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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. - PowerPoint PPT Presentation

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  • iGEM 2006 PRESENTATIONThe NCBS Team :Adil, Aparna, Ashesh, Dhanya, Krithiga, Ruchi, Sugat and Mukundwww.ncbs.res.in/events/ssb/

  • The Living Networks2DChemotaxisUV SwitchCell-CycleSynchronization

  • 2-D CONTROL OVER CHEMOTAXISCan we achieve 2-D control by hacking into this system?Chemosensory receptors :Tar or Tsr

  • Tri-Gradient SystemGlucose(Default)UU1250 : Gift from Sandy Parkinson

  • Motion-control Strategies

  • ExperimentsInduction Results

  • Assays for Chemotaxis

  • Setting up GradientsBridge Setup (Microscopic)SOURCE (Rhodamine)SINKSlant Plate (Macroscopic)KMnO4 gradientTieman et al (1996)J BacteriologyInoculation site

  • Assays for ChemotaxisAgar Plug Assay (Microscopic)SpacerAgar + 10mM ChemoattractantE. coliSuspension0min29minYu & Alam (1997) FEMS Microbiol Lett

  • 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 interactionsWhat next?

  • SYNCHRONIZATION OF CELL CYCLE OSCILLATORS

  • A multi-cell system:

    Core oscillator:The E.coli cell cycleOscillator coupling:Vibrio quorum sensing machineryCell cycle modulation: DnaA sequestration

  • Design and Features:Modular assembly allows us to test and modify intermediates independently.Whacky Idea

  • Multiple controls to tweak the system and multiple readouts

    pTet - aTc controlpLac - IPTG controlpBAD - arabinose controlRFPCFPYFP

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

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

  • Cell cycle modulation: DnaA sequestrationWe 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.

  • ConclusionsA part with oscillatory PoPs output LuxI-CFP fusion is functionalOscillations are transmitted without degradation through quorum sensingpX alone is not sufficient to affect cell cycle progression

  • Network Construction by DNA self-assembly

  • Acknowledgements

    Our iGEM ambassador - ReshmaM. M. Panicker for M13 strategyAll Living Networks workshop participantsCloning services provided by Bangalore GeneiFunding provided by NCBS

    Living Networks 2.0: Sticky pieces!June 2007

    Focus on protein and DNA self-assembly