2007 Summer Undergraduate Research Program

Aarti Chawla

This summer I worked with Kari Severson, a graduate student in the Knight lab. One of the aims of the Knight lab is to understand the mechanism by which intestinal bacteria promote gut-associated lymphoid tissue (GALT) development. There are a number of processes by which the intestinal microflora stimulate GALT development, but this summer I focused on one of these potential mechanisms: M cell transport.

M cells are specialized epithelial cells that transcytose bacteria from the lumen into their pocket where they can facilitate interaction with the immune cells present there. It has earlier been shown by Rhee et al. that certain bacterial species induce rabbit GALT development better than others, in particular a combination of B. subtilis and B. fragilis induce B cell proliferation and follicle formation much better than either bacterium does individually. I hypothesized that there is a correlation between bacterial uptake by the M cells and GALT development. To test this hypothesis, the goal of my project was to generate M cells in vitro using a previously described system, and upon generating M cells, test for bacterial uptake using B subtilis and/or B. fragilis. My data showed that neither B. subtilis nor B. fragilis alone were sampled by M cells, but B. fragilis was taken up by M cells in the presence of B. subtilis. Although preliminary, these data suggest that B. subtilis may facilitate the uptake of B. fragilis through M cells, and offer an interesting possibility as to how commensal bacteria may work together to promote rabbit GALT development.

I worked in Dr Nancy Zeleznik-Le laboratory this summer. My summer research project was involved with beginning to explore a novel mechanistic pathway that might be relevant to MLL leukemia. Dr Zeleznik-Le had recently determined that expression of a particular microRNA (small non-coding RNA), mir196b, was regulated by MLL. This same microRNA was also over-expressed in MLL fusion leukemias. I cloned several different sizes of precursors for this microRNA into an expression vector. We first used PCR amplification to obtain the fragments to clone, then ligated them into a retroviral vector, and screened for plasmids containing the insert. I was successful in obtaining the correct clones and prepared the DNA for subsequent functional analyses. 

Niyati Parikh

I had the wonderful opportunity to work in Dr. Alan Wolfe's lab this summer. My project focused on investigating the relationship between the pta-ack pathway and the cpxAR pathway in E.coli bacteria. The cpxAR pathway enables bacteria to respond to various stress signals from the envelope surrounding the bacteria. The pta-ack pathway produces acetyl phosphate and acetate. During the course of the ten weeks, I identified a linkage between central metabolism and the cpxAR signaling pathway. By performing epitasis experiments (making combinations of double mutants in two different pathways and determining what is required for activation of the cpxAR pathway), I discovered that pta-ack may be another input into cpx signaling. Contributing to the discovery of this alternate mechanism by which cpx signaling may be activated was a unique opportunity and I enjoyed learning skills and techniques involved in research. 

 Caitlin Paul

This summer I worked with Venkata Yeramilli, a graduate student in the Knight lab. One of the focuses of the Knight lab is to understand the mechanism by which B cells are stimulated in the Gut Associated Lymphoid Tissues (GALT). My project for the summer was to test if a cytokine known as B cell activating factor (BAFF) can stimulate B cells isolated from rabbit GALT. To address this question, I cloned, expressed and purified rabbit BAFF from E.coli. I then set up in vitro cultures with splenocytes and B cells isolated from GALT to test if BAFF can induce these cells to undergo proliferation. From my preliminary experiments, it appeared that BAFF only induced proliferation in splenocytes, but not in B cells isolated from GALT. Perhaps BAFF induces a survival signal for GALT B cells. I really enjoyed my time in the lab this summer and I learned so much during this 10 week program.


 

Enya Qing

This summer I had an amazing time working on a combined project between the labs of Dr. Karen Visick and Dr. Alan Wolfe. The goal of my project was to address the role of cyclic-di-GMP, a novel second messenger, in the regulation of flagella assembly by Vibrio fischeri. Previous studies by the two labs had shown that the bacteria’s flagellation was dependent on the presence of magnesium. They also found out that loss of the MifA protein, which synthesizes cyclic-di-GMP, restores some motility to cells grown in the absence of magnesium. These data suggested that c-di-GMP produced by MifA inhibits flagellation; however, it is unknown at what stage in flagellar biogenesis this inhibition occurs. In my project, I searched for and isolated mutants that could overcome MifA-mediated inhibition. Some of these mutations mapped to mifA itself, while others mapped elsewhere on the V. fischeri chromosome. Although I was not able to identify the chromosomal mutations I obtained, my work revealed that such an approach is feasible. 

I spent this summer working at Dr. Karen Visick's lab, which studies the symbiosis between Euprymna scolopes (a Hawaiian squid) and Vibrio fischeri (a bioluminescent bacterium). Previous experiments from the Visick lab supported the model that an interaction between a sensor kinase and a response regulator promotes transcription of the symbiosis polysaccharide (syp) cluster, a group of genes necessary for the colonization of E. scolopes by V. fischeri. The goal of my project was to determine which response regulators were required (or partially required) for interacting with a sensor kinase, called RscS, for the induction of this syp cluster and for the regulation of downstream phenotypes (including attachment to glass biofilms, pellicles, and wrinkled colonies). By the end of the summer, I had tested 36 out of the 40 putative response regulators and determined that SypG was the only response regulator involved in syp transcription, while a number of other response regulators, including SypG, were involved in downstream phenotype regulation. The future direction of my project is to investigate whether RscS indeed donates its phosphate to SypG, as expected from my results..