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.

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.

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 1:Streaking plates

Figure 3: Sean Reim with Thermo-cyclers

Gene Transfer Study as Related to Bile Salt Sensitivity in Mutant Escherichia coli StrainsSean 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.

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.

APPROACH

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.

RESULTS AND DISCUSSION

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. 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.

BW25113 JW1271 JW1272 JC3272F JC3272ILL BW25113 JW1271 JW1272 JC3272F JC3272I

Figure 4: Sean Reim withThermo-cyclers

Figure 6: Jim Bidlack withGel Imaging Machine

Figure 5: Sean Reim withGel Imaging Machine

Figure 2: Sean and Madeline Pipetting

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