Elucidation of the Bile Salt Sensitivity Gene Locus in Escherichia coli Angeline Satchell, Anna Graves, Sandra Leke-Tambo, Rachael Scott, Jonna Whetsel, and Jim Bidlack Department of Biology, University of Central Oklahoma, Edmond, OK Abstract Scientific research is being practiced to isolate, clone, and sequence the yciS gene that is bile salt resistant and the yciM gene that is bile salt sensitive in Escherichia coli. With the strains BW25113, JW1271, JW1272, JC3272F, and JC3272I, we are amplifying the DNA using the polymerase chain reaction (PCR), then by gel electrophoresis to analyze the DNA fragments, and followed by DNA sequencing to verify the correct series of DNA and identify any possible contamination. The team has been successful at. amplifying some strains of the E. coli. The next step will be sequencing followed by inserting plasmid into the respective part of the strains of E. coli Abstract Scientific research is being practiced to isolate, clone, and sequence the yciS gene that is bile salt resistant and the yciM gene that is bile salt sensitive in Escherichia coli. With the strains BW25113, JW1271, JW1272, JC3272F, and JC3272I, we are amplifying the DNA using the polymerase chain reaction (PCR), then by gel electrophoresis to analyze the DNA fragments, and followed by DNA sequencing to verify the correct series of DNA and identify any possible contamination. The team has been successful at. amplifying some strains of the E. coli. The next step will be sequencing followed by inserting plasmid into the respective part of the strains of E. coli Materials and Methods Cultures: The strains used in this experiment included: JC3272I, JC3272F, BW25113, JW1271, JW1272 stored in freezer at -80 o C. DNA Extraction: The strains of E. coli were plated on Petri dishes with LB broth. PCR Mixture: A PCR Kit was purchased for this protocol. The mixture includes : water, 10x buffer, DNTP, Magnesium, YCIRO, YCIFI, E. coli DNA and Taq Polymerase Thermal cycler: The thermal cycler was used after the PCR procedure was complete to amplify the DNA. Electrophoresis: A agarose gel was prepared with 190 mL of DI water and 10 mL of TAE Buffer. After the gel was made, the gel loading dye was added to the amplified PCR products. This was followed by the products being pipetted into the wells of the agarose gel for electrophoresis. Materials and Methods Cultures: The strains used in this experiment included: JC3272I, JC3272F, BW25113, JW1271, JW1272 stored in freezer at -80 o C. DNA Extraction: The strains of E. coli were plated on Petri dishes with LB broth. PCR Mixture: A PCR Kit was purchased for this protocol. The mixture includes : water, 10x buffer, DNTP, Magnesium, YCIRO, YCIFI, E. coli DNA and Taq Polymerase Thermal cycler: The thermal cycler was used after the PCR procedure was complete to amplify the DNA. Electrophoresis: A agarose gel was prepared with 190 mL of DI water and 10 mL of TAE Buffer. After the gel was made, the gel loading dye was added to the amplified PCR products. This was followed by the products being pipetted into the wells of the agarose gel for electrophoresis. Literature Cited Bidlack, J.E., and P.M. Silverman An active type IV secretion system encoded by the F plasmid sensitizes Escherichia coli to bile salts. J. Bacteriol. 186: Baba, T., T. Ara, M. Hasegawa, Y. Takai, Y. Okumura, M. Baba, K. A. Datsenko, M. Tomita, B. Wanner, and H. Mori Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Molec. Syst. Biol. 2: Literature Cited Bidlack, J.E., and P.M. Silverman An active type IV secretion system encoded by the F plasmid sensitizes Escherichia coli to bile salts. J. Bacteriol. 