RESULTS AND DISCUSSION Bile Salt Sensitivity and Gene Transfer in Escherichia coli Philip Grider and Jim Bidlack Department of Biology, University of Central Oklahoma, Edmond, OK 73034 ABSTRACT This is a continuation of research focused on a newly-discovered mutant strain of Escherichia coli that demonstrates sensitivity to bile salts. During the process of isolating this mutation, we found that it is at or near a previously described gene locus. Hence, this research may help to further elucidate the function of gene(s) in this region of the bacterial chromosome. Five strains of E. coli (BW25113, JC3272F, JC3272I, JW1271 and JW1272) are currently being analyzed for bile salt sensitivity and for the presence of a gene locus that encodes for bile salt sensitivity. Amplification of DNA, through use of selective primers, is being performed by polymerase chain reaction (PCR). Initial results revealed successful amplification of the desired locus in three of the five strains, but additional research is needed to successfully amplify the remaining two strains. Once amplification of all strains is complete, DNA fragments will be cloned and inserted back into appropriate strains to confirm that this gene locus exists and to further understand how it is expressed. INTRODUCTION   Genetic variants of the bacterium, Escherichia coli, are being investigated to learn how bacteria may become bile salt resistant or sensitive through a process called transformation. Gene mapping and polymerase chain reaction can be used to isolate and amplify DNA sequences for bile salt resistance/sensitivity. Once amplified, purified sequences can then be inserted into plasmids and used in transformation experiments to further characterize the gene locus. This research will provide scientists with better understanding of the genetic makeup and mechanisms of gene transfer in bacteria and will help future scientists to develop drug treatments for antibiotic resistant bacteria. Figure 1: Gel Electrophoresis Equipment with gel MATERIALS AND METHODS   Cultures: Five E. coli strains were utilized in this experiment: BW25113, JW1271, JW1272, JC3272F, and JC3272I. DNA Extraction: Using an inoculating loop, randomly selected colonies were placed into microcentrifuge tubes containing 100 µL of TAE. PCR mixture: PCR mixture included: DI H2O, Mg2+, Primers, Extracted DNA, Taq polymerase, and dNTP’s (Figure 6). Carolina blue loading dye was added to the amplified PCR samples for electrophoresis. Thermal Cycler: A pre-set program was used on the Thermo PCR Sprint Thermal Cycler (Figure 8) to amplify the targeted locus. SDS Polyacrylamide Electrophoresis: A gel was prepared using a mixture of 1.5 g agarose and 150 mL of TE Buffer. This mixture was heated until clear, with 5 µL of EtBr then added. The gel and amplified PCR mixtures were ran on a FisherBiotech Model H5 Horizontal Gel Electrophoresis System (Figure 1). Gel Imaging: The Kodak Gel Logic 100 gel imager was used to identify and document the results from electrophoresis. JW1272 JW1271 100 10-2 10-4 10-6 8μl 4μl 2μl 1μl 8μl 4μl 2μl 1μl Ladder JW1272 JW1271 BW25113 JC3272I JC3272F Ladder Ladder Control Control Ladder JC3272F JC3272I BW25113 JW1271 JW1272 1.8 kbp 1.8 kbp Figure 4: Bile Salt Plate showing differential selection Figure 6: Pipetting the PCR Mixture 100 10-2 10-4 10-6 JC3272F JC3272I BW25113 JW1271 JW1272 Figure 2: Gel image showing gene locus and desired banding Figure 3: Mg2+ concentrations test to amplify JW1271 & JW1272 Figure 5: LB Plate showing growth at all concentrations Figure 7: Serial Dilutions & Plating Figure 8: Loading PCR Thermal Cycler RESULTS AND DISCUSSION Preliminary results have shown to be successful both in differentiating resistance/sensitivity to bile salts (Figures 4 and 5), as well as in the amplification of the desired gene locus using PCR and gel electrophoresis (Figures 2 and 3). Results indicate JC3272F and BW25113 show resistance to bile salts, while JC3272I is sensitive. JW1271 and JW1272 were engineered to have only specific fragments of the gene for resistance, so the consequence of JW1271 showing no resistance to bile salts in any trial to date leads us to speculate that the resistant gene is more likely to be found closer to the fragment remaining on JW1272. Testing also indicates higher PCR amplification success rates of the 1.8 kbp gene sequence in the two knockout strains (JW1271 & JW1272) by decreasing Mg2+ concentrations below the 2 μL initially utilized to 1 μL (Figure 3). Further analysis will be done to confirm the 1 μL Mg2+ concentrations are sufficient in amplification of all strains. Amplification and purification of the 1.8 kbp locus of all strains will be completed allowing for cloning and testing for transformation. This project will help further our understanding of gene transfer and, perhaps, development of pharmaceuticals to offset antibiotic resistance in bacteria. LITERATURE CITED Bidlack, J.E., and P.M. Silverman. 2004. An active type IV secretion system encoded by the F plasmid sensitizes Escherichia coli to bile salts. Journal of Bacteriology 186:5202-5209. ACKNOWLEDGEMENTS Funding for this project was provided by the University of Central Oklahoma CURE-STEM program and Office of Research & Grants. We thank Dr. Philip Silverman of the Oklahoma Medical Research Foundation for his research and guidance necessary for the current progress of this experiment. We also thank our Research Group for much needed assistance and support (Figure 9). Figure 9: Research Group