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Cling- E. coli : Bacteria on target

Cling- E. coli : Bacteria on target

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Cling- E. coli : Bacteria on target

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  1. Cling-E. coli :Bacteria on target Harvard iGEM 2007 Kevin Shee Perry Tsai Shaunak Vankudre George Xu Ellenor Brown Stephanie Lo Alex Pickett Sammy Sambu

  2. The motivationTo develop a system for directing bacteria to a target of interest and effecting downstream activity

  3. Potential Targets and Applications Bind Proteins Bind Toxins Bind Viruses Bind Tissue Bind DNA Bind Other Cells Bind Surface

  4. Bacterial targeting Quorum-sensing Fec signal transduction Quorum-sensing Fec signal transduction

  5. Surface Engineered Bacteria Engineered to Bind and Signal Fusion Protein OmpA – C terminal insertion OmpA-Loop1 insertion AIDA-1 – N terminal insertion FecA – loop insertion Membrane Protein

  6. Selecting/enriching for surface engineered bacteria Direct Selection Direct magnetic beads Indirect selection MACS FACS Figure here to illustrate Direct selection and indirect selection

  7. Indirect Bead Assays Direct Bead Assays Cell Sorting with his and strep2

  8. After Separation <NEW TITLE> Test: Cell Sorting with AIDA1 + sender constructs (with his and strep2) Before Separation strep2 his

  9. Results: (MORE DESCRIPTIVE TITLE HERE) Initial cultures Enriched cultures through selection Successful selection of E.coli expressing His or Strep2 on surface

  10. Bacterial targeting Quorum-sensing Fec signal transduction

  11. Reporter luxI/luxR Quorum Sensing Receiver + R OHHL Sender

  12. Receiver Sender Cell-Cell Signaling Constructs • Receivers (luxR + Reporter) • GFPReceivers • tetR controlled (Bba_T9002) • Quorum controlled (Bba_R0062 + Bba_C0261 + Bba_E0240) • mRFPReceivers • tetR controlled (Bba_F2620 + Bba_I13507) • Quorum controlled (Bba_R0062 + Bba_C0261 +Bba_I13507) • mCherryReceivers (Bba_F2620 + Bba_J06702) • Senders (bicistronic luxI + Reporter) • mRFPSender • tetR controlled (Bba_S03623 + Bba_I13507) • lacI controlled (Bba_S03608 + Bba_I13507) • Quorum controlled (Bba_R0062 + Bba_A340620 + Bba_I13507) • GFPSender • tetR controlled (Bba_S03623 + Bba_E0240) • lacI controlled (Bba_S03608 + Bba_E0240) • Quorum controlled (Bba_R0062 + Bba_A340620 + Bba_E0240) • mCherrySender • tetR controlled (Bba_S03623 + Bba_J06702) • Single Cell • Constitutive (Bba_J23039 + Bba_T9002) • Quorum Controlled (Bba_R0062 + Bba_A340620 + Bba_C0261 + Bba_E0240) • Construction Intermediates

  13. Receiver Sender Switch-like Quorum Response

  14. Selection with Direct Magnetic Beads Control: no selection Experimental: Selection with beads

  15. 60-fold Enrichment through Direct Magnetic Beads Control: no beads Selection with streptactin beads

  16. Receiver Sender The plate-drop experiment

  17. Bacterial targeting Quorum-sensing Fec signal transduction

  18. Motivation: Fec System • Goal: Direct cell signaling • Method: Re-engineer an existing signaling pathway • Fec system: • well-characterized • substrate specific

  19. Overview of Fec System Braun et al. “Gene Regulation by Transmembrane Signaling.” Biometals 2006 Apr;19(2):103-13.

  20. Fec: Motivation and Methods Re-engineer FecA: Mutate loop 7 and/or loop 8 Structural Evidence: L7 moves up to 11Å, helix unwinds L8 moves up to 15Å Assume signaling will occur with binding. Loops 7 & 8 Ferguson AD et al. “Structural Basis of Gating by the Outer Membrane Transporter FecA. Science 2002 Mar 1: 295(5560) 1715-9

  21. Direct Signaling from the Outer Membrane: the Fec System Advantages of Direct Signaling from the Outer Membrane: Substrate Specificity The FecIRA system is the only well-characterized signaling scaffold in Gram-negative bacteria FecA is an iron transporter and signal transducer on the outer membrane of E. Coli K-12 When ferric citrate binds, FecA activates periplasmic FecR, which then activates the sigma factor FecI, resulting in gene expression The system is repressed by the Fur repressor in iron-rich conditions Braun et al. “Gene Regulation by Transmembrane Signaling.” Biometals 2006 Apr;19(2):103-13.

  22. Results Wild Type Induction of FecA with Sodium Citrate and a GFP Reporter shows approximately 2000 RFU increase MACS Results Results from Nickel and His Fluorescence Assays

  23. CONCLUSION Targeting: AIDA Intercellular signaling: QS Intracellular signaling: Fec

  24. Future Directions Targeting: AIDA Intercellular signaling: QS Intracellular signaling: Fec

  25. ACKNOWLEDGEMENTS Advisors George Church Debra Auguste Jagesh V. Shah William Shih Pamela Silver Alain Viel Tamara Brenner Teaching Fellows Nicholas Guido Bill Senapedis Mike Strong Harris Wang Funding HHMI Harvard Provost Harvard Life Sciences Division Harvard School of Engineering and Applied Sciences

  26. N terminus modification of AIDA1

  27. CSR by gene Design Fusion of tags & randomers to extracellular portion of OmpA loop 1 (loop insertion) PCR product insertion (950 bps) Insertion of ds oligos

  28. CSR by gene design

  29. Bacterial Targeting: Cell Surface Reengineering (CSR) CSR by PCR product digestion & ligation: Fusion of peptides to the C terminus of OmpA Fusion of peptides to the N terminus of AIDA1 <OmpA AIDA1 structures> Fusion of tags & randomers to extracellular portion of OmpA loop 1 (loop insertion) PCR product insertion (950 bps) Insertion of ds oligos