Monday, August 2, 2010
Pacific Concourse (Hyatt Regency San Francisco)
We have developed an automated high-throughput screen (HTS), in collaboration with the DeLisa lab at Cornell, to look for YebF secretion mutants in E. coli. YebF, a 10.8KDa protein of unknown function, is secreted to the periplasm similar to TypeII secretion systems but also undergoes extracellular secretion via some unknown pathway. A transposon Tn5-mediated mutagenesis library was created and screened to identify genetic elements crucial for YebF secretion. Visualization and detection of secretion mutants is accomplished through fluorescence based FlAsH labeling technology by Invitrogen. A primary screen of over ~10,000 mutants was carried out and revealed several candidates exhibiting increased and decreased YebF secretion via fluorescence. Further verification and secondary screening via Western analysis has confirmed changes in secretion of a few of these candidates. Ultimately, our goal is to reveal genetic elements responsible for YebF secretion. In addition, future efforts include applying similar screening methodologies to Type II secretion system mutants in order to elucidate the mechanism of payload specificity.