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http://openwetware.org/wiki/IGEM:IMPERIAL/2006
Engineering a Engineering a Molecular Predation OscillatorMolecular Predation Oscillator
Thanks to staff from Imperial College for the support over the summer.
Funding
• European Commission• Imperial College Deputy
Rectors Fund• Faculty of Engineering• Faculty of Natural
Sciences
Acknowledgements
Students
• Christin Sander • Deepti Aswani • Farah Vohra • Jiongjun Bai • John Sy • John Chattaway • Jonathan Wells • Tom Hinson
Advisors
• Prof. Richard Kitney • Prof. Paul Freemont • Dr. David Mann • Kirsten Jensen • Vincent Rouilly • Chueh-Loo Poh • Matthieu Bultelle
Team Members
Project Summary Our Favourite PartsAchievements
Our Full System Set-Up
well mixed cultureIn chemostat
[AHL]
Cell population
time
time
Prey molecule generator
Predator molecule generator
Output signal
Wash-out
reservoir
In-flow
Design
• Based on Lotka-Volterra predation dynamics• Use of quorum sensing/quenching BioBricks available from the Registry• Population wide oscillations of AHL in a chemostat• Design broken down into two cell system to introduce higher flexibility
Modelling
• Derivation of the complete dynamical model • Full theoretical analysis and detailed simulations• Existence of oscillations with controllable frequency, amplitude and profile
Implementation
• Standard assembly using BioBricks• Successful building of oscillator components• Contributions to the Registry by adding tested, functional and intermediate parts• Quality control procedure
Testing/Validation
• Definition of testing protocols to satisfy component specifications• Analysis of experimental data• Characterization of the different test constructs for extracting parameters
Specifications
• Stable oscillations for more than 10 periods• High Signal to Noise Ratio• Controllable frequency and amplitude• Modular design for easy connectivity• Full documentation for quality control
Parameters:
a, b, c : Population-dependent a0, b0, c0: Constants d1, d2, e : Wash-out related
• We have used the traditional engineering approach to build a stable and flexible molecular oscillator
• Our original design relies on population dynamics and was inspired by the Lotka-Volterra predation model
• Every step of the development cycle (Specifications, Design, Modelling, Implementation, Testing/Validation) has been fully documented on our OWW site
• Derivation of the complete dynamical model, describing the main biochemical reactions driving our oscillator.
• Full theoretical analysis and detailed computer simulations, validating our design with regard to our specifications.
• Successful building and characterization of functional parts, providing the building blocks for the final oscillator.
PoPs Blocker
• Use of Cre-Recombinase• PoPs control mechanism defining an irreversible switch
Predator Sensing
• Characterization of a new AHL sensing part
Prey Molecule Generator
• Production of AHL via positive feedback loop
Oscillator
On/Off
Frequency tuningAmplitude tuning
Output signal
Oscillator
On/Off
Frequency tuningAmplitude tuning
Output signal
Oscillator
On/Off
Frequency tuningAmplitude tuning
Output signal
Test part
Predictive model transfer function
Experimental data
Fitting model to data
Parameter extractions
[AHL]
[GF
P]
Prey Generator
Prey Sensing
PoPs
AHL
a
a0
Predator Generator
Predator Sensing
PoPs
AHL
c
c0
Predator Killing
AHL
time
b
b0
Our Full System Set-Up
The Prey Generator
LuxI LuxR
RequiredDynamic
UsefulBioBricks
FinalConstruct LuxILuxR
tetR pLux
E.Coli
LuxILuxR
LuxR
Self promoted expression of A
AHLAHL
tetR pLux
The Predator Generator
LuxR aiiANatural
degradation
Degradation of A by B
Expression of B promoted by A/B interaction
Degradation of B
AHLAHL
Lactonase
Killing
LuxR
LuxR
SensingLuxR aiiA
E.ColiAHL
AHL
AHL
pLux
pLux
AHL
well mixed cultureIn chemostat
[AHL]
Cell population
time
time
LuxILuxRtetR pLux
LuxILuxRtetRtetR pLuxpLux
pLuxLuxR aiiA
pLuxpLuxLuxR aiiA
Prey molecule generator
Predator molecule generator
Output signal
Wash-out
reservoir
In-flow
Change in population ratio
J37019
J37032
J37033
J37034
Sensing PreyT9002
Final PreyJ37015
Built Sequenced DocumentedTested Characterized
Built BuiltSequenced Sequenced
Intermediate Parts
Cre/Lox J37027
Sensing PredatorJ37016
J37023
Functional Parts
AHL
LuxRdt
d[LuxR]
dt
d[LuxR]
Gene Expression Enzymatic Reaction
AHLe AHLe
LuxRd1 LuxRd1
Washout
dt
d[AHL]
dt
d[AHL] AHLa
AHLa
0 AHLa
AHLa
0
AHLb
AHLaiiAb
0
AHLb
AHLaiiAb
0
LuxRAHLc
LuxRAHLc
0 LuxRAHLc
LuxRAHLc
0
Self promoted expression of
AHL
Degradation of AHL
Degradation of AHL by aiiA
Production of LuxR
Degradationof LuxR
aiiAdt
d[aiiA]
dt
d[aiiA] LuxRAHLc
LuxRAHLc
0 LuxRAHLc
LuxRAHLc
0 aiiAd2 aiiAd2
Production of aiiA
Degradation of aiiA
Oscillations with limit cycles No oscillations
Prey
Predator
Prey
Time
Predator
Prey
Prey
Time