Lab Report 3

Lab Report 3: Transformation of E. coli Cell With Yeast Shuttle Vector pRS426

By Manuel Molina Villalba Group Members: Gabriel Fernando, Elizabeth Blake April 1, 2010 University of Pennsylvania CBE 480-001 Laboratory in Biotechnology and Genetic Engineering

Introduction Experiments were undertaken using a yeast shuttle vector, pRS426, in order to assess the efficiency and success of transferring the vector from S. cerevisiae, yeast, to E. coli bacteria. This is an important procedure that makes it possible to exploit the differences between eukaryotic and bacterial cells in many genetic engineering applications. The shuttle vector was isolated from yeast cell cultures using a QIAprep® Spin MiniPrep Kit. The pRS426 vector was then mixed with Chemically Competent JM109 E. coli cells to transform these via a heat-shock procedure. The presence of the Lac-Z operon was exploited in a blue-white screening procedure that was used to identify JM109 cells that possibly transformed successfully. Cells that produce blue colony forming units were cultured for further experimentation. A final isolation of the pRS426 vector from the blue-white screened E. coli cell culture was undertaken to confirm the presence of the vector by use of flash gel electrophoresis.

Materials and Methods pRS426 Vector The vector pRS426 is a phagemid vector that is 5726 base pairs long [4]. Figure 1 displays many of the vector’s features. Some of the more important features of this vector are, in order: ori(f1) - Lac Z - T7 promoter - MCS(KpnI-SacI) - T3 promoter-LacI - ori(pMB1) - ampR - ori (2 µm) - URA3 [4]. pRS426 contains 21 restriction sites that can be used in many genetic engineering applications. It contains a 2µm origin of replication site. The vector has REP3 and FRT sequences that give it a high propagation number in yeast, which is about 20 per haploid cell [4]. Non-selective growth of host cells containing this vector experiences loss of about 4.4 ± 1.4% of progeny per doubling

through mitotic segregation [4]. One appropriate type of selective medium lacks uracil. Since this vector can express uracil production, the vector will be placed in a host strain that is deficient in this amino acid. The host cell will then be grown in a defined, uracillacking medium. The presence of the Lac Z operon makes this a suitable vector for bluewhite screening. For the applications of these experiments, blue colonies denote the desired cells since these indicate the possible presence of the pRS426 vector or any other vector containing the Lac Z with the α-fragment. Figure 1. Map of pRS426 Vector[1]: Major features of this vector include 21 restriction sites, 2µm origin of replication, high copy number in yeast cells (REP3 and FRT sequences). Other major features in order include ori(f1) - lacZ - T7 promoter - MCS (KpnI-SacI) - T3 promoter lacI - ori(pMB1) - ampR - ori (2 micron) URA3. The type of selective medium used in the subsequent experiments lacked uracial, since the vector can express the production of uracil. The Lac Z operon also contains the α-fragment that is important in the use of the blue-white screening technique. For the applications of this report the blue colony forming units (CFU’s) from the screening are desired. These colonies indicate that the CFU’s consist of cells containing the Lac Z operon with a α-fragment.

Growth Conditions for S. cerevisiae and E. coli Yeast, S. cerevisiae, cell cultures containing the pRS426 vector were grown in two different defined mediums. One was a selective medium that lacked the uracil amino acid. This medium consists of 6.7 g YNB without amino acids, 20 g d-glucose, 20 g Bacto agar, 1.4 g Sigma drop-out mix, 532 mg histidine, leucine, tryptophan mix and additional H2O until a 1L volume is reached. The histidine, leucine, and tryptophan mix consists of 76 mg histidine, 76 mg tryptophan and 380 mg leucine. The other medium was a YEPD medium that consists of 5 g of yeast extract, 10 g of peptone and 10 g of dglucose. These cells were incubated at 37˚C in suspension. Blue colonies that resulted

from the blue-white screening of the transformed E. coli cells were inoculated in 10mL of Luria Bertani (LB) medium† and incubated with shaking at 37˚C overnight. pRS426 Vector Isolation From Yeast Yeast cell cultures were separated from their respective mediums by centrifugation at about 4000 rpm for 5 minutes and discarding the supernatant. Yeast cell pellets were isolated from 13mL and 5mL of yeast suspension containing selective medium and the suspension containing YEPD medium, respectively. Each plasmid vector was isolated from its respective cell pellet by following the “Isolation of Plasmid DNA from Yeast Using the QIAprep® Spin Miniprep Kit” protocol[2].

