the effect of cyclic peptides on sigma e regulation in escherichia coli
DESCRIPTION
The Effect of Cyclic Peptides on Sigma E Regulation in Escherichia coli. Catherine Shea and Shruti Panchavati Dr. Sarah Ades Lab. Part of Dr. Ades’ lab studying σE in E. coli σE is an essential sigma factor necessary for cell envelope homeostasis - PowerPoint PPT PresentationTRANSCRIPT
The Effect of Cyclic Peptides on Sigma E Regulation in Escherichia
coli
Catherine Shea and Shruti PanchavatiDr. Sarah Ades Lab
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
Cyclic Peptide Inhibitors• Created a SICLOPPS library through intein-catalyzed cyclic peptide production
Naumann 2008
σE Pathway
• σE transcribes the sRNA rybB• rybB works with the Hfq protein• Targets the mRNA of OmpC for degradation
σE rybB + HfqOmpCmRNA
Let’s Find Inhibitors!
• Set up an artificial system with two plasmids
rpoErybB
Plasmid 1 Plasmid 2
OmpC yfp
Plasmid 1 Action
rpoErybB
σE
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 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
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
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• ΔydcQ allows cells to live without sigma E
• Strain 6491: ΔydcQ• Strain 6716: ΔrybB
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
- .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
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
Observation under Florescence Microscope
“OFF” screening strainrpoE-rybB plasmid + ompC-yfp reporter
“ON” screening strainVector + ompC-yfp reporter
“ON” screening strain 17 + .0002% arabinose“OFF” screening strain
F3 + arabinose“OFF” strain
E15 - arabinose
E15 + arabinose
832 - arabinose
832 + arabinose
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
??