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

6. 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. Indirect Bead Assays Direct Bead Assays Cell Sorting with his and strep2

8. After Separation <NEW TITLE> Test: Cell Sorting with AIDA1 + sender constructs (with his and strep2) Before Separation strep2 his

9. Results: (MORE DESCRIPTIVE TITLE HERE) Initial cultures Enriched cultures through selection Successful selection of E.coli expressing His or Strep2 on surface

10. Bacterial targeting Quorum-sensing Fec signal transduction

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

12. Receiver Sender Cell-Cell Signaling Constructs • Receivers (luxR + Reporter) • GFPReceivers (Bba_T9002) • mRFPReceivers (Bba_F2620 + Bba_I13507) • mCherryReceivers (Bba_F2620 + Bba_J06702) • Senders (bicistronic luxI + Reporter) • mRFPSender • tetR controlled (Bba_S03623 + Bba_I13507) • lacI controlled (Bba_S03608 + Bba_I13507) • GFPSender • tetR controlled (Bba_S03623 + Bba_E0240) • lacI controlled (Bba_S03608 + 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

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

16. The plate-drop experiment BBa_S03623 – BBa_I13507 BBa_T9002 T9002 S23I07 OHHL Receiver -> GFP Red OHHL sender

17. Plate Drop Experiment with Enriched Sender

18. Bacterial targeting Quorum-sensing Fec signal transduction

19. 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.

20. Fec: Motivation and Methods Structural information suggests possibility of maintaining signaling with changed binding. L7 moves up to 11Å, helix unwinds L8 moves up to 15Å Select binding targets by inserting random library, controls known to bind nickel and streptavidin into loops 7 and 8. Even if signaling cannot be maintained, binding of controls proves that FecA can be used as scaffold for surface expression of peptides Computational approach in collaboration with the lab of Costas Maranas, Penn State Dept of Chemical Engineering. Ferguson AD et al. “Structural Basis of Gating by the Outer Membrane Transporter FecA. Science 2002 Mar 1: 295(5560) 1715-9

21. 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

22. Biobricking the Fec System • Construct Features: • Swappable FecA - FecA is flanked by Nhe1 and AflII sites to allow the easy mutagenesis and replacement of FecA. • Variable Promoters - each component will be on a separate constitutive promoter. • The optimization of GFP expression using promoters of different strengths is planned.

23. Biobricking the Fec System • Mutagenesis of Fec promoter to weaken gene expression, providing a range of sensitivity. • Mutagenesis of the Fec promoter to remove FUR repressor binding site, allowing easier assays.

24. CONCLUSION Targeting: AIDA Intercellular signaling: QS Intracellular signaling: Fec

25. Future Directions Targeting: AIDA Intercellular signaling: QS Intracellular signaling: Fec

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