Thursday, September 27, 2007

Research Synopsis

So my grad school application process continues. This is part of my personal is a synopsis of my research. Let me know what you think, any changes I should make, etc. Thanks in advance!

For the past two and a half years, I have had the privilege to work as an undergraduate researcher in the lab of Dr. Graham Hatfull at the University of Pittsburgh. Dr. Hatfull is one of a unique group of researchers adamantly recruiting young students into biological research. Students as young as 6th and 7th grade are able to work in his lab through his Phagehunting program. Although I did not begin my work in the program at that young of an age, many would consider my start as a college freshman early compared to most.

I began my research in January of my freshman year. The first project was to use bioinformatic methods to analyze and annotate the genome of a Bacillus phage, MP15. Utilizing programs such as DNA Master, Glimmer, and GeneMark, I called the most likely protein coding regions in the genome. These putative genes were then compared to GenBank databases to assign potential functions and evaluate the novelty of the phage’s genome. Not surprisingly, this phage proved to be highly novel; its genome had a large amount of genes with no significant sequence similarity to those genes in the databases.

Having enjoyed my first research experience, I then continued work in the lab, moving from bioinformatics into wet lab projects. I became part of a larger project to isolate and characterize novel mycobacteriophages on Mycobacterium smegmatis. I isolated, amplified, and concentrated a novel mycobacteriophage named BPs. Using Sanger sequencing procedures, the phage’s genome was sequenced. The final sequence was analyzed and annotated. Lysogeny experiments showed BPs to form stable lysogens in M. smegmatis, likely integrating at a tRNA^Arg gene in the host. Furthermore, host range studies showed that BPs was able to infect not only its initial host, but also M. chelonae, M. bovis BCG, and notably, M. tuberculosis (M. tb.).

Following up this host range study of BPs, I began to characterize the ability of over two hundred mycobacteriophages (published and unpublished) to infect M. chelonae, M. bovis BCG, and M. tb. Dr. Hatfull approached me with a few ideas for projects that could stem from the large amount of host range work I was performing. One of these projects involved the discovery of a phage capable of performing generalized transduction in M. tb.

Generalized transduction occurs when a phage capsid accidentally packages host DNA, resulting in a viral particle capable of inserting that DNA into a new cell. This mechanism has been exploited in a variety of bacterial systems. Currently, there are only two published phages with the ability to transduce Mycobacteria; Bxz1 and I3. However, neither of these is able to infect or transduce M. tb. My goal was to find which of our published phages could infect M. tb. and which could function as transducing phages. I developed and performed transduction assays with two different genetic markers. At the same time, I finalized the host range of our published phages.

The results showed that 11 of our 30 published phages were able to transduce M. smegmatis and that 7 of these 30 were able to infect M. tb. However, no phages appear in both groups. Bioinformatic analysis of putative genes showed that there was no particular gene present only in the group of M. tb. infecting phages. Rather, the only genetic element common among M. tb. infectors is the presence of cohesive genetic termini. Those phages capable of mediating generalized transduction all have terminally redundant genetic termini. Currently, I am asking whether or not the presence of terminally redundant ends prevents phage from infecting M. tb.

The nature of a phage’s genomic ends determines how the DNA circularizes upon infection. Phages with cohesive termini circularize via complementation; while phages with terminal redundancy circularize by homologous recombination. M. tb. is notorious for their large amount of illegitimate recombination, which potentially may inhibit phages with terminal redundancy from circularizing and subsequently replicating. Previous studies have shown that recombination proteins are necessary for certain phages with terminal redundant ends, such as Salmonella phage P22, to circularize and replicate. Previous work with mycobacteriophages has discovered functionally analogous proteins for recombination in Mycobacteria. These proteins, encoded by phage Che12, have been successfully utilized to promote homologous recombination in both M. smegmatis and M. tb. My current work is establishing whether the presence of these proteins can overcome a recombination deficiency in M. tb. and allow terminally redundant phages cause infection. If this is true, generalized transduction in M. tb. may be a possibility.

Aside from learning laboratory techniques and critical thinking, this research experience has provided me with the opportunity to present my work at a wide variety of scientific meetings. These meetings include: the 2005 Molecular Genetics of Bacteria and Phages Meeting, the 106th and 107th Annual American Society for Microbiology General Meetings, along with meetings hosted by the University of Pittsburgh.

My work with phage has opened my mind to the world of microbial interactions. The constant flux between host and pathogen, on any scale, is vast and decidedly interesting. Studying these interactions can provide the focus for a wide range of projects. Microbial interactions have components in vaccine development, industrial fermentations, novel antimicrobial development, genetic tool creation, and evolutionary studies. It is my hope that graduate school will allow me to delve deeper into the interactions between microbes and humans, themselves, or their environment.

This is the first 2/3 of a ~1000 word essay. The remaining will be geared towards which ever school I am applying too. Thanks for your opinions.

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