Transcript Solar Poster 2005 - University of Central Oklahoma

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
Figure 1: Gel Electrophoresis
Equipment with gel
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.
JW1271
8μl 4μl 2μl 1μl
8μl 4μl 2μl 1μl
100 10-2 10-4 10-6
Ladder
JW1272
Control
Control
Ladder
Ladder
JC3272F
JC3272I
BW25113
JW1271
JW1272
Ladder
MATERIALS AND METHODS
JC3272F
Cultures: Five E. coli strains were utilized in this experiment:
BW25113, JW1271, JW1272, JC3272F, and JC3272I.
JC3272I
BW25113
DNA Extraction: Using an inoculating loop, randomly selected colonies
were placed into microcentrifuge tubes containing 100 µL of TAE.
JW1271
JW1272
1.8 kbp
1.8 kbp
Figure 4: Bile Salt Plate
showing differential selection
100
10-2
10-4
10-6
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.
Figure 6: Pipetting the PCR Mixture
Thermal Cycler: A pre-set program was used on the Thermo PCR
Sprint Thermal Cycler (Figure 8) to amplify the targeted locus.
JC3272F
JC3272I
BW25113
JW1271
JW1272
Figure 2: Gel image showing
gene locus and desired
banding
Mg2+
Figure 3:
concentrations
test to amplify JW1271 &
JW1272
Figure 5: LB Plate showing
growth at all concentrations
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.
Figure 7: Serial Dilutions & Plating
Figure 8: Loading PCR Thermal Cycler
RESULTS AND DISCUSSION
LITERATURE CITED
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.
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 186:52025209.
ACKNOWLEDGEMENTS
Figure 9: Research Group
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).