IGEM2006 Imperial Powerpoint

Slide1: 

iGEM Jamboree MIT – Nov 4th 2006

Slide2: 

Engineering a Molecular Predation Oscillator

Slide3: 

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

Project Ideas: 

Feasibility Originality of Design BioBrick Availability Future Impact Project Ideas

What is an Oscillator?: 

What is an Oscillator? Engineering Biology

The Engineering Approach: 

The Engineering Approach Standard Engineering Practice Certified

The Main Challenges : 

The Main Challenges Main challenges of past oscillators: Figure Reference : Michael B. Elowitz & Stanislas Leibler Nature 2000 Unstable Noisy Inflexible Repressilator

Our Initial Design Ideas: 

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

The Lotka-Volterra Model: 

X Y bXY The Lotka-Volterra Model aX cXY dY : Prey : Predator X Y

Typical LV Simulations: 

Typical LV Simulations Small Amplitude Graph of Prey vs. Time Large Amplitude Low Frequency High Frequency prey prey prey prey time time time time

Required Biochemical Properties: 

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

Slide12: 

Molecular System B A 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

Quorum sensing/quenching: 

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

Designing the Prey Generator: 

Designing the Prey Generator Required Dynamic Useful BioBricks Final Construct Self promoted expression of A

Designing the Predator Generator: 

Degradation of B Degradation of A by B Expression of B promoted by AB interaction Designing the Predator Generator Required Dynamic Useful BioBricks Final Construct

Slide16: 

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

Full System set-up: 

Full System set-up Well mixed culture In chemostat Wash-out reservoir In-flow

Full System set-up: 

Full System set-up Well mixed culture In chemostat Wash-out reservoir In-flow

Path to Our Goal: 

Specifications oscillator Based on Lotka-Volterra Modelling Lotka-Volterra Based on Quorum Sensing Path to Our Goal Start!

Modelling the Full System: 

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

Modelling the Full System: 

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

Modelling the Full System: 

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

Modelling the Full System: 

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

Modelling the Full System: 

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

Full System Simulations: 

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

Typical System Behaviours: 

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

Modelling the Full System: 

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

Path to Our Goal: 

Specifications oscillator Based on Lotka-Volterra Modelling Lotka-Volterra Based on Quorum Sensing Modelling Full system Path to Our Goal Start!

Breaking Down the Complexity: 

Breaking Down the Complexity Prey Sensing PoPs AHL a a0

Characterization Predator Sensing: 

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

Characterization Predator Sensing: 

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

Implementation: 

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

On Our Experience: 

Implementation Modelling Design Specifications Testing/Validation On Our Experience

Path to Our Goal: 

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!

Contributions to the Registry: 

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

Our Wiki: 

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

Thank You: 

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