2011 - 20th Annual NC State Undergraduate Research Spring Symposium

Session Time : 4/12/11 10:30 AM - 4/12/11 11:45 AM

Content Area : Molecular & Structural Biochemistry

Student Presenters :
Julia Carina Frei Biochemistry

Mentors and/or Co-Authors :
John Cavanagh Biochemistry

Abstract Title : Troubleshooting Expression and Purification of Bacillus subtilis Regulatory Protein, YwcC

Abstract :
Biofilms are multicellular communities encased in an extracellular matrix and are the basis of nearly 80% of infections, especially antibiotic resistant infections. Almost all known bacteria species are capable of utilizing biofilms for defense. In particular, the bacterium, Bacillus subtilis, is capable of forming robust biofilms and can be used to study the molecular machinery that regulates biofilm formation. The TetR-like transcriptional regulator, YwcC, has been shown to play a role in biofilm formation through its repression of slrA. SlrA is an antirpressor protein for SinR, the master regulator of biofilm formation in Bacillus. When YwcC is active, it binds to the slrA promoter and prevents transcription, which allows SinR to repress biofilm-related genes. However, once YwcC has been inactivated, SlrA is freely expressed and binds to SinR, allowing biofilm formation. This semester I have worked to develop a protocol for YwcC that would express the protein in concentrations high enough for NMR studies and for its purification. Using the original His-tag construct, I was unable to generate a stable sample despite trying various methods, because the protein would either aggregate or precipitate out of solution. Next, I attempted to create an expression system using a vector that would yield a GST-fused form of the protein. This construct was completed successfully, but it did not produce a fused GST-YwcC product. Currently, I am working on a system that will yield the protein without an affinity tag.