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

  3. The vision:Bacterial targeting via membrane display

  4. The vision:Inter-cellular activation viaLux quorum-sensing

  5. The vision:Intra-cellular activation viaFec signal transduction

  6. 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

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

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

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

  10. Direct Selection using Magnetic Beads After magnetic selection

  11. Direct Selection using Magnetic Beads

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

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

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

  15. PCR products Lane1: Ladder Lane2: 1st 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. E X S P N Gene Design

  18. X P N P Gene Design: Operations M M

  19. X S N N Gene Design: Operations His/strep tags OR randomers M M M M

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

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

  22. Cell-Cell Signaling:Constructs Sender Receiver Single Cell Construct – “JT” Two Cell Construct Receiver Sender

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

  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 5uL of ligation reaction (4500 !! ng DNA) were transformed into chemically competent BL21 Gold DE3 Cells. uL transformant vs. colony count

  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.