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

2. Harvard iGEM 2007 Introduction The motivation To develop a system for directing bacteria to a target of interest and effecting downstream activity

3. Harvard iGEM 2007 Introduction Bind Proteins Bind Other Cells Bind Tissue Bind Surface Bind DNA Bind Viruses Bind Toxins Potential Targets and Applications

4. Harvard iGEM 2007 Introduction Quorum-sensingFec signal transduction Bacterial targeting Quorum-sensingFec signal transduction

5. Harvard iGEM 2007 Bacterial Targeting Surface Engineered Bacteria Engineered to Bind and Signal Fusion Protein Membrane Protein OmpA – C terminal insertion OmpA-Loop1 insertion AIDA-1 – N terminal insertion

6. Harvard iGEM 2007 Bacterial Targeting Selecting/enriching for surface engineered bacteria Magnetism Activated Cell Sorting (MACS) Fluorescence Activated Cell Sorting (FACS) – His tag + nickel beads – Strep2 tag + streptavidin beads Fluorescence Bead Assays Magnetic Bead Assays

7. Harvard iGEM 2007 Bacterial Targeting His/Strep2 –tagged bacteria are enriched by MACS Magnetic Bead Assays

8. Harvard iGEM 2007 Bacterial Targeting Test: Cell Sorting with AIDA1 + sender constructs (with his and strep2) - Assay Results his strep2 Before Separation After Separation

9. Harvard iGEM 2007 Bacterial Targeting 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. Harvard iGEM 2007 Quorum Sensing Bacterial targeting Fec signal transductionQuorum-sensing

11. Harvard iGEM 2007 Quorum Sensing luxI/luxR Quorum Sensing Sender + R OHHL Receiver

12. Harvard iGEM 2007 Quorum Sensing Receivers (luxR + Reporter) –GFP Receivers tetR controlled (Bba_T9002) Quorum controlled (Bba_R0062 + Bba_C0261 + Bba_E0240) –mRFP Receivers tetR controlled (Bba_F2620 + Bba_I13507) Quorum controlled (Bba_R0062 + Bba_C0261 + Bba_I13507) –mCherry Receivers (Bba_F2620 + Bba_J06702) Senders (bicistronic luxI + Reporter) –mRFP Sender tetR controlled (Bba_S03623 + Bba_I13507) lacI controlled (Bba_S03608 + Bba_I13507) Quorum controlled (Bba_R0062 + Bba_A340620 + Bba_I13507) –GFP Sender tetR controlled (Bba_S03623 + Bba_E0240) lacI controlled (Bba_S03608 + Bba_E0240) Quorum controlled (Bba_R0062 + Bba_A340620 + Bba_E0240) –mCherry Sender 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 Cell-Cell Signaling Constructs Receiver Sender

13. Harvard iGEM 2007 Quorum Sensing Switch-like Quorum Response Sender Receiver

14. Harvard iGEM 2007 Quorum Sensing Selection with Direct Magnetic Beads Control: no selection Experimental: Selection with beads

15. Harvard iGEM 2007 Quorum Sensing 60-fold Enrichment through Direct Magnetic Beads Control: no beadsSelection with streptactin beads

16. Harvard iGEM 2007 Quorum Sensing Enriched senders activate quorum response Receiver Sender

17. Harvard iGEM 2007 Fec signal transduction Bacterial targeting Quorum-sensingFec signal transduction

18. Harvard iGEM 2007 Fec signal transduction Motivation: Fec System Goal: Direct cell signaling Method: Re-engineer an existing signal transduction pathway Fec system: – well-characterized – substrate specific

19. Harvard iGEM 2007 Fec signal transduction Overview of Fec System Braun et al. “Gene Regulation by Transmembrane Signaling.” Biometals 2006 Apr;19(2):103-13. Ferric citrate

20. Harvard iGEM 2007 Fec signal transduction Overview of Fec System Ferric citrate Loops 7 & 8

21. Harvard iGEM 2007 Fec signal transduction Overview of Fec System Braun et al. “Gene Regulation by Transmembrane Signaling.” Biometals 2006 Apr;19(2):103-13. Ferric citrate

22. Harvard iGEM 2007 Fec signal transduction Fec: Motivation Re-engineer FecA: Mutate loop 7 and/or loop 8 Structural Evidence: –L7 moves up to 11Å, helix unwinds –L8 moves up to 15Å Ferguson AD et al. “Structural Basis of Gating by the Outer Membrane Transporter FecA. Science 2002 Mar 1: 295(5560) 1715-9 Loops 7 & 8

23. Harvard iGEM 2007 Fec signal transduction 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

24. Harvard iGEM 2007 Fec signal transduction Wild-Type GFP Expression

25. Harvard iGEM 2007 Fec signal transduction Troubleshooting and Next Steps Problems: –Growth media –Toxicity: membrane disruption? Goals: –Nickel and Streptavidin Binding –Finding new targets with signaling Random library Computational Approach

26. Harvard iGEM 2007 Conclusion 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

27. Harvard iGEM 2007 Conclusion 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

28. Harvard iGEM 2007 Conclusion 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

29. Harvard iGEM 2007 Bacterial Targeting N terminus modification of AIDA1

30. Harvard iGEM 2007 Bacterial Targeting C terminus modification of OmpA Post-Assay Plates Pre-assay plates

31. Harvard iGEM 2007 Bacterial Targeting 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

32. Harvard iGEM 2007 Bacterial Targeting CSR by gene design

33. Harvard iGEM 2007 Bacterial Targeting 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 Fusion of tags & randomers to extracellular portion of OmpA loop 1 (loop insertion) – PCR product insertion (950 bps) – Insertion of ds oligos