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