Benjamin Van Leeuwen and Dr. Allen Buskirk, Department of Chemistry and Biochemistry
Dr. Buskirk discontinued my previous project on discovering novel ribosomal stalling sequences shortly after the start of the semester because of its lack of relevance to more current ribosomal research trends. While there are still an unknown number of stalling sequences that have yet to be discovered, the current research has moved away from that field into more novel ribosomal research. Therefore, I was not able to finish my project as planned and presented in my last application.
However, I worked on a new project with some very interesting biological and medicinal implications. TrmD and Trm5 are analogous tRNA methtransferases located in bacterial and human cells, respectively. The genes encoding these proteins are essential. They each catalyze the m1G37 base 3’ to the anticodon. While both perform essentially the same function, the way in which they do this is completely different. Therefore, testing various methylation inhibitors on each of the plasmids in order to discover one that inhibits the function of the bacterial trmD gene, while having no effect on the human trm5 gene, could lead to the eventual development of a new antibiotic that would exclusively target and kill bacterial cells while leaving human cells alone (Lahoud, Goto-ito and Yoshida).
We have been collaborating with a small molecule lab at Thomas Jefferson University in Philadelphia for this project. Unfortunately, the plasmids that they had been using were flawed. Basically, they had created plasmids that express TrmD and Trm5 without including an off mechanism. This resulted in low levels of protein expression when almost no expression was expected. This made even the most basic experiments nearly impossible. In order to fix this, I will be designing new plasmids that will include an IPTG inducible promoter, which will make translation possible only if the plasmid is grown in an IPTG medium. I will then test the plasmids to ensure that the cells that contain them are unable to grow in the absence of IPTG. This will allow the Philadelphia lab to perform the experiments mentioned above.
Once I prepared the primers needed for PCR amplification of the trmD and trm5 genes I PCR amplified these genes and prepared to ligate them into the pTrc99b plasmid, which includes the IPTG inducible promoter. To ensure that the plasmids were complete and that the genes had been successfully ligated, I sequenced them so that we know that everything should have theoretically worked. After ensuring that the new plasmid was correct, I grew both of the strains in media both with and without IPTG. If the system worked, then there should have been no growth on the plates containing no IPTG. Based on the success of this system, I would have then been able to work with the Philadelphia lab on developing an assay in a trmD knockout bacterial strain. As mentioned above, there are important biologically significant outcomes of the work we are doing with the lab in Philadelphia.
This turned out to be a much more complex project then I thought it would. The first time that I tried PCR amplification, there was very little product. After some fiddling with the annealing temperatures I was able to get a good amount of the trmD product. However, I kept having problems with the trm5. It did not seem to want to amplify. I spent the majority of my research time working through ways to try and get the gene to amplify. All that I tried was not working, so after consulting with a graduate student in the lab, I decided to move on with the trmD.
I was able to get a good amount of the trmD from PCR amplification. After dpn-1 treatment, I was able to ligate it into the pTRC-99b plasmid. Once ligated, I transformed the prepared plasmid into DH10B competent cells and plated them onto AMP plates. Luckily the transformation worked. The next step will be to prepare the cells from the colonies on the transformation plates so that they can be used in the creation of a knockout assay for the trmD gene. Because I have graduated and because Dr. Buskirk has moved on to other projects, this will be one that will have to be finished by the lab in Philadelphia. The success of this project will depend on whether or not they believe it still holds biologically relevant outcomes.