Transcript Solar Poster 2005 - University of Central Oklahoma

Gene Transfer Study as Related to Bile Salt Sensitivity in Mutant Escherichia coli Strains
Sean Reim, Adelee Traylor, and Jim Bidlack
Department of Biology, University of Central Oklahoma, Edmond, OK 73034
ABSTRACT
Biomedical investigations are being pursued to determine if
bile salt sensitivity in Escherichia coli can be attributed to a
gene locus within the bacterial genome. Techniques such as
polymerase chain reaction (PCR), agarose gel electrophoresis,
and DNA sequencing are being implemented in order to track
down, sequence, and identify this gene locus. Currently, an
undergraduate student is learning these techniques with new
equipment purchased by the UCO Biology Department and
through Oklahoma’s IdeA Network of Biomedical Research
Excellence (INBRE) program. After several months of rigorous
scientific analysis, our team successfully isolated and
amplified the desired gene locus in three of the five mutant E.
coli strains under investigation. These strains are as follows:
BW25113, JC3272F, and JC3272I. Further investigation is
needed in order to determine why PCR was unable to amplify
the remaining E. coli strains (JW1271 and JW1272) for the
desired gene locus. With successful amplification of some
mutant strains, sequencing of the amplified gene locus in
those strains can now begin. Once sequencing is complete,
our team will move forward to the next phase of this
experiment, which is to determine whether this gene is
expressed as a dominant or recessive allele.
APPROACH
RESULTS AND DISCUSSION
A total of five E. coli strains (Figure 1) underwent DNA amplification by means of
polymerase chain reaction in a thermo-cycler (Figures 2, 3, and 4). Once
amplification was complete, PCR samples were subjected to agarsose gel
electrophoresis in order to produce a gel, which was then imaged (Figure 5 and 6)
for future reference. Once amplification was considered satisfactory, the amplified
DNA from the original PCR samples were cleaned and processed using a PCR
clean-up kit. These newly cleaned samples are awaiting shipment to an
independent lab for sequencing. If a mutation is confirmed through sequencing, the
section of the genome containing the mutation will be placed into the appropriate
vectors for cloning. Once cloning has taken place the DNA will be transferred into a
selected line of cells. The cloned DNA will contain an antibiotic marker, thus
enabling us to easily determine whether the DNA was successfully inserted into the
cell plasmid. Once the cells have successfully taken up the mutant DNA, it will be
determined whether the gene for bile salt sensitivity is dominant or recessive. This
determination of expression will be done by inserting a plasmid for bile salt
resistance into a bacterium that is known to be bile salt sensitive. If the bile salt
sensitive bacteria then show signs of becoming resistant post insertion of a bile salt
resistant plasmid, then we know the gene to be dominant.
Cultures of each strain were plated and colony isolates were successfully obtained (Figure 1).
Selected colonies were uniform in color, morphology, and size. Hence, the same protocol for each
PCR trial was implemented for each strain of E coli. After each PCR trial, samples were added to a
loading dye (Carolina blue) and subjected to electrophoresis. Upon completion of electrophoresis,
the gel was analyzed using UV light provided by a gel imaging machine. (Figures 5 and 6). The
results of each trial were digitally recorded onto a designated computer and analyzed for significant
data. A lambda ladder was used for each trial in order to properly assess the band size. Several
trials were performed on each of the five strains and only the gels containing successful
amplification of the desired band size of 1.8 Kbp are shown. Figure 7: Black arrows indicate where
successful amplification of the desired locus has correlated to the appropriate banding region in the
strain BW25113. Black arrows in Figure 8 indicate where successful amplification of the desired
locus has correlated to the appropriate banding region in strains JC3272F and JC3272I.
Figure 1:
Streaking plates
Figure 2: Sean and Madeline
Pipetting
Figure 3: Sean Reim with
Thermo-cyclers
Multi-drug resistance (MDR) among organisms is fast becoming an increasing challenge to the
treatment of those at risk of contracting a bacterial infection. It is important to continue pursuing
research that will aid in preventing the phenomenon of MDR and its spread to other species; which,
as of yet, do not possess the capability of MDR. Our research has produced the isolation and
amplification of a gene locus (approximately 1.8 Kbp in size) which seems to be linked to bile-salt
sensitivity in 3 mutant E. coli strains (BW25113, JC3272F and JC3272I). This suggestion is based
on the results of previous research conducted by OMRF investigators. Bile-salt sensitivity is unique
in that it is thought to be induced by disabling the gene responsible for bacterial secretion systems
(i.e. efflux pumps). This particular gene locus is a part of an operon known to control transport
proteins responsible for the construction of bacterial secretion systems. Once disabled, bacteria
lack the ability to pump toxic substances back into their external environment; thus making them
susceptible to the effects of various toxic substances, which in most cases results in the death of
the bacterium. BW25113 is a generic E. coli strain known to contain the desired targeted locus and
was thus used as a control in this experiment. The successful amplification of mutant strains
JC3272F and JC3272I will be sequenced and the subsequent information used to set up a cloning
experiment in order to determine whether this gene is in fact responsible for bile-salt sensitivity.
