2008 ASM General Meeting, Boston MA

On June 1st through 5th, I have the distinct pleasure of attending my third ASM General Meeting, in as many years. My poster titled "Novel Generalized Transducing Phages of the Mycobacteria" is number M-005. Feel free to come and chat about esoteric microbiology topics.

I plan on writing on a handful of interesting topics presented.

It should be a great time for all!

Walking the Line Between Grades and Experience: My Life as an Undergraduate Researcher (Part 2)

Continued from Part 1

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I finished my sophomore year with a B-average, and went into the summer with my mind set on losing myself in my research again. I was doing some really cool assays with M. tb and M. bovis BCG (I was the only undergraduate in the lab taking advantage of our facilities). This work spring boarded me into a highly competative fellowship from HHMI. Since this was quite a some of money, and it had to be renewed every semester (including summer's), I was dead set on putting out high quality and large quantity data.

So I did. My junior year (filled with Physics and Biochemistry) was spent skipping classes to put out data. And I did, I revised current protocols for a generalized transduction assay, assayed more than 2 dozen phages for this ability, screened over 200 phages for the ability to infect M. tb. (Manuscript in process) My work in the lab from my junior year was, in my opinion the best I had put out.

However, that summer when I went to renew my fellowship for my senior year, I received surprsing news. I would not be accepted into the program that fall because my grades were raising "red flags." It was in the opinion of the comittee that if I was to head to graduate school (like I planned) I would need to get my grades up. I was startled.

I had heard that experience was the primary factor in determining eligibility to PhD programs. I guess Cs in two semesters each of Physics and Biochemistry didn't look so good, not to mention that O. Chem 1 was repeated, along with its lab.

So, they cut off my funding so that I would spend more time studying, to lower the red flags.

Now, I am not a stupid person. Although, some may say that my grades don't demonstrate the intelligence required for higher learning. I took a step back from my research senior year, and concentrated on bringing my grades up. Each semester this past year (which were both heavy on senior-level biology courses) I earned a GPA of 3.8. Somehow, my final cumulative GPA was ~3.4, despite some very low semesters in the past.

I had to do this to show that I had learned the foundational knowledge of biology. I had already proved I knew my way around the lab, I had to show that I was doing more than going through the motions.

I brought my grades up to a point where there are no longer any red flags that shadow my lab accomplishments. I still managed to put out great work in the lab (soon-to-be first author on two papers), although I will concede that I did not finish as much research as I would have liked.

However, I'm now heading to my top choice of the graduate schools I applied to (Emory University, PhD Program in Microbiology and Molecular Genetics). My words of advice are to gain as much experience as possible, nothing is held in higher regard. But, to do so at the expense of grades...not such a good idea.

Although it worked out better than I could have imagined, there was a strong possibility that it would not have worked out this way. But now, I am onward to a place where building experience is the primary focus, and so I am quite excited to continue on my science career.

The End

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Revisit Part 1 Here

Walking the Line Between Grades and Experience: My Life as an Undergraduate Researcher (Part 1)

From the moment I began looking for a university to attend after high school, I knew I wanted to do biology research. "Experience is key" I was told, in order to do anything after receiving a 4yr degree. So although I was unsure what it was I truly wanted to pursue after getting my BS in Microbiology, I KNEW that I would need research experience to succeed.

And so, I sent out my first requests to volunteer in research labs in October of my freshman year. No one would take a 1st year with no foundational knowledge....except one lab. The following semester, a post-doc in Graham Hatfull's lab, by the name of Marisa Pedulla, took me on board with a group of other students, although I was the youngest, to give us our first taste of research. We set out to perform bioinformatic analysis on a group of 6 Bacillus phage genomes that the Pittsburgh Bacteriophage Institute had recently finished sequencing. I was given the tools to uncover the patterns within the genetic code to find the most likely genes, hypothesize their function, and deduce evolutionary relationships.

I was hooked. It fascinated me to be able to unlock the secrets that patterns of four simple letters could hold.

I spent way to much time in front of a computer screen that semester, engrossed in sequence, running a million BLAST searches or ClustalW alignments at once. Suffice to say, my grades dropped. I had started my 1st year with a B average (not bad for an incoming freshman acclimating to a life 5hrs from childhood friends and family), and ended it with a C average. I dropped the ball. But I thought, "This experience will make up for a loss in grades." Little did I know....

I was asked to continue working on bacteriophage genetics in the Hatfull Lab that summer. So as an 18yr old just finished with one year of college, I began life as an adult. I rented out my own place--while all my friends had packed up to move back home with parents for the summer, I was packing boxes to take to a one-bedroom place a few blocks from the lab.

That summer I again engrossed myself in my work and work for the Hatfull Lab. (I should mention that some of this work is in the process of being published...). Summer went by quite fast, I visited my friends and family from home only twice because I was so over my head in what I was doing at the lab. Too soon, the summer ended and my second year began.

I was still working in the lab--only now I was required to work 10hrs a week. This soon became 20-30hrs a week because I became excited when things went well on some sequencing projects I was doing. Little did I know that I would be smacked over the head by two little words. Organic Chemistry. The bane of many undergraduate biology major's existances. I did not spend as much time studying for that course as I should have, and it showed in my grades. D+ in O. Chem 1--meaning I would have to retake the course. Suffice to say, I got an A in our honors genetics class (mainly because it was so applicable to what I was doing in the lab). But again, I my grades really did not accuratly portay my potential and what I knew. Again I thought, "It's my experience that will count, not my grades."

