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Cling- E. coli : Bacteria on target

This study focuses on developing a system for directing bacteria to a specific target and analyzing downstream activities. Potential targets include proteins, toxins, viruses, tissues, and DNA. Various techniques like cell sorting and magnetic bead assays are used for selecting and enriching bacteria. Quorum sensing and Fec signal transduction pathways are also explored for cell-cell signaling. The Fec system, a well-characterized pathway, is re-engineered to achieve direct cell signaling. Future directions include optimizing targeting peptides and further characterizing the quorum sensing and Fec signal transduction pathways.

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

  6. Selecting/enriching for surface engineered bacteria • Magnetism Activated Cell Sorting (MACS) • Fluorescence Activated Cell Sorting (FACS) • His tag + nickel beads • Strep2 tag + streptavidin beads Magnetic Bead Assays Fluorescence Bead Assays

  7. His/Strep2 –tagged bacteria are enriched by MACS Magnetic Bead Assays

  8. After Separation Before Separation Test: Cell Sorting with AIDA1 + sender constructs (with his and strep2) - Assay Results strep2 his

  9. Results: Cell Selection Assays are a Success! Selecting for surface engineered bacteria (such as his and strep2) is possible through either direct or indirect cell separation.

  10. Bacterial targeting Quorum-sensing Fec signal transduction

  11. 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 Sender

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

  16. Receiver Sender Enriched senders activate quorum response

  17. Bacterial targeting Quorum-sensing Fec signal transduction

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

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

  20. Overview of Fec System Ferric citrate Loops 7 & 8

  21. Constructs • From Braun lab (Germany) • Fec knock-out strain, AA93 • FecIRA plasmid • Fec promoter, GFP plasmid • pCola Duet Vector • Allows regulated expression of Fec genes under T7 promoter

  22. Wild-Type GFP Expression

  23. Troubleshooting andNext Steps • Problems: • Growth media • Toxicity: membrane disruption? • Goals: • Nickel and Streptavidin Binding • Finding new targets with signaling • Random library • Computational Approach

  24. Conclusions • Targeting • His and Strep2 tags on AIDA, targeting bacteria to nickel and streptavidin was successful • Quorum sensing • Constructed one-cell system • Characterized two-cell system • Combined with targeting • Fec signal transduction • Characterized Fec system

  25. Future Directions • Bacterial targeting • Trying out new targeting peptides (calmodulin) • Optimizing the random library approach in selecting for targeting peptides • Quorum sensing • Characterizing one-cell system • Optimizing quorum response after targeting • Fec signal transduction • Effect signal transduction with targeting • Application

  26. 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

  27. N terminus modification of AIDA1

  28. 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

  29. CSR by gene design

  30. 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

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