1. Harvard iGEM 2007 Introduction 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 targeting bacteria to a specific substrate and effecting a cellular response

3. Harvard iGEM 2007 Introduction Bind Proteins Bind Other Cells Bind Tissue Bind Surface Bind DNA/RNA 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 Introduction 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 Introduction Selecting/enriching for surface engineered bacteria Tags –6xHis + nickel beads –Strep2 + streptavidin beads Assays –Magnetic Activated Cell Sorting (MACS) –Fluorescence Activated Cell Sorting (FACS) Fluorescence Bead Assays Magnetic Bead Assays

7. Harvard iGEM 2007 Introduction His/Strep2 –tagged bacteria are enriched by MACS (after MACS selection)

8. Harvard iGEM 2007 Introduction Results: Cell Selection Assays are a Success! AIDA was re-engineered to target nickel and streptavidin with 6xHis and Strep2 tags respectively, and selecting for surface-engineered bacteria was accomplished through magnetic activated cell sorting.

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

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

11. 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

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

13. Harvard iGEM 2007 Quorum Sensing Selection with Magnetic Beads Sender AIDA-1 – N terminal insertion

14. Harvard iGEM 2007 Quorum Sensing 60-fold Selection through Magnetic Beads Control: no beadsSelection with streptavidin beads Green (untagged) Red (tagged)

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

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

17. 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

18. 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

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

20. Harvard iGEM 2007 Fec signal transduction Constructs From Braun lab (U. Tuebingen, Germany) –Fec knock-out strain, AA93 –FecIRA plasmid –Fec promoter, GFP plasmid pColA Duet Vector –Allows regulated expression of Fec genes under T7 promoter

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

22. 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

23. 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

24. 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

25. 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 Special thanks to… Volkmar Braun (University of Tuebingen) Costas Maranas (Penn State University)

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

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

28. 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

29. Harvard iGEM 2007 Bacterial Targeting CSR by gene design

30. 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