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Cling- E. coli : Bacteria on target

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. The motivation. To develop a system for directing bacteria to a target of interest and effecting downstream activity.

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Cling- E. coli : Bacteria on target

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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 transmembrane signalling 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 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 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 signal

  8. Selecting/enriching for surface engineered bacteria • Direct Selection • Direct magnetic beads • Indirect selection • MACS • FACS

  9. Direct Selection using Magnetic Beads After magnetic selection

  10. Direct Selection using Magnetic Beads

  11. Cell-Cell Signaling:luxI/luxR Quorum Sensing Reporter Target (bead) Receiver + Sender R OHHL

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

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

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

  15. Direct Magnetic Beads: Good Enrichment

  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. 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.

  19. 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

  20. 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

  21. 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.

  22. 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.

  23. CONCLUSION To be added

  24. 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

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