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. The motivation Bacterial targeting is necessary for spatially- specific activity in the body or in nature Post-targeting activity and transmembrane signalling are the next step in engineering genetic circuits that interface extracellular and intracellular environments To develop a system for directing bacteria to a target of interest and effecting downstream activity

3. The vision: Bacterial targeting via membrane display

4. The vision: Inter-cellular activation via Lux quorum-sensing

5. The vision: Intra-cellular activation via Fec signal transduction

6. 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

7. AIDA-1 his or AIDA-1 strep2 Surface Engineered Bacteria Engineered to Bind and Signal Kan Sender LuxI RFP Amp Amp and Kan Positive Signal Background Co-transform

8. AIDA-1 his or AIDA-1 strep2 Surface Engineered Bacteria Engineered to Bind and Signal Kan Sender LuxI RFP Amp Amp and Kan Positive Signal Background Co-transform signal

9. Direct Selection Talon and Streptactin Magnetic Beads Indirect Selection MACS Magnetic Beads And Antibodies Streptactin Magnetic beads -bind strep2 (strep binding peptide) or Talon Magnetic beads -bind polyhistidine tag 2 Methods for selecting/enriching for surface engineered bacteria MACS microbeads -binds Mouse IgG antibodies Y Anti-Strep2 tag Mouse IgG antibody Anti-his tag antibody Y Y Y Y Y Labeled cells retained Unlabeled cells Washed away

10. After magnetic selection Direct Selection using Magnetic Beads

12. Fusion at C terminus: Bead Assay (His tag) Beads Pre-assay plates

13. Loop Insertion PCR product digestion & ligation – Primer design – Digest-Ligate-Transform motif Gene design – Insertion points created for inserting synthetic constructs

14. Loop Insertion: PCR products ES Pre-loop1 OmpA OmpA Portion with Modified loop1 E P E|P digested vector XP Complete Plasmid M

15. PCR products Lane1: Ladder Lane2: 1 st portion OmpA Lane 3: strep2-OmpA portion 2 Lane 4: 6xHis-OmpA portion 2

16. PCR: final plasmid as a template Red line indicates the 1 kb band

17. Gene Design EPXSN

18. Gene Design: Operations N P XP M M

19. N N XS M M M M His/strep tags OR randomers

20. PCR Results Red line indicates the 1 kb line (7/8)

21. Cell-Cell Signalling Receiver Sender R OHHL Target (bead) Reporter luxI/luxR Quorum Sensing +

22. Cell-Cell Signaling: Constructs SenderReceiver Single Cell Construct – “JT” Two Cell Construct ReceiverSender

23. Sharp increase in fluorescence indicates quorum activity Amount of sender cells added Fluorescence per cell

24. Testing for self-induction: Fluorescence over OD at various times

25. Direct Magnetic Beads: Good Enrichment

26. Plate Drop Experiment with Enriched Sender

27. OmpA C-Terminus Library 5uL50uL200uL Vector233lots 10mer14149lots 15mer6275lots uL transformant vs. colony count 5uL of ligation reaction (4500 !! ng DNA) were transformed into chemically competent BL21 Gold DE3 Cells.

28. 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.

29. 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

30. 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

31. 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. Biobricking the Fec System

32. 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. Biobricking the Fec System

33. CONCLUSION To be added

34. ACKNOWLEDGEMENTS Thank you.