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The Effect of Cyclic Peptides on Sigma E Regulation in Escherichia coli

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

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The Effect of Cyclic Peptides on Sigma E Regulation in Escherichia coli

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  1. The Effect of Cyclic Peptides on Sigma E Regulation in Escherichia coli Catherine Shea and Shruti Panchavati Dr. Sarah Ades Lab

  2. 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 Project Summary

  3. Cyclic Peptide Inhibitors • Created a SICLOPPS library through intein-catalyzed cyclic peptide production Naumann 2008

  4. σE Pathway • σE transcribes the sRNA rybB • rybB works with the Hfq protein • Targets the mRNA of OmpC for degradation OmpC mRNA rybB + Hfq σE

  5. Let’s Find Inhibitors! • Set up an artificial system with two plasmids rybB OmpCyfp rpoE Plasmid 1 Plasmid 2

  6. Plasmid 1 Action σE rybB rpoE rybB

  7. Plasmid 2 Action Hfq OmpCyfp OmpC/yfp mRNA rybB RNase RNase

  8. Possible Outcomes • If rybB is present in the cell, OmpC/yfp will be degraded • If pathway is blocked, OmpC/yfp will not be degraded

  9. 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

  10. Selecting for the SICLOPPS plasmid Bright cell growing on Kan/Amp/Chlor plate = contains all three plasmids Chlor plate Miniprep Transform into DH5α Test in screening strain Miniprep Chlor plate Kan/Amp plate

  11. Previous FACS Results The image shows fluorescence of control (OFF) strain 6802 (screening strain without ydcQ deletion) ompC-yfp repressed by rybB

  12. D13 + arabinose E8 + arabinose E15 + arabinose F3 + arabinose

  13. 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

  14. SigmaE sRNA rpoE Ompc-yfp SICLOPPS rybB Amp Kan Chl Plasmid 1 Reporter

  15. 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 E15 E15 D13 D13 6491 6716 F3 F3 E8 E8

  16. 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

  17. Preparation of New Strains • Screening Strain SGWMH(Q)VS 16 SGWSW(Q)EP 17 SGWSER(Q)T 18 SGWAD(Q)CK 19 • Chromosome of Screening Strain • Deletion of digestive enzymes • Deletion of ydcQ

  18. Observation under Florescence Microscope “OFF” screening strain rpoE-rybB plasmid + ompC-yfp reporter “ON” screening strain Vector + ompC-yfp reporter

  19. “ON” screening strain “OFF” screening strain 17 + .0002% arabinose

  20. F3 + arabinose “OFF” strain E15 - arabinose E15 + arabinose 832 - arabinose 832 + arabinose

  21. 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 ? ? sigmaE rybB ompc-ypf

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