Peter Janzen and Dr. Eric Wilson, Department of Microbiology and Molecular Biology

The purpose of this project was to see if a genetic alteration to gram-negative bacteria would affect the phenotypic ability to resist the attachment of chemokines. In other words, we were trying to see if we could change the structure of a bacterium by altering the DNA. As a result of that alteration, would our immune systems fight the infection more easily with a chemokines response.

In order to test these questions I used bacteria that had defects in the genetic code. The mutations were created by transposons placed in the ypTB 1005, ypTB 1884, and ypTB 3041 genes. This is a diversion from the original plans which omitted the last two mutants and included three additional mutants. The reason for this was due to my slow work the first semester as I learned and mastered laboratory techniques. The other three genes of interest were reassigned and I continued work on the aforementioned mutants.

Unfortunately at this date I have not completed my research but great progress has been made. It would be easiest to discuss my research gene by gene as follows:

ypTB 1005

This gene helps code for the lipopolysaccharide (LPS) chains found on the membranes of the bacteria. The mutant containing the transposon was chosen for study because a binding assay showed a high concentration of bound chemokines. I was able to develop primers allowing for successful PCR (a technique that allows for multiple copies of DNA to be created). Once the gene was replicated I was able to successfully ligate the gene in a plasmid. By doing this a working copy of the gene is now ready to be inserted into the original mutant that has the defective copy. Due to some difficulties that I cannot explain at this time, I have not been able to successfully transform, or insert the plasmid into the mutant.

ypTB 1884

This gene helps code for the efflux pump of Yersinia. An efflux pump helps remove unwanted or destructive molecules from the bacterium. Once again this mutant with the transposon in this gene was chosen due to the high concentration of bound chemokine observed from a binding assay. This gene has been difficult to replicate through PCR. I have used several primers, many of them unable to create sufficient copies of the gene. I recently was able to create one that works and will soon ligate into a plasmid.

ypTB 3041

This gene has had the most success. This also helps code for the efflux pump. I was able to create primers that led to successful PCR. This gene was then ligated into a plasmid and then successfully transformed into the Y. pseudotuberculosis mutant. In simpler terms I was able to reinsert a working copy of the gene into the mutant that had the defective copy. At this point, the bacteria are waiting to undergo another binding assay. Hopefully this assay will yield results similar to the wild-type unaltered bacteria. This will show that the gene really does play an important role on chemokine binding.

As explained I have had a lot of bumps in the road over the past year while becoming proficient in laboratory techniques and procedures. I have plans to continue to work with Dr. Wilson in finishing the work that was started. I am guessing that work will continue to progress for all three genes in the reinsertion experiment. After that is completed the next step will be to try and completely remove the gene from the genetic code and observe if the new phenotype resembles the original mutant phenotypes.