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Evaluation of the Bile Salt Sensitivity Gene Locus in Escherichia coli Anna Graves, Angeline Satchell, Sandra Leke-Tambo. Rachael Scott, Jonna Whetsel, and James E. Bidlack University of Central Oklahoma, Edmond, OK 73034 Abstract Results Conclusions The data collected have helped confirm that there is bile salt sensitivity in the JC3272I E. coli strain. Research will continue to identify if a mutation has occurred at or near the yciS and yciM genes using PCR to isolate the proposed sight of mutation, and subsequent sequencing will create confirmation. We hope to publish these results and will pursue subsequent spot testing to create higher quality data. This project focuses on a chromosomal mutation which may cause bile salt sensitivity in Escherichia coli. Previous experiments have shown that the mutation is located at or near the yciS and yciM genes. The current focus of the research is to confirm that the mutated strain of E. coli shows bile salt sensitivity. To demonstrate bile salt sensitivity, a process similar to a minimum bactericidal concentration test is performed on five strains of E. coli, including the mutated strain, a wild type strain, a common laboratory strain, and two knockout strains - one lacking the yciS and another lacking the yciM gene. A sister project is currently evaluating the sequence of the mutation using a polymerase chain reaction to pinpoint and replicate the sight of the mutation. Once both components of the project are complete, the mutated sequence will be cloned and placed into a plasmid in an attempt to transform a resistant strain into a sensitive strain. The results from this experiment will provide information for modified descriptions of the yciS and yciM genes. Acknowledgment • Figure 1 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. Hypothesis There is a base pair insertion at or near the yciS and yciM genes that causes a bile salt sensitivity in Escherichia coli Literature Cited Bidlack JE, Silverman PM. An active type IV secretion system encoded by the F plasmid sensitizes Escherichia coli to bile salts. J Bacteriol. 2004 Aug;186(16):5202-9. PubMed PMID: 15292121; PubMed Central PMCID: PMC490876. Figure 2 Discussion Materials & Methods • • • • • Bile Salt Treated Agar Plates: 1.5 grams Bile Salt 40 grams LB Agar Mix 1000 ml distilled water Dilutions of five strains of E.coli are made in a range between 1.0 – 0.000001. These dilutions are tested on both a 1.5% bile salt environment, and on a standard LB agar plate. Figure 1: LB Agar Plate, dilution columns from left to right, 1, .01, .0001, .000001. In order from top row to bottom JC3272I, JC3272F, BW25113, JW1271, JW1272. The strain which we believe is mutated is JC3272I, and strains with gene knockout where mutation is thought to occur, JW1271, and JW1272. Each strain is growing vibrantly in a LB Agar Plate Figure 2: Growth shown is on a 1.5% bile salt agar plate, dilution columns from left to right, 1, .01, .0001, .000001. In order from top to bottom: BW25113, JW1271, JW1272, JC3272 F, and JC3272I. JC3272I, and strains with the gene knockout show poor growth. While the lab strain, BW25113, and wild strain, JC3272F, show normal growth rates. Heewook Lee, Ellen Popodi, Haixu Tang, and Patricia L. Foster (2012) Rate and molecular spectrum of spontaneous mutations in the bacterium Escherichia coli as determined by whole-genome sequencing. PNAS.109(41) E277E2783, doi:10.1073/pnas. 1210309109 Retrieved May 2013 from http://www.pnas.org/content/109/41/E2774 Boesenberg-Smith, K. A., Pessarakli M. M., Wolk D. M. (2012, January). Assessment of DNA Yield and Purity: an Overlooked Detail of PCR Troubleshooting. Clinical Microbiology Newsletter. Retrieved May 2013 from agtc.wayne.edu/pdfs/assessment_of_dna_yield_and_purity_an_ove rlooked_detail _of_pcr_troubleshooting.pdf Merritt, E. M., Donaldson. R. J. (2009). Effect of bile salts on the DNA and membrane integrity of enteric bacteria. Journal of Medical Microbiology.58(12):1533-1541. http://jmm.sgmjournals.org/content/58/12/1533.full University of Miami. (2011). GenePage for the yciM gene of Escherichia coli K-12. Ecogene. Retrieved May, 2013 from http://www.ecogene.org/?q=gene/EG12691 Williams J. A., Carnes A. E., Hodgson C. P. (2009) Plasmid DNA vaccine vector design: impact on efficacy, safety and upstream production. Biotechnol Adv. 27(4):353–370. doi:10.1016/j.biotechadv.2009.02.003. Yoshida N, Sato M. Plasmid uptake by bacteria: a comparison of methods and Efficiencies. (2009) Appl Microbiol Biotechnol. 2009 Jul;83(5):791-8. doi:10.1007/s00253-009-2042-4. Epub. Review. PubMed PMID: 19471921.