synchronized chemotactic oscillators

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Synchronized Chemotactic Oscillators. S.M.U.G. Summer Synthetic Biology Competition Massachusetts Institute of Technology November 6, 2004. Motivation. Our goal: an interesting, complex system something cool. But how to make it happen? We focused on implementing modularity - PowerPoint PPT Presentation

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  • Synchronized Chemotactic OscillatorsSummer Synthetic Biology CompetitionMassachusetts Institute of TechnologyNovember 6, 2004S.M.U.G.

  • MotivationOur goal: an interesting, complex system something cool. But how to make it happen?We focused on implementing modularityBreaking biological systems into modular piecesAt a low level, this is BioBricksBuilding a modular system allowed efficient division of labor key for a team this large

  • A Chemotactic OscillatorAttractant PlugBacterial Swimming PoolChemoattractant Gradient

  • A Chemotactic OscillatorBacteria are added to the swimming pool

  • A Chemotactic OscillatorChemotaxis is enabled, indicated by green bacteriaBacteria start swimming up the gradient towards the attractant plug

  • A Chemotactic OscillatorThe bacteria congregate around the attractant plug

  • A Chemotactic OscillatorEach bacteria has an internal oscillator, driving switch between: chemotaxis enabled chemotaxis disabled

  • A Chemotactic OscillatorThe bacteria communicate their internal oscillator phase with each other using cell-to-cell signaling

  • A Chemotactic Oscillator enabling the entire population to change state synchronously

  • A Chemotactic OscillatorIn red bacteria, chemotaxis is disabledBacteria start to randomly move away from the attractant plug

  • A Chemotactic OscillatorEventually the bacteria are dispersed around the swimming pool

  • A Chemotactic OscillatorAgain the bacteria communicate with each other using cell-to-cell signaling

  • A Chemotactic OscillatorThe inter-cellular signaling molecule diffuses throughout out the swimming pool to the entire population

  • A Chemotactic OscillatorState changes: chemotaxis is enabled

  • A Chemotactic Oscillator... and onward they swim, doomed to a fate worse than that of Sisyphus

  • A Chemotactic OscillatorFutile, this wretched swimming!

  • Top-Level System DiagramOscillatorModuleChemotaxisModuleIndividualsOsc PhasePopulationsOsc PhaseChemotaxisEnableCell BoundaryCell-to-CellSignalingModule

  • Top-Level System DiagramOscillatorModuleChemotaxisModuleIndividualsOsc PhasePopulationsOsc PhaseChemotaxisEnableCell BoundaryCell-to-CellSignalingModule

  • OutlineOverviewMotivationSystem DescriptionHow we got there: Synopsis of Summer ActivitiesModule DiscussionCell-to-cell Signaling ModuleOscillator ModuleChemotaxis ModuleModule IntegrationFinal Remarks

  • Learning about SynthBioPreliminary discussion and design workPrevious Class Experiences at MITMuch design, little implementationJuneJulyAugustCool ideas, but we wondered: Can we do this?Had to hit the lab

  • Introduction to a Biology LabObjective during this period was parts characterizationAchieved useful work on RBS characterizationAttempted to build sets of linked invertersBegan work on cell-to-cell signallingJuneJulyAugust So...you're saying that was supposed to be refrigerated?Agarose gels, Agar gels...what's the difference?

  • Finalizing DesignBrainstormed several comprehensive and full-system designsChoosing one design gave us purpose and focus in labJuneJulyAugust

  • Thereafter: Making it HappenFinal elementIntegrating modules is surely easier said than doneHow to prepare the experimental setup for our work?Barry, Jason, Fred, and Vikki will now discuss these topics. Chris will offer final remarks.

  • Cell-to-Cell Signaling ObjectivesDecompose signaling into elementsDesign, build and test elementsExplore how elements might function as part of the full systemCell-to-CellSynchronizationModuleIndividualsOsc PhasePopulationsOsc PhaseChemotaxisEnableCell Boundary

  • The Lux SystemLux box withleft and right promotersluxRluxCDABEGluxILHR

  • The Lux SystemLux box withleft and right promotersluxRluxCDABEGluxILRHLuciferaseLuxRDimerizationHRHHRRR

  • The Lux SystemLux box withleft and right promotersluxRluxCDABEGluxILRHLuciferaseHLuxRDimerizationHRHHRRRRRH

  • Utilizing Existing ComponentsLux box withleft and right promotersluxRluxCDABEGluxILRHLuciferaseHLuxRDimerizationHRHHRRRRRH

  • Utilizing Existing ComponentsLux box withleft and right promotersluxRluxCDABEGluxILRHLuciferaseHLuxRDimerizationHRHHRRRRRH

  • Utilizing Existing ComponentsLux box withleft and right promotersluxRluxCDABEGluxILRHLuciferaseHLuxRDimerizationHRHHRRRRRH

  • Receiver DesignBBa_I13270RHRHHRRRRRH

  • Receiver BuildingVaried Upstream Promoter - Ptet, luxPLHigh (100-200) and Low Copy (10-20) PlasmidUsed YFP Output Device as a PoPS ReporterBuilt in DH5alpha using standardized assembly, Transformed into MC4100 and HCB1103BBa_I13273