The kit consisted of the

appropriate buffers to lyse the yeast cells, precipitate undesired cell material and a QIAprep-Spin Column to separate the plasmid from the undesired cell material. The cell pellet in acid-washed 450µm-600µm glass beads was vortexed to lyse cells by shearing. 10-30 µL of plasmid solution was isolated from the 5mL and 13mL cell suspension sample. E. coli Transformation by Heat Shock 50 µL of thawed chemically competent E. coli cells were mixed with 3µL of vector that was isolated from the yeast cells suspended in selective medium. The same mixture was made for the vector that was isolated from the yeast cells suspended in YEPD medium. The cell mixtures were kept in ice for 10 minutes. After the 10 minutes elapsed, the cell mixtures were heat-shocked at 42˚C for 45-50 seconds. The cell mixtures were then kept in ice for 2 minutes. 510 µL of SOC† medium at 37˚C was added to each mixture, and the resulting mixtures were incubated at 37˚C for 60 minutes with shaking. Blue-White Screening †

See Appendix A.1

10 µL of a mixture of 1µL of cells transformed with the vector isolated from the yeast in the selective medium, and 1 mL of LB medium was plated on an LB-Amp plate. 20µL of 50 mg/mL X-Gal and 100µL of 1M IPTG inducer were also added to the LB-Amp Plate. The same plate was made for cells transformed with the vector isolated from the yeast in the YEPD medium. As a control 100µL and 400µL samples from each cell mixture were plated on different LB-Amp plates. The plates were then incubated at 37˚C overnight, in preparation for blue-white screening. The blue-white screening technique was used to determine if the pRS426 vector is present in the transformed E. coli cells. The pRS426 vector contains a Lac promoter, operator and the α- part of the ß-galactosidase gene (Lac Z’). ß-galactosidase is expressed in α-complementation, which means that an E. coli deletion mutant with the Ω part Lac Z’M15 codes the second part of the ß-galactosidase gene. Upon screening, the Lac promoter is induced with IPTG, and the X-gal substrate is added to an agar plate with cells. Blue colonies will form as a result of X-gal being cleaved by functional ß-galactosidase. Blue colonies will confirm the presence of an uninterrupted Lac Z operon, indicating the possibility that desired vector is in the transformed cells that is being screened. If the α part (Lac Z’) of the gene is not expressed, ß-galactosidase will be non-functional with only the Ω- part. The resulting colony will be white, indicating that the Lac Z operon is missing or interrupted. pRS426 Vector Isolation From E. coli An E. coli cell pellet was separated from 5mL of E. coli cell suspension by centrifuging at 4000 rpm for 5 minutes and discarding the supernatant. After centrifuging, the plasmid was isolated from the cell pellet by using the Qiagen plasmid prep kit according to the manufacturers protocol [3]. The kit consisted of the appropriate buffers to lyse cells,

precipitate undesired cell material and a QIAprep-Spin Column to separate the plasmid from the undesired cell material. 10-30µL of plasmid solution was isolated from a 5mL cell suspension. Flash Gel Electrophoresis The flash gel electrophoresis technique was done on a gel from Lonza. Each of the plasmid vectors was run at 250 V for 8 minutes. A 1 kb DNA Ladder from NE Biolabs was used as a reference to find the mass of each band in order to deduce the concentration of plasmid. The flash gel ladder was used to measure migration distances in order to deduce the base pair of the vector. 4:6 vector samples consisting of 4µL of plasmid vector sample isolated from yeast, 1µL of TAE buffer and 1µL 6x Flash Gel Loading dye were run through the gel. A 3:6 vector sample that consisted of 3µL of plasmid vector sample isolated from E. coli, 2µL of TAE Buffer and 1µL of 6x Flash loading gel was also run through a gel.

Results and Discussion The chemically competent E. coli had a poor transformation efficiency. For 28 LB-Amp plates that were plated with transformed E. coli cells for blue-white screening, there was a yield of two blue colonies. Successive experiments were made on inoculations from these two colonies. Efficiency calculations on this data may be unreliable due to the small size of the data and the lack of distribution of blue colonies amongst the plates. From Figure 1 it can be estimated that the length of the vector lies between 1461 and 6576 base pairs. From Figure 2 it can be estimated that the length of the vector lies between 3949 Base pairs to 9502 base pairs in length. Super-imposing these to limits to find an area in which the vector lies in both have ranges we get an overall range or of

3949 and 6576 base pairs, which is a smaller range that is close to the expected size of the vector. Figure 1 Flash Gel of Yeast Shuttle Vector pRS426, Separated from Yeast: Lane 1: 5 µL 1 Kb DNA Ladder. Lanes 2 & 4: 4 µL pRS426 DNA isolated from yeast, 1 µL TAE Buffer, and 1 µL Flash Gel loading dye. Lane 9: 5 µL 1 Kb DNA Ladder. The length of the vector is estimated to be between 1461 Base pairs to 6576 base pairs in length. Isolation of the vector was difficult. The isolation may have yielded small amounts of the desired vector, decreasing the transformation efficiency.

