igem Jamboree MIT Nov 4th 2006 Imperial College
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1 igem Jamboree MIT Nov 4th 2006
2 Engineering a Molecular Predation Oscillator
3 igem Imperial Biomedical Engineers Electrical Engineer Biochemist Biologists Biomedical Engineers Biochemists Dr Mann
4 Project Ideas Bio-Memory Oscillator Bio-Clock Feasibility Originality of Design BioBrick Availability Future Impact
5 Our Definition What is an Oscillator? Device producing a periodic variation in time of a measurable quantity, e.g. amplitude. Engineering Biology
6 The Engineering Approach Specifications Testing/Validation Design Implementation Standard Engineering Certified Practice Modelling
7 The Main Challenges Fluorescence Main challenges of past oscillators: Unstable Noisy Inflexible Repressilator Requirement for a typical Our Specifications: engineering oscillator Sustained Stability: >10 Oscillations periods High SNR: Signal High to Noise Ratio Controllable Flexibility: Controllable Oscillations Amplitude and Frequency Standardized Device for Modular Design Easy Connectivity Easy Connectivity Time (min.) Figure Reference : Michael B. Elowitz & Stanislas Leibler Nature 2000
8 Our Initial Design Ideas Based on Large populations of molecules to reduce influence of noise Oscillations due to population dynamics A well characterized model Molecular Predator - Prey
9 The Lotka-Volterra Model d d X Y ax Prey Growth cxy Predator Growth Prey bxy Killing by Predator dy Predator Death Population Size X :Prey Y : Predator Time
10 Typical LV Simulations Graph of Prey vs. Time Low Frequency High Frequency prey prey Small Amplitude time time prey prey Large Amplitude time time
11 Required Biochemical Properties d A Self promoted Prey Growth expression of A A Prey Killing by of Predator A by B B A d B Expression of B Predator promoted Growth by AB interaction B A Predator Death of B B Population Size A B Time
12 Molecular System Prey Generator Cell Self promoted expression of A A Cell-cell communication Constraint: Chemostat of A by B A Flexibility: B Ratio of populations A A of B B B Predator Generator Cell Expression of B promoted by AB interaction
13 Quorum sensing/quenching Vibrio fischeri Bacillus + plux LuxI aiia BBa_C0062 Forms a complex with to activate plux BioBricks available BBa_C0061 Makes plux BBa_R0062 plux Promoter BBa_C0160 Degrades ptet BBa_F2620 plux ->Pops Receiver
14 Designing the Prey Generator Required Dynamic Self promoted expression of A Useful BioBricks LuxI C0061 tetr F2620 plux Final Construct LuxI tetr plux LuxI
15 Designing the Predator Generator Required Dynamic Expression of B promoted by AB interaction of A by B of B Useful BioBricks plux R0062 C0062 aiia C0060 Natural degradation Final Construct Lactonase Sensing plux aiia Killing
16 System Overview Prey Generator Cell Pool of will oscillate LuxI ptet plux LuxI Predator Generator Cell Lactonase aiia plux
17 Full System set-up reservoir LuxI tetr plux In-flow Cell population Input signal Change in population ratio Prey molecule generator time Wash-out [] Output signal plux aiia Well mixed culture In chemostat time Predator molecule generator
18 Full System set-up reservoir LuxI [] Output signal tetr plux In-flow Prey molecule generator time Wash-out Cell population plux aiia Well mixed culture In chemostat Change in population ratio time Predator molecule generator
19 Path to Our Goal Start! Specifications Design Modelling Implementation Testing Specifications oscillator Based on Lotka-Volterra Based on Quorum Sensing Modelling Lotka-Volterra Modelling Full system
