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 FecA – loop insertion

6. Harvard iGEM 2007 Bacterial Targeting 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. Harvard iGEM 2007 Bacterial Targeting Cell Sorting with his and strep2 Indirect Bead Assays Direct Bead Assays

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

9. Harvard iGEM 2007 Bacterial Targeting Results: (MORE DESCRIPTIVE TITLE HERE) Successful selection of E.coli expressing His or Strep2 on surface Initial culturesEnriched cultures through selection

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

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

12. Harvard iGEM 2007 Quorum Sensing Receivers (luxR + Reporter) –GFP Receivers (Bba_T9002) –mRFP Receivers (Bba_F2620 + 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) –GFP Sender tetR controlled (Bba_S03623 + Bba_E0240) lacI controlled (Bba_S03608 + 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 The plate-drop experiment Receiver Sender

17. Harvard iGEM 2007 Two Component System Bacterial targeting Quorum-sensingFec signal transduction

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

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

20. Harvard iGEM 2007 Two Component System 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. 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

21. Harvard iGEM 2007 Two Component System 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. Harvard iGEM 2007 Two Component System 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. Harvard iGEM 2007 Conclusion CONCLUSION Targeting: AIDA Intercellular signaling: QS Intracellular signaling: Fec

24. Harvard iGEM 2007 Conclusion Future Directions Targeting: AIDA Intercellular signaling: QS Intracellular signaling: Fec

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

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