The Effect of Cyclic Peptides on Sigma E Regulation in Escherichia coli
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The Effect of Cyclic Peptides on Sigma E Regulation in Escherichia
coli
Catherine Shea and Shruti PanchavatiDr. Sarah Ades Lab
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Project Summary
• Part of Dr. Ades’ lab studying σE in E. coli
•σE is an essential sigma factor necessary for cell envelope homeostasis
• Complete inhibition of σE causes cell death
• It would be beneficial to find σE inhibitors when trying to discover new antibiotics
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Cyclic Peptide Inhibitors• Created a SICLOPPS library through intein-catalyzed cyclic peptide production
Naumann 2008
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σE Pathway
• σE transcribes the sRNA rybB• rybB works with the Hfq protein• Targets the mRNA of OmpC for degradation
σE rybB + HfqOmpCmRNA
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Let’s Find Inhibitors!
• Set up an artificial system with two plasmids
rpoErybB
Plasmid 1 Plasmid 2
OmpC yfp
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Plasmid 1 Action
rpoErybB
σE
rybB
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Plasmid 2 Action
rybB
Hfq
OmpC yfp
OmpC/yfp mRNA
RNase RNase
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Possible Outcomes
• If rybB is present in the cell, OmpC/yfp will be degraded
• If pathway is blocked, OmpC/yfp will not be degraded
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Inhibitors Found:
• Plasmid 1 (rpoE σE rybB) 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
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Selecting for the SICLOPPS plasmidBright cell growing on Kan/Amp/Chlor plate = contains all three plasmids
Miniprep Transform into DH5α
Chlor plate
Chlor plate
Kan/Amp plate
MiniprepTest in screening strain
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Previous FACS Results•The image shows fluorescence of control (OFF) strain
•6802 (screening strain without ydcQ deletion)
•ompC-yfp repressed by rybB
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D13 + arabinose E8 + arabinose
E15 + arabinose F3 + arabinose
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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• ΔydcQ allows cells to live without sigma E
• Strain 6491: ΔydcQ• Strain 6716: ΔrybB
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Amp
rpoE rybB
SigmaE sRNA
Kan
Ompc-yfp
Chl
SICLOPPS
Plasmid 1 Reporter
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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
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Observation under Microscope
- .0002% arabinose + .0002% arabinose(Longer and Fatter)
Problem with growth at 37 degrees Celsius. Toxic intermediates are produced during formation. Fixed by growth at 30 degrees Celsius
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Preparation of New Strains
• Screening Strain
16
17
18
19
• Chromosome of Screening Strain• Deletion of digestive enzymes• Deletion of ydcQ
SGWMH(Q)VS
SGWSW(Q)EP
SGWSER(Q)T
SGWAD(Q)CK
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Observation under Florescence Microscope
“OFF” screening strainrpoE-rybB plasmid + ompC-yfp reporter
“ON” screening strainVector + ompC-yfp reporter
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“ON” screening strain 17 + .0002% arabinose“OFF” screening strain
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F3 + arabinose“OFF” strain
E15 - arabinose
E15 + arabinose
832 - arabinose
832 + arabinose
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Conclusion
• 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
ompc-ypfrybBsigmaE
??