Again, I knew so little.


Continue Reading Part 2 Here

I Got You Phage

Back in August of 2006, I wrote and performed this song for a lab meeting to summarize my work I had done that summer and the previous year (All regarding a bacteriophage called BPs).

Some of the lyrics are hard to catch, you can find them below the embed. Enjoy!


Lyrics
Will I find you? Well, I don't know.
I won't find out until my smeg will grow.
I scraped the dirt right off my shoe
You were there and phage, I found you.

(Chorus)
Phage, I got you phage. I got you phage. I got you phage.

They say you won't kill TB, you'll only infect Bovis BCG
I can tell from your lack of spots
You won't infect all the bacteria we got

(Chorus)

You have recE in your genes
You have Halo on your team
And when your cruising round the town
Bateria die when you're around.

So let them say your tail's too long
I don't care, I wrote the song
So put your little head by mine
It has the smallest genome we can find

(Chorus)

I found you from dirty sludge
I found you while eating fudge
I got micrographs of you
I still got lysogen work to do
I want you to integrate, I'll watch it when you replicate
I found you, I won't let go
I found you, I love you so

Phage
I got you phage
I got you phage I got you phage I got you phage
~End Lyrics~

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There will hopefully be some more microbiology songs coming soon (as soon as I get a camera up and running). With titles such as:
1. It's Gonna Be There (The E. coli song)
2. Brillant Dance of the Starvation Response
3. Phage in the Soil

Just For Phun!

I found this in my photo archives, I thought some of you may enjoy it! (2005)

RecQ's Role in Illegitimate Recombination

Genetic recombination is a ubiquitous event that occurs in every species; it is necessary for the production of unique gametes, it is involved in DNA damage repair, it provides a mechanism for evolution, and it is a required action for many viruses and phages to undergo nucleic acid replication.(My discussion here will focus entirely on recombination in prokaryotes and their viruses)

There are three different types of recombination: homologous recombination--where DNA of significant sequence similarity recombines, non-homologous recombination--where DNA without significant sequence similarity recombines (usually along gene boundaries), and finally there is illegitimate recombination--where DNA recombines randomly.

Although DNA is surprisingly fluid, there are enzymes that mediate recombination--by initiating DNA binding, strand invasion, and stabilizing ssDNA intermediates. Also, of important note, is that organisms have varying degrees of recombination levels. A classical example occurs within the Mycobacteria. Mycobacterium smegmatis has relatively low levels of illegitimate recombination (IR), while M. tuberculosis is notorious for high levels of IR compared to homologous recombination. This raises a question that can be phrased in a few ways. "What enzymes are responsible for IR?" or perhaps, "What enzymes for homologous recombination are lacking?"

I performed a cursory search of the Rec-type proteins in both M. smegmatis and M. tuberculosis, and found that M. smegmatis contained a RecQ homologue, while M. tuberculosis did not. A literature searched showed that RecQ is a DNA helicase that is involved in the suppression of IR (Hanada and colleagues in this paper published back in 1997) in E. coli.

The paper describes a rather elegant experiment to see the frequency of IR by utilizing a specialized transducing phage. Briefly, a specialized transducing phage is a lysogenic phage that, upon excision from the chromosome, accidentally packages host DNA located directly adjacent to the integrated. This event is usually very rare and is catalyzed by an IR event. Therefore, any changes that increase IR frequency will increase the frequency of transducing particles. In this instance, the authors used Lambda Phage, and a screened for the production of transducing particles.

The authors found that in a recQ minus strain, the number of transducing particles increased 10-100x higher than wild type (depending on conditions and marker used). This increase could be removed by the complementation of recQ. Furthermore, the authors found that this pathway was independent of the well characterized RecA pathway by creating a recQ / recA double mutant and finding no difference in IR from the recQ mutant.

They discuss further associations with the RecJOF pathways, however, delving further into a discussion on Rec pathways is beyond the scope of what I can discuss here. (More information can easily be found by searching Google for "Rec proteins")

Since this study was published back in 1997, it made me curious as to whether anyone has studied the lack of recQ in M. tuberculosis, its presence in M. smegmatis, and their drastic differences in IR. As it turns out, no one has (to my knowledge).

I am currently interested in examining recombination functions necessary for phage replication in the Mycobacteria. You can read about my current tuberculosis research here and I will post a recent update here (coming soon). I'm currently working on knocking out recQ in M. smegmatis and expressing recQ in M. tuberculosis and asking if there are effects on phage infection. There is already a system in place that overcomes IR in M. tuberculosis to allow allelic exchange, ( Nat Methods. 2007 Feb;4(2):147-52.) and I am testing this system as well.

For more information on Mycobacterial Genetics, I suggest the following book which is the most recent text on the subject.

Molecular Genetics Mycobacteria

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Hanada, K., Ukita, T., Kohno, Y., Saito, K., Kato, J., Ikeda, H. (1997). RecQ DNA helicase is a suppressor of illegitimate recombination in Escherichiacoli. Proceedings of the National Academy of Sciences of the United States of America, 94(8), 3860-3865.

Research Synopsis

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

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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.

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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.