Further experiments will show how this gene operates within the operon and whether bile-salt
sensitivity is dominant or recessive.
INTRODUCTION
In recent years bacteria have gained the attention of the scientific
community with their ability to become multidrug resistant (MDR).
Any elucidation into these mechanisms of resistance would prove
monumental in terms of medical costs and potential human lives
saved. Efflux pumps (bacterial secretion systems) play an
important role in a bacterium’s ability to become MDR. Efflux
pumps enable bacteria to extrude toxic substrates from within the
cell and into their external environment. It is estimated that roughly
5-10% of bacterial genomes are committed to transport, with a
significant proportion of these being involved with efflux pumps.
Genes controlling the assembly of pili through the complex
interactions of these proteins can be found in plasmids. Those
bacteria carrying the efflux pump genes in their chromosome gain
the inherent advantage of increasing their chances of survival in
non-suitable environments. Efflux genes are part of an operon
with a single regulatory gene in control of expression. An increase
in expression of these genes is coupled to a resistance of various
substrates (antibiotics, bile salts etc…)
Previous research, through the collaborative efforts of OMRF and
UCO, produced an effective screening technique for inhibitors of
bacterial secretion systems.
These procedures exploit the
sensitivity of certain strains of E. coli to bile salts. The screening
technique provides a means by which we can hypothesize and test
possible mechanisms for gene transfer in bacterial mutants.
Further investigation through the use of these techniques is
needed in order to provide a conceptual model of how multiple
proteins interact to enable F-pilus formation.
Figure 4: Sean Reim with
Thermo-cyclers
Figure 5: Sean Reim with
Gel Imaging Machine
Figure 6: Jim Bidlack with
Gel Imaging Machine
L
BW25113
JW1271
JW1272
JC3272F
L BW25113
JC3272I
JW1271
JW1272
JC3272F
JC3272I
MATERIALS AND METHODS
Cultures: Two separate sets of mutant strains were kept in a -80˚ C freezer. Each set of mutants
were stored in glycerol. The strains used in this experiment included: BW25113, JW1271, JW1272,
JC3272F, and JC3272I.
DNA Extraction: Cultures were plated approximately every two weeks. Three colonies from each
strain were selected for amplification based upon morphology. Using an inoculating loop, selected
colonies were placed into a microcentrifuge tube containing 100 µL of TAE. Each DNA extraction
tube was subsequently vortexed and stored properly in a 4˚F fridge for subsequent analysis.
PCR mixture: A PCR mixture was prepared based upon a predetermined set of protocols. The
mixture included: DI H2O, Mg2+, Primers, Extracted DNA, Taq polymerase, and dNTP’s.
Thermocycler: We used a pre-set program on the thermocycler in order to most efficiently amplify
the targeted locus.
SDS Polyacrylamide Electrophoresis: A gel was prepared using 2 g of agarose, 180 mL of DI
H2O, and 20 mL of TAE. This mixture was heated until clear and allowed to slightly cool before
adding 10 µL of EtBr. A loading dye (Carolina blue) was added to the amplified PCR samples and
pipetted into the wells of the set gel for electrophoresis.
Gel Imaging: A gel imager was used to document the results from electrophoresis. These results
were recorded as image files onto the hard drive of the lab’s computer. A hard copy produced by
the gel imager was printed as a Polaroid image and stored in a laboratory notebook solely
dedicated to this experiment.
Figure 7: Amplification of targeted gene locus in
strains JC3272F and JC3272I
Figure 8: Amplification of targeted gene locus in
3 samples of strain BW25113
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.
M. A. Webber and L. J. V. Piddock. 2003. The importance of efflux pumps in
bacterial antibiotic resistance. Journal of Antimicrobial Chemotherapy 51: 9-11.
ACKNOWLEDGEMENTS
Funding for this project was provided by Oklahoma’s IdeA Network of Biomedical
Research Excellence (INBRE) program and UCO’s Office of Research & Grants.
We thank Dr. Philip Silverman at the Oklahoma Medical Research Foundation for
his guidance and much-needed information to implement experimental
procedures.