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iGEM 2006 PRESENTATION

iGEM 2006 PRESENTATION. The NCBS Team : Adil, Aparna, Ashesh, Dhanya, Krithiga, Ruchi, Sugat and Mukund. www.ncbs.res.in/events/ssb/. The Living Networks. UV Switch. 2D Chemotaxis. Cell-Cycle Synchronization. Bray D, Cell Movements (Garland, 1992). 2-D CONTROL OVER CHEMOTAXIS.

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iGEM 2006 PRESENTATION

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  1. iGEM 2006 PRESENTATION The NCBS Team : Adil, Aparna, Ashesh, Dhanya, Krithiga, Ruchi, Sugat and Mukund www.ncbs.res.in/events/ssb/

  2. The Living Networks UV Switch 2D Chemotaxis Cell-Cycle Synchronization

  3. Bray D, Cell Movements (Garland, 1992) 2-D CONTROL OVER CHEMOTAXIS Chemosensory receptors : Tar or Tsr Can we achieve 2-D control by hacking into this system?

  4. Constructs IPTG aTC + + Serine (Inducible) Aspartate (Inducible) tar cfp tsr rfp Plac Ptet Tsr Tar CFP RFP serine aspartate Transform tar, tsr null strain UU1250 Tri-Gradient System Glucose (Default) UU1250 : Gift from Sandy Parkinson

  5. Response to a single gradient Response to a dual gradient Receptor Concentration Distance Moved? All-or-none response Linear response Motion-control Strategies

  6. ExperimentsInduction Results

  7. Predicted Outcome Serine Aspartate Assays for Chemotaxis

  8. Setting up Gradients Bridge Setup (Microscopic) Slant Plate (Macroscopic) Inoculation site KMnO4 gradient SINK Tieman et al (1996) J Bacteriology SOURCE (Rhodamine)

  9. Assays for Chemotaxis Agar Plug Assay (Microscopic) Spacer 0 min Agar + 10mM Chemoattractant 29 min E. coli Suspension Yu & Alam (1997)FEMS Microbiol Lett

  10. What next? • Yet to establish that the constructs rescue chemotaxis • Resolving cross-induction issue • Fine tuning of Plug Assay • Dual gradient experiments • Construction of chemotaxis model incorporating receptor interactions

  11. SYNCHRONIZATION OF CELL CYCLE OSCILLATORS

  12. AI LuxI LuxI AI AI cell cycle oscillator cell cycle oscillator cell cycle modulation DnaA DnaA cell cycle modulation A multi-cell system: Core oscillator: The E.coli cell cycle Oscillator coupling: Vibrio quorum sensing machinery Cell cycle modulation: DnaA sequestration

  13. Oscillatory sender module Receiver module Cell cycle modulation AI degradation module Oscillation readout module Design and Features: Modular assembly allows us to test and modify intermediates independently. Whacky Idea

  14. Multiple controls to tweak the system and multiple readouts pTet - aTc control CFP pLac - IPTG control pBAD - arabinose control YFP RFP

  15. Core oscillator: The E.coli cell cycle To test oscillations, we have used cell length as a correlate of cell cycle phase. Pcya produces an oscillatory PoPs output

  16. Oscillator coupling: Vibrio quorum sensing machinery We tested Propagation of the oscillatory signal using YFP expression as readout. The oscillatory signal can be propagated through the quorum sensing circuit without degradation.

  17. Phase contrast image DNA is stained with DAPI. Cell cycle modulation: DnaA sequestration We tested effect of DnaA sequestration on growth rate of cells, cell morphology and DNA localization in those cells. Presence of DnaA binding sites did not affect growth rate of cells, though cells grew bigger and had DNA accumulated at central region.

  18. Conclusions • A part with oscillatory PoPs output • LuxI-CFP fusion is functional • Oscillations are transmitted without degradation through quorum sensing • pX alone is not sufficient to affect cell cycle progression

  19. Network Construction by DNA self-assembly

  20. Acknowledgements • Our iGEM ambassador - Reshma • M. M. Panicker for M13 strategy • All Living Networks workshop participants • Cloning services provided by Bangalore Genei • Funding provided by NCBS Living Networks 2.0: Sticky pieces! June 2007 Focus on protein and DNA self-assembly

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