Catherine Shea and Shruti Panchavati Dr. Sarah Ades Lab
The Effect of Cyclic Peptides on Sigma E Regulation in Escherichia coli Catherine Shea and Shruti Panchavati Dr. Sarah Ades Lab Project Summary Part of Dr. Ades lab studying E in E. coli E is an essential sigma factor necessary for cell envelope homeostasis Complete inhibition of Ecauses cell death It would be beneficial to find E inhibitorswhen trying to discover new antibiotics Cyclic Peptide Inhibitors
Created a SICLOPPS library through intein-catalyzedcyclic peptide production Naumann 2008 E Pathway + Hfq rybB E E transcribes the sRNA rybB
rybB works with the Hfq protein Targets the mRNA of OmpC for degradation +Hfq OmpC mRNA rybB E Lets Find Inhibitors! Set up an artificial system with two plasmids
rybB OmpC yfp rpoE Plasmid 1 Plasmid 2 Plasmid 1 Action E rybB rpoE rybB Plasmid 2 Action rybB Hfq OmpC yfp OmpC/yfp mRNA RNase RNase Possible Outcomes If rybB is present in the cell, OmpC/yfp will be degraded If pathway is blocked, OmpC/yfp will not be degraded Inhibitors Found: Plasmid 1 (rpoE ErybB) contains a gene for Ampicillin resistance Plasmid 2 (OmpC/yfp) contains a gene for Kanamycin resistance SICLOPPS library plasmid contains a gene for Chloramphenicol resistance Bright cells growing on Kan/Amp/Chlor plates contained all three plasmids and inhibited the pathway and were selected for further study Selecting for the SICLOPPS plasmid
Bright cell growing on Kan/Amp/Chlor plate = contains all three plasmids Chlor plate Miniprep Transform into DH5 Test in screening strain Miniprep Chlor plate Kan/Amp plate Previous FACS Results The image shows fluorescence of control (OFF) strain 6802 (screening strain without ydcQ deletion) ompC-yfp repressed by rybB D13 + arabinose E8 + arabinose E15 + arabinose F3 + arabinose Recent Findings Specific genetic background for optimal success
Remove enzymes which digest arabinose Bacteria normally digest arabinose and a large amount of arabinose is deadly to cells Deletion of ydcQ gene ydcQ allows cells to live without sigma E Strain 6491: ydcQ Strain 6716: rybB Amp rpoE rybB SigmaE sRNA Kan Ompc-yfp Chl SICLOPPS Plasmid 1 Reporter Preparation of strains
Transformed strains with cyclic peptides E15 SGWEYVRP, D13 SGWSAYTL, F3 SGWLGPQR, E8 SGWRSVWA Streaked on Kan, Amp, Chl plates to screen for sensitivity Added lacZ gene and performed beta-glucosidase to test for high sigma E level 6491 E15 D13 F3 E8 6716 E15 D13 F3 E8 Observation under Microscope
% arabinose % arabinose (Longer and Fatter) Problem with growth at 37 degrees Celsius. Toxic intermediates are produced during formation. Fixed by growth at 30 degrees Celsius Preparation of New Strains
16 17 18 19 SGWMH(Q)VS SGWSW(Q)EP Screening Strain SGWSER(Q)T SGWAD(Q)CK Chromosome of Screening Strain Deletion of digestive enzymes Deletion of ydcQ Observation under Florescence Microscope
OFF screening strain rpoE-rybB plasmid + ompC-yfp reporter ON screening strain Vector + ompC-yfp reporter ON screening strain OFF screening strain % arabinose F3 + arabinose OFF strain E15 - arabinose E15 + arabinose 832 - arabinose 832 + arabinose Conclusion ompc-ypf rybB sigmaE ? While cyclic peptides here have shown to inhibit the system, the mechanism of action is still unclear Next step is to test what site the cyclic peptides block