1. Imperial College iGEM Jamboree MIT – Nov 4th 2006

2. Imperial College Engineering a Molecular Predation Oscillator

3. Imperial College Biomedical Engineers Biochemists iGEM 2006 @ Imperial Biologists Biochemist Biomedical Engineers Electrical Engineer Dr Mann

4. Imperial College Feasibility Originality of Design BioBrick Availability Future Impact Project Ideas Bio-Clock Bio-Memory Oscillator

5. Imperial College What is an Oscillator? EngineeringBiology Device producing a periodic variation in time of a measurable quantity, e.g. amplitude. Our Definition

6. Imperial College Implementation ModellingDesign Specifications Testing/Validation The Engineering Approach Standard Engineering Practice Certified

7. Imperial College Sustained Oscillations High Signal to Noise Ratio Controllable Oscillations Standardized Device for Easy Connectivity Requirement for a typical engineering oscillator Our Specifications: Stability: >10 periods SNR: High Flexibility: Controllable Amplitude and Frequency Modular Design Easy Connectivity The Main Challenges Main challenges of past oscillators: Figure Reference : Michael B. Elowitz & Stanislas Leibler Nature 2000 Unstable Noisy Inflexible Time (min.) Fluorescence Repressilator

8. Imperial College Our Initial Design Ideas Large populations of molecules to reduce influence of noise Oscillations due to population dynamics A well characterized model Molecular Predator - Prey Based on

9. Imperial College X Y bXY The Lotka-Volterra Model d dt d Time Population Size aX cXYdY Prey Killing by Predator Prey Growth Predator GrowthPredator Death : Prey : Predator XYXY

10. Imperial College Typical LV Simulations Small Amplitude Graph of Prey vs. Time Large Amplitude Low FrequencyHigh Frequency prey time

11. Imperial College Prey Killing by Predator Prey Growth A Required Biochemical Properties Time Population Size A B B Predator GrowthPredator Death A A A BB B d dt d Self promoted expression of A Degradation of A by B Expression of B promoted by AB interaction Degradation of B

12. Imperial College Molecular System A B A A B B Self promoted expression of A Expression of B promoted by AB interaction Degradation of A by B Degradation of B A Cell-cell communication Constraint: Chemostat Flexibility: Ratio of populations Prey Generator Cell Predator Generator Cell

13. Imperial College Quorum sensing/quenching LuxR LuxI pLux aiiA Vibrio fischeri + AHL Bacillus pLux Promoter Forms a complex with AHL to activate pLux Makes AHL Degrades AHL BioBricks available LuxI LuxR aiiA pLux AHL->Pops Receiver LuxR pTet pLux BBa_C0062 BBa_C0061 BBa_C0160BBa_R0062 BBa_F2620

14. Imperial College Designing the Prey Generator Required Dynamic Useful BioBricks Final Construct LuxI LuxR tetRpLux Self promoted expression of A LuxI C0061 LuxR tetR pLux F2620 LuxI LuxR AHL

15. Imperial College Degradation of B Degradation of A by B Expression of B promoted by AB interaction AHL AHL Lactonase AHL Killing LuxR AHL Sensing Designing the Predator Generator Required Dynamic pLux LuxRaiiA Natural degradation LuxR C0062 pLux R0062 aiiA C0060 Useful BioBricks Final Construct

16. Imperial College Prey Generator Cell System Overview LuxI pLux LuxR pTet LuxI LuxR AHL LuxR pLux LuxR aiiA LuxR AHL Lactonase AHL Pool of AHL will oscillate Predator Generator Cell

17. Imperial College Full System set-up Well mixed culture In chemostat LuxI LuxR tetRpLux LuxRaiiA Prey molecule generator Predator molecule generator Wash-out reservoir In-flow time Change in population ratio [AHL]Output signal Cell population time Input signal

18. Imperial College Full System set-up Well mixed culture In chemostat LuxI LuxR tetRpLux LuxRaiiA Prey molecule generator Predator molecule generator Wash-out reservoir In-flow [AHL] Cell population time Output signal Change in population ratio

19. Imperial College Specifications Design Implementation Testing Modelling Specifications oscillator Based on Lotka-Volterra Modelling Lotka-Volterra Based on Quorum Sensing Modelling Full system Path to Our Goal Start!

