing, the following protein bands detected confirmed in conjunction with observations observations using UV light the high levels of expression of GFP in arabinose media and professional cells. Each solution had sharp bands above the 25kDa; GFP is 27kDa in size (Figure 2). Lactose, fructose, professional plasmid, and LB/ampicillin had fainter bands above the 25kDa marker suggesting detection of proteins greater in size relative to GFP and of a different identity. SDS-PAGE data alone only suggests the presence of a protein with a similar size to GFP in cells grown in arabinose media; it does not conclusively identify GFP as the protein detected.

Ryan Hahn Biology 302 AA Differential GFP Production Production in E. coli Growth Medias 70000     ) 60000    m    n    5 50000    8    4     (

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Figure 3: The following chart shows different GFP production levels in Escherichia coli colonies in the following growth medias in the lab group versus the class average: LB+amp. ( 1), LB+amp.+arabinose ( 2  2),LB+amp.+fructose ( 3 ), and LB+amp.+professional plasmid (8).  3), LB+amp.+lactose ( 4  4), LB+amp.+ arabinose professional cells (6 ),  After cell lysing using using lysozyme to preserve GFP function, the cell cell solutions were measured by a spectrophotometer spectrophotometer  for fluorescence intensity. intensity. Higher intensity indicates indicates higher levels of GFP activity. activity.

Using spectrophotometry, GFP expression measured to be qualitatively higher in arabinose media than other sugar media (Figure 3). Cultures with GFP had higher emission data values, indicating that the solution was more fluorescent. Data confirmed phenotypic comparisons between treatments. Class averages for each treatment were higher for nearly all treatments but the group measurements followed the same trends as the class averages showing internal consistency.

Conclusion

GFP has a wide range of applications that allow for the confirmation of bacterial transformations. Phenotypic observations between different growth media alone are inconclusive alone but produce trends in unempirical physical measurements and characteristics. SDS-PAGE when compared with UV light observations confirmed the presence of GFP in cells and spectrophotometry measured the levels of GFP expression in each treatment. Synthesizing data from each protocol, it is concluded that arabinose sugar induces the transcription of GFP. This indicates that the GFP gene is under the control of the arabinose  promoter. Lactose Lactose and and fructose show levels levels of expression expression similar similar to the base base level of the LB/AMP LB/AMP media media with no sugar substrate, substrate, indicating these sugars are not able to induce significant levels of GFP expression. expression . Due to the availability and versatility of GFP as a reporter gene, future medical research may  be able to to utilize GFP to research research diseases diseases whose whose genetic genetic origins still puzzle puzzle scientists. scientists.

References

Ryan Hahn Biology 302 AA 1: Baeshen, Nabih A., Mohammed N. Baeshen, Abdullah Sheikh, Roop S. Bora, Mohamed Morsi M Ahmed, Hassan A I Ramadan, Kulvinder Singh Saini, and Elrashdy M. Redwan. "Cell factories for insulin production." Microbial Cell Factories 13, no. 1 (2014). doi:10.1186/s12934-014-0141-0. doi:10.1186/s12934-014-0141-0. 2: Patrick, Marcy. "Plasmids 101: Green Fluorescent Protein (GFP)." Plasmids 101: Green Fluorescent Protein (GFP). Accessed February 04, 2017. http://blog.addgene.org/plasmids-101-green-fluorescent protein-gfp. 3: Alberts, Bruce, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter. Molecular Biology of the Cell. 6th ed. New York: Garland Science, 2015. 4: Martin-Morris, Linda. Laboratory Manual Biology 302. Winter 2017. Seattle, WA: Professional Copy Print, 2017.