Mary Gates Endowment for Students

Courtney Wigmore

Project

Expression, Purification, and Fixation of RNA polymerase B-Subunit (RpoB) supporting BAC-CUTAC

I am aiming to help modify Cleavage Under Targeted Accessible Chromatin (CUTAC) which allows investigators to measure which sections of DNA are actively being bound by the RNA polymerases which is required for the transcription of mRNA from DNA; however, it was designed for eukaryotic samples. Currently CUTAC works well with mice and humans to target RNA polymerase, allowing us to see what genes are being expressed. This is what Dr. Glenny’s project is focused on, together she and I will adapt CUTAC to measure RNA polymerase binding in bacteria. This is important because it would allow us to gain gene expression data from the microbial community in the lung. To adapt CUTAC for bacteria we need to develop an antibody that recognizes microbial RNA polymerase. RNA polymerase subunit-β (RpoB) is a highly conserved subunit of RNA polymerase and will help us know transcriptional rates of bacterial DNA within a community. The project I will undertake is to express, purify, and formalin-fix RpoB.

What I learned

I have learned a lot of technical lab skill such as making cell cultures, DNA extraction, PCR, creating plasmids, transforming cells, and protein purification. I also have developed better critical thinking and communication skills in working with others in the lab. Learning from mistakes and working with others to find a better method to reach our goals was very important in this project. For the application, I gained better writing skills, and valuable feedback from mentors so I can improve on communicating scientific ideas.

Advice for future applicants

Never be afraid to ask for help or peer review on the paper and start early. The suggestions from others and help on slowly writing the paper was very helpful for me. There are also opportunities to work with others applying to the endowment, those workshops were helpful because you can talk with the reviewers of the applications and get advice on what yours may need.

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