  • Receiver Testing1. Hi/Lo ratio2. Switch Point3. Response Time

  • Receiver TestingHi/Lo ratio 17Switch Point 2nMI13273 - pSB1A2

  • Receiver TestingI13273 - pSB1A2 - Growth Defects

  • Receiver TestingI13273 - pSB3K3 - Growth Restored

  • Receiver TestingI13273 - pSB3K3 - Response Time

  • SendersLHluxI

  • TransmissionDiffusion rateDegradation due to dilution (e.g. in chemostat)Degradation due to raised pHActive enzymatic degradation - aiiA

  • Transmission60 minute incubation of HSL at various pHUse that HSL to activate receivers at neutral pHpH Dependent Degradation

  • TransmissionaiiA Enzymatic intracellular Degradation of HSL

  • Future WorkDevelop the ability to adjust receiver transfer function parameters at willComplete characterization of existing sender device using the receiver deviceBuild and test the sender device used in the synchronized oscillatorContinue to test the aiiA degradation mechanismTest under different operating conditions - microfluidic chemostat, microscope slides etc.

  • Oscillator ModuleStand-alone OscillatorRelaxation OscillatorRing OscillatorSynchronized OscillatorSynchronatorsSynchronized Ring OscillatorFuture Work

    Cell-to-CellSynchronizationModuleIndividualsOsc PhasePopulationsOsc PhaseChemotaxisEnableCell BoundaryOscillatorModule

  • Input/Outputcell boundary

    OscillatorDevice

    PoPS

  • Lux/aiiA Relaxation OscillatorP LuxRRLAConstitutivePromoterLuxRLuxIaiiALuxR is constitutively expressed, while LuxI and aiiA are regulated by a LuxR activated promoter

  • Lux/aiiA Relaxation OscillatorRLHALuxRDimerizationHRConstitutivePromoterRLuxRLuxIaiiALuxR forms a dimer while LuxI synthesizes HSLP LuxR

  • Lux/aiiA Relaxation OscillatorRHLHALuxRDimerizationHRHHRRConstitutivePromoterRRRHLuxRLuxIaiiALuxR and HSL bind to form the transcriptional activator providing positive feedbackP LuxR

  • Lux/aiiA Relaxation OscillatorRHLHALuxRDimerizationHRHHRRConstitutivePromoterRRRHLuxRLuxIaiiALuxR and HSL bind to form the transcriptional activator providing positive feedbackP LuxR

  • Lux/aiiA Relaxation OscillatorRHLALuxRDimerizationDegradationof HSL by aiiAHRHHRRConstitutivePromoterRRRHLuxRLuxIaiiAaiiA degrades HSL providing negative feedbackP LuxR

  • Lux/aiiA Relaxation OscillatorRHLALuxRDimerizationDegradationof HSL by aiiAHRHHRRConstitutivePromoterRRRHLuxRLuxIaiiAaiiA degrades HSL providing negative feedbackP LuxR

  • Simplified Relaxation OscillatorInitial modeling work used a system of continuous differential equations to examine a simplified oscillatorFolds the positive feedback into a single Protein A ignoring the details of LuxI, HSL, and LuxREven with these simplifications, the model can give insight into what experimental constructs would be useful when building the actual Lux/aiiA oscillatorPromoterProtein AProtein B

  • State Space AnalysisIntersection of nullclines yields system equilibrium pointEqulibrium point changes withProtein B degradation rate

  • Preliminary Modeling ResultsA vs B State SpaceConcentration of A vs Time

  • Experimental WorkModeling work suggested possible test constructsExperimental work on the Lux/aiiA relaxation oscillator was put on hold Initial results on aiiA were discouragingNot enough degradation tags were available to effectively tune the aiiA degradation rate

  • Input/Outputcell boundary

    OscillatorDevice

    PoPS

  • Ring Oscillator

  • Input/Output

    OscillatorDevice

    Average oscillation phase of populationIndividual oscillation phasecell boundaryPoPssenderreceiverPoPsAHL

  • Synchronized Oscillator OptionsRepressilator & Synchronization DeviceFunctional oscillatorNeed to design synchronization deviceSynchronator4 designs available from the MIT 2003 Synthetic Biology courseDesigned to synchronize, completely built, but untested and uncharacterizedSee-ya-latorModeled after Yankees ALCS performance

  • Synchronator Designs gfpDesign 1Design 3gfpDesign 2Design 4gfp

  • Synchronator Design 2gfp

  • Synchronator Designs gfpDesign 1Design 3gfpDesign 2Design 4gfp

  • Oscillator Lockdown ExperimentIPTGATCgfpAdd IPTG = GFP highAdd ATC = GFP highAdd HSL = GFP low

  • Synchronator Lock-Down

    Chart3

    987.7610.142005.88763.51

    763.842057.262549.431433.01

    01540.47724.86736.53

    2188.46741.642243.88736.53

    Control

    IPTG

    ATC

    HSL

    Fluorescence Units

    Normal

    synchronator 1synchronator 2synchronator 3synchronator 4

    timepointtime(hr)Synch 1 (re-gate) - meanstdSynch 1 (re-gate) - covSynch 1 - meanstdSynch 1 - cvSynch 2 (re-gate) - meanstdSynch 2 (re-gate) - covSynch 2 - meanstdSynch 2 - cvSynch 3 (re-gate) - meanstdSynch 3 (re-gate) - covSynch 3 - meanstdSynch 3 - cvSynch 4 (re-gate) - meanstdSynch 4 (re-gate) - covSyn