20 Modelling the Full System d Self promoted expression of of by aiia of d Expression of of d aiia Expression of aiia of aiia
21 Modelling the Full System d [ ] Self promoted expression of of by aiia of d [ ] Expression of of d [ aiia ] aiia Expression of aiia of aiia
22 Modelling the Full System Gene Expression d [ ] a a 0 [ ] + [ ] of by aiia of d [ ] [ ][ ] [ ][ ] Production c of c + 0 of d [ aiia ] aiia [ ][ ] [ ][ ] Production c of c0 + aiia of aiia
23 Modelling the Full System Gene Expression Enzymatic Reaction d [ ] a a 0 [ ] + [ ] b [ aiia][ ] b + [ ] 0 of d [ ] [ ][ ] [ ][ ] Production c of c + 0 of d aiia ] aiia [ [ ][ ] [ ][ ] Production c of c0 + aiia of aiia
24 Modelling the Full System Gene Expression Enzymatic Reaction d [ ] a a 0 [ ] + [ ] b [ aiia][ ] b + [ ] 0 e[ ] of d [ ] [ ][ ] [ ][ ] Production c of c + 0 d 1 [ ] d aiia ] aiia [ [ ][ ] [ ][ ] Production c of c0 + aiia d [ aiia] 2
25 Full System Simulations Graph of Prey vs. Time Low Frequency High Frequency prey prey Small Amplitude time time prey prey Large Amplitude time time
26 Typical System Behaviours Oscillations with limit cycles Predator Predator No oscillations Prey Prey Prey Prey Time Time
27 Modelling the Full System Population dependent d [ ] Gene Expression a a 0 [ ] + [ ] Enzymatic Reaction b [ aiia][ ] b + [ ] 0 e[ ] of d [ ] d aiia ] aiia [ ][ ] [ ][ ] Production c of c + [ [ ][ ] [ ][ ] 0 Production c of c0 + aiia Wash-out related d 1 [ ] d [ aiia] 2 Constant
28 Path to Our Goal Start! Specifications Design Modelling Implementation Testing Specifications oscillator Specifications test constructs Based on Lotka-Volterra Based on Quorum Sensing Design test constructs Modelling Lotka-Volterra Modelling Full system Modelling Test construct Build test constructs Characterized test constructs
29 Breaking Down the Complexity Prey Generator Predator Generator Prey Sensing Predator Sensing Predator Killing PoPs a a0 PoPs c c0 time b b0
30 Characterization Predator Sensing Test part Predictive model transfer function [GFP] Experimental Data Average with variance and curve fit Experimental data []
31 Characterization Predator Sensing Test part Predictive model transfer function [GFP] Average with variance and curve fitting Experimental data Fitting model to data Parameter extractions []
32 Implementation Registry Catalogue Parts Prey J37034 RS+J37034 Prey with Riboswitch + Assembly process J37023 J37024 AiiA Test Construct J37025 Final predator J37033 J37019 Sensing predator J37031 Sensing predator J
33 On Our Experience Specifications Testing/Validation Design Implementation Modelling
34 Path to Our Goal Start! Specifications Design Modelling Implementation Testing Specifications oscillator Specifications test constructs Based on Lotka-Volterra Based on Quorum Sensing Design test constructs Modelling Lotka-Volterra Modelling Full system Modelling Test construct Modelling characterized sys Build test constructs Build full system Characterized test constructs Characterized Full system Our Goal!
35 Contributions to the Registry Functional Parts Built Sequenced Tested Characterized Documented Final Prey J37015 Sensing Prey T9002 Sensing Predator J37016 Cre/Lox J37027 Intermediate Parts Built Sequenced Built Sequenced J37033 J37034 J37019 J37032 J37023
36 Our Wiki Documentation Communication Organization
37 Thank You Acknowledgements: Prof. Tony Cass Dr. Anna Radomska Dr. Rupert Fray Dr. David Leak Dr. Mauricio Barahona Dr. Danny O'Hare Dr. Geoff Baldwin Susan E. Wryter David Featherbe Ciaran Mckeown James Mansfield Funding: European Commission Deputy Rectors Fund Faculty of Engineering Faculty of Natural Sciences
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