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Remote Control of Bacterial Chemotaxis

Remote Control of Bacterial Chemotaxis. UCSF iGEM Team 2006. Patrick Visperas Matthew Eames Eli Groban Ala Trusina. Christopher Voigt, Tanja Kortemme, Chao Tang, Chris Rao. Goal. Start. Remote Control of a Bacterial Machine. How Chemotaxis is Observed. Goulian Motility Assay (U Penn).

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Remote Control of Bacterial Chemotaxis

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  1. Remote Control of Bacterial Chemotaxis UCSF iGEM Team 2006 Patrick Visperas Matthew Eames Eli Groban Ala Trusina Christopher Voigt, Tanja Kortemme, Chao Tang, Chris Rao

  2. Goal Start Remote Control of a Bacterial Machine

  3. How Chemotaxis is Observed Goulian Motility Assay (U Penn) How Bacterial Chemotaxis Works Bacteria Swim Up Gradients Glide CCW Tumble ? CW concentration gradient No gradient

  4. No CheW A No Swimming P~ Y Y The Regulatory Network W A P~

  5. Binding Partners • Orthogonal binding pair Tsr-CheW (Liu et al, 1991) • We mapped these mutations onto the Tar-Chew complex Tar CheW

  6. Tsr mutation can be mapped to Tar Gradient Aspartate • point mutation in Tar* reduces motility to approximately 40% that of wild-type

  7. Part Design • New orthogonal signaling pairs “Codon randomization” is used to reuse scaffolds without fear of recombination

  8. UU1250 (Parkinson, U Utah) tar tsr tap trg aer cheW UUDcheW cheW* tar* tar tsr tap trg aer cheW Chassis Engineering • Build E. coli strain lacking all sensors and CheW, but retaining the other chemotaxis proteins Wild-type E. coli tar tsr tap trg aer cheW

  9. INPUT IRR IRL IRL IRR FimE % bacteria RED 0 0.3 1.2 5 [arabinose] (mM) GREEN Device Characterization: The Fim-Switch [ara] OUTPUT MCS Ptrc* gfp rfp FimE T. Ham, et al (2006) Tested in UU1250 2 Possible Problems - ara 1. The plasmid is unstable 2. The strain contains FimE/ FimB + ara

  10. System Design (11 kB) Constructed via DNA synthesis (DNA 2.0) Phe Asp - arabinose

  11. System Design (11 kB) Constructed via DNA synthesis (DNA 2.0) Phe Asp Phe Asp + arabinose - arabinose

  12. Results GradientGradient Aspartate Phenylalanine

  13. Conclusions • Orthogonal pairs are a rapid method to built signaling pathways that can operate simultaneously • Chassis developed to rapidly screen for new protein-protein interactions • Limited by the switch performance in this chassis • Directional control could also be achieved by non-chemical inputs (light, radiation, etc)

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