Figure 2 Flash Gel of Yeast Shuttle Vector pRS426, Separated from E. Coli: Lane 1: 5 µL 1 Kb DNA Ladder. Lanes 6 & 8: 3 µL pRS426 DNA isolated from E. coli, 2 µL TAE Buffer, and 1 µL Flash Gel loading dye. Lanes 2-5 & 7: 3 µL pUC19 DNA, 2 µL TAE Buffer, and 1 µL Flash Gel loading dye. This vector is estimated to be 2897 Base Pairs in length. Lane 9: 5 µL Flash Gel DNA Ladder. The length of the vector is estimated to be between 3949 Base pairs to 9502 base pairs in length. Isolation of the vector was less difficult relative to isolation from yeast. There was a significant amount of vector in the E. coli cells.

From Figure 1 it can be concluded that isolation of a plasmid from yeast cells is more difficult to achieve than for E. coli cells. The isolation of the pRS426 vector proved to be more difficult for yeast cells than for E. coli. The poor isolation of the pRS426 vector may have led to the poor transformation that was experienced by the E. coli cells. Yeast cells have more residual DNA than Bacteria, and therefore the plasmid may have failed to separate in the QIAprep-Spin Column. A different separation column setup should be considered. From Figure 2, it can be concluded that it is more difficult to separate pRS426 from E.

coli than pUC19. The same protocol for plasmid isolation was followed for both vectors, but the lanes for the pUC19 plasmid, lanes 2-5 and 7, show clear and defined bands while the pRS426 plasmid sample smeared within the lane, lanes 6 and 8. Although the lanes have material smeared within the lane, there is pRS426 band outlined between [BP lengths]. This shows that the lysis of the E. coli cells containing pRS426 was successful and had similar results to that the of E. coli cell lysate containing pUC19. The smeared lanes may contain other residual DNA and cell fragments that may have failed to separate in the QIAprep-Spin Column. This means that separation of the pRS426 plasmid vector from the cells’ residual DNA fragment may be improved by using a different column set up. Options that can be considered for changing the set up include using a different column, using the same column with a different length or changing the amounts of solution that flow through. From the foregoing experiments we can consider the causes of having very low transformation efficiencies. A low transformation efficiency can result from a low yield of isolated plasmid: a lower concentration of plasmid decreases the probability of a vector permeating the cells membrane during the transformation process. From the super-imposed range of base pair lengths, it seems that the unknown vector may be pRS426. From the gel results, we can consider what factors facilitate the isolation of a plasmid: an appropriate separation column setup for the vector of interest should always be sought after if better results are to be obtained.

Acknowledgements Many thanks to everyone that helped guide our group through the experiments. Special thanks to Ken Chen, or lab coordinator, for helping us set up experiments. Also, thanks

to Dr. Miriam Wattenbarger for her guidance throughout our experiments.

References 1. Lab Life. http://www.atcc.org/catalog/numSearch/numResults.cfm?atccNum=77107 2. Qiagen Sample and Assay Technologies. www.qiagen.com. 2001. User developed Protocol: Isolation of plasmid DNA from yeast using the QIAprep® Spin Miniprep Kit. www.qiagen.com/literature/handbooks/default.asp. 3. Qiagen Sample and Assay Technologies. www.qiagen.com. 2006. Protocol: Plasmid DNA Purification Using the QIAprep Spin Miniprep Kit and a Microcentrifuge. QIAprep® Miniprep Handbook. 2 ed. 22- 24. 4. Saccharomyces Genome Database (SGD) project. Vector Data Base. http://genome-www.stanford.edu/vectordb/vector_descrip/PRS426.html.

Appendix A.1: Recipes for Luria Bertani and YEPD Medium LB (Luria-Bertani) Liquid Medium Tryptone Yeast Extract NaCl

10 g/L 5 g/L 10 g/L

H2O 900 mL **Adjust to pH 7.5 with 1 M NaOH. Approximately 1 mL/L broth.

LB Agar Add 15 g/L of agar to the LB liquid medium. Autoclave.

LB-Amp Medium Add 100 µg/ml of Ampicillin for LB-Amp medium.

Ampicillin Make up 500 mg of Ampicillin in 20 mL of H2O (25 mg/mL) and filter sterilize, dividing into 1 mL aliquots. Store frozen at -20 to -80°C. Add to cold liquid medium or 45°C medium containing agar to prevent thermal degradation. Typical concentrations for selecting cells containing plasmids are 50 to 100 µg/mL which are prepared by adding 2 to 4 mL of stock to a liter of medium. Plates containing ampicillin can be kept at 4°C for 2 weeks; in an incubator at 37°C, the ampicillin will be degraded in 24 hours. The frozen stock can be kept for several months.

YEPD Medium Yeast Extract Peptone D-glucose (Dextrose) **Dissolve in 500 ml of H2O. Autoclave.

5g 10 g 10 g