186: Baba, T., T. Ara, M. Hasegawa, Y. Takai, Y. Okumura, M. Baba, K. A. Datsenko, M. Tomita, B. Wanner, and H. Mori Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Molec. Syst. Biol. 2: Introduction Bacteria have the ability to adapt to drugs or chemicals and build up multidrug resistance (MDR). In this examination of Escherichia coli counteraction to bile salt, we will show that bile salt sensitivity is associated with the two genes, yciS and yciM. This experiment will show a new method of controlling antibiotic resistance in bacteria with the results giving us a better way of perceiving gene transfer in Escherichia coli. Introduction Bacteria have the ability to adapt to drugs or chemicals and build up multidrug resistance (MDR). In this examination of Escherichia coli counteraction to bile salt, we will show that bile salt sensitivity is associated with the two genes, yciS and yciM. This experiment will show a new method of controlling antibiotic resistance in bacteria with the results giving us a better way of perceiving gene transfer in Escherichia coli. Approach We are using the PCR procedure in a thermal cycler to isolate the DNA for bile salt sensitivity of the five stains of E. coli: BW25113,JC3272F, JC3272I, JW1271, JW1272. The genes yciS and yciM can be found within a 1.8 Kbp fragment of the E. coli chromosome. The restriction enzymes BamHI and SmaI will be used to cut the DNA before and after the genes yciS and yciM. The genes will be then inserted into plasmid puc19 and cloned to obtain an abundant supply of the bile salt sensitive JC3272I genes and resistant from JC3272F genes. The bile salt resistant strain JC3272F, along with the plasmid, will then be tested for bile salt sensitivity. The bile salt sensitive strain JC3272I, along with the plasmid, will also be tested for bile salt sensitivity. Results will explain how bile salt sensitivity is expressed in E. coli. Approach We are using the PCR procedure in a thermal cycler to isolate the DNA for bile salt sensitivity of the five stains of E. coli: BW25113,JC3272F, JC3272I, JW1271, JW1272. The genes yciS and yciM can be found within a 1.8 Kbp fragment of the E. coli chromosome. The restriction enzymes BamHI and SmaI will be used to cut the DNA before and after the genes yciS and yciM. The genes will be then inserted into plasmid puc19 and cloned to obtain an abundant supply of the bile salt sensitive JC3272I genes and resistant from JC3272F genes. The bile salt resistant strain JC3272F, along with the plasmid, will then be tested for bile salt sensitivity. The bile salt sensitive strain JC3272I, along with the plasmid, will also be tested for bile salt sensitivity. Results will explain how bile salt sensitivity is expressed in E. coli. Results and Discussion The E. coli were plated and DNA was extracted successfully. The PCR procedure was done on each strain of E. coli. The gel loading dye was added before doing the gel electrophoresis. The gel was analyzed using the gel imaging machine If our results show that the bile salt sensitive strains can show resistance or bile salt resistant strains can show sensitivity, the genes yciM and/or yciS are the phenotype engaged. Knowing the genes we would be one step closer to being able to make antibiotic free types of medication using E. coli. The bile salt resistant strains could have plasmids inserted to combat diseases. Results and Discussion The E. coli were plated and DNA was extracted successfully. The PCR procedure was done on each strain of E. coli. The gel loading dye was added before doing the gel electrophoresis. The gel was analyzed using the gel imaging machine If our results show that the bile salt sensitive strains can show resistance or bile salt resistant strains can show sensitivity, the genes yciM and/or yciS are the phenotype engaged. Knowing the genes we would be one step closer to being able to make antibiotic free types of medication using E. coli. The bile salt resistant strains could have plasmids inserted to combat diseases. Acknowledgments We thank The University of Central Oklahoma CURE-SSS- STEM program and the Office of Research and Grants for providing funding for this research project. We also would like to thank Dr. Philip Silverman of the Oklahoma Medical Research Foundation for his guidance and scientific research. Figure 4. Image of strains: JC3272I, JC3272F, BW25113, JW1271, JW1272 amplified. Figure 1. Plating strains of E. coli on Petri Dishes with LB broth. Figure 2. Following PCR procedure using PCR kit and E. coli. Figure 3. Preparing for gel electrophoresis.