Cling- E. coli : Bacteria on target

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