20. Imperial College d dt Modelling the Full System Expression of aiiA Degradation of AHL Self promoted expression of AHL Degradation of AHL by aiiA AHL LuxR aiiA d dt d Expression of LuxR Degradation of aiiA Degradation of LuxR

21. Imperial College Modelling the Full System aiiA Expression of aiiA Degradation of AHL Self promoted expression of AHL Degradation of AHL by aiiA AHL LuxR Expression of LuxR Degradation of aiiA Degradation of LuxR

22. Imperial College Modelling the Full System aiiA Production of aiiA Degradation of AHL Degradation of AHL by aiiA Production of LuxR AHL LuxR Gene Expression Degradation of aiiA Degradation of LuxR

23. Imperial College Modelling the Full System aiiA Production of aiiA Degradation of AHL Production of LuxR AHL LuxR Gene Expression Enzymatic Reaction Degradation of aiiA Degradation of LuxR

24. Imperial College Modelling the Full System aiiA Production of aiiA Degradation of AHL Production of LuxR AHL LuxR Gene Expression Enzymatic ReactionDegradation

25. Imperial College Full System Simulations Small Amplitude Graph of Prey vs. Time Large Amplitude Low FrequencyHigh Frequency prey time

26. Imperial College Typical System Behaviours Oscillations with limit cyclesNo oscillations Prey Predator Prey Time Predator Prey Time

27. Imperial College Modelling the Full System aiiA Production of aiiA Degradation of AHL Production of LuxR AHL LuxR Gene Expression Enzymatic ReactionDegradation   AHLb aiiAb  0 Population dependent Constant Wash-out related

28. Imperial College Specifications Design Implementation Testing Modelling Specifications oscillator Based on Lotka-Volterra Modelling Lotka-Volterra Based on Quorum Sensing Modelling Full system Path to Our Goal Start! Specifications test constructs Design test constructs Modelling Test construct Build test constructs Characterized test constructs

29. Imperial College Breaking Down the Complexity Prey Sensing Prey Generator PoPs AHL a a0 Predator Killing AHL time b b0 Predator Generator Predator Sensing PoPs AHL c c0

30. Imperial College Characterization Predator Sensing Test part Predictive model transfer function Experimental data [AHL] [GFP] Experimental Data Average with variance and curve fit

31. Imperial College Characterization Predator Sensing Test part Predictive model transfer function Experimental data Fitting model to data Parameter extractions [AHL] [GFP] Average with variance and curve fitting

32. Imperial College Implementation Prey Prey with Riboswitch Sensing predator Final predator J37034 J37033 J37019 J37031 J37032 + + + + + + + + + + + RS+J37034 J37025 J37024 AiiA Test Construct J37023 + + + Registry Catalogue Parts Assembly process

33. Imperial College Implementation Modelling Design Specifications Testing/Validation On Our Experience

34. Imperial College Specifications Design Implementation Testing Modelling Specifications oscillator Based on Lotka-Volterra Modelling Lotka-Volterra Based on Quorum Sensing Modelling Full system Specifications test constructs Design test constructs Modelling Test construct Build test constructs Characterized test constructs Modelling characterized sys Build full system Characterized Full system Path to Our Goal Our Goal! Start!

35. Imperial College Contributions to the Registry J37019 J37032 J37033 J37034 Sensing Prey T9002 Final Prey J37015 BuiltSequenced Documented Tested Characterized Built Sequenced Intermediate Parts Cre/Lox J37027 Sensing Predator J37016 J37023 Functional Parts

36. Imperial College Our Wiki Documentation Communication Organization http://openwetware.org/wiki/IGEM:IMPERIAL/2006

37. Imperial College 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 Imperial College Deputy Rectors Fund Faculty of Engineering Faculty of Natural Sciences