iGEM Jamboree MIT – Nov 4th 2006

1. iGEM Jamboree MIT – Nov 4th 2006

2. Engineering a Molecular Predation Oscillator

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

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

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

6. Design Specifications Modelling Implementation Testing/Validation The Engineering Approach Standard Engineering Practice Certified

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

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

9. Prey Growth Prey Killing by Predator Predator Growth Predator Death Population Size d d dt dt Time The Lotka-Volterra Model X aX bXY Y cXY dY X Y :Prey : Predator

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

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

13. AHL + LuxR pTet pLux Quorum sensing/quenching Vibrio fischeri Bacillus LuxR LuxI LuxI LuxR aiiA aiiA pLux BioBricksavailable Forms a complex with AHL to activate pLux Makes AHL BBa_C0062 BBa_C0061 Degrades AHL pLux pLux Promoter BBa_R0062 BBa_C0160 AHL->Pops Receiver BBa_F2620

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

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

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

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

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

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

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

21. Modelling the Full System Gene Expression Degradation of AHL by aiiA Degradation of AHL AHL Degradation of LuxR Production of LuxR LuxR Production of aiiA Degradation of aiiA aiiA

22. Modelling the Full System Gene Expression Enzymatic Reaction Degradation of AHL AHL Degradation of LuxR Production of LuxR LuxR Production of aiiA Degradation of aiiA aiiA

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

24. Full System Simulations Graph of Prey vs. Time Low Frequency High Frequency prey prey Small Amplitude time time prey prey Large Amplitude time time

25. Typical System Behaviours Oscillations with limit cycles No oscillations Predator Predator Prey Prey Prey Prey Time Time

26. Population dependent [ ] [ ] b aiiA AHL [ ] + b AHL 0 Wash-out related Constant Modelling the Full System Gene Expression Enzymatic Reaction Degradation Degradation of AHL AHL Production of LuxR LuxR Production of aiiA aiiA

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

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

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

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

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

32. On Our Experience Specifications Design Testing/Validation Implementation Modelling

33. Specifications Design Modelling Implementation Testing Path to Our Goal Start! 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 Our Goal!

34. Contributions to the Registry Functional Parts Built Sequenced Tested Characterized Documented Final PreyJ37015 Sensing PreyT9002 Sensing PredatorJ37016 Cre/Lox J37027 Intermediate Parts Built Sequenced Built Sequenced J37019 J37033 J37032 J37034 J37023

35. Our Wiki • Documentation • Communication • Organization http://openwetware.org/wiki/IGEM:IMPERIAL/2006

36. 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 Thank You Funding: • European Commission • Imperial College Deputy Rectors Fund • Faculty of Engineering • Faculty of Natural Sciences