Transcript Slide 1

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 73034
Abstract
Approach
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
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.
Escherichia coli Strain
Sensitivity to Bile Salts
BW25113
Resistant
JW1271
Sensitive
JW1272
Sensitive
JC3272F
Resistant
JC3272I
Sensitive
Figure 2. Following PCR procedure using
PCR kit and E. coli.
Figure 1. Plating strains of E. coli
on Petri Dishes with LB broth.
Materials and Methods
Figure 3. Preparing for gel
electrophoresis.
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.
Cultures: The strains used in this experiment
included: JC3272I, JC3272F, BW25113, JW1271,
JW1272 stored in freezer at -80o 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.
Figure 4. Image of strains: JC3272I, JC3272F, BW25113, JW1271,
JW1272 amplified.
Acknowledgments
We thank The University of Central Oklahoma CURE-SSSSTEM 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.
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. J. Bacteriol. 186:5202-5209.
Baba, T., T. Ara, M. Hasegawa, Y. Takai, Y. Okumura, M. Baba, K. A. Datsenko, M. Tomita, B. Wanner,
and H. Mori. 2006. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the
Keio collection. Molec. Syst. Biol. 2:2006.0008.