AHL - iGEM 2006
AHL - iGEM 2006
AHL - iGEM 2006
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<strong>iGEM</strong> Jamboree<br />
MIT – Nov 4th <strong>2006</strong><br />
Imperial College
Engineering a Molecular Predation Oscillator<br />
Imperial College
Biomedical<br />
Engineers<br />
Biomedical<br />
Engineers<br />
<strong>iGEM</strong> <strong>2006</strong> @ Imperial<br />
Electrical<br />
Engineer<br />
Imperial College<br />
Biochemist<br />
Dr Mann<br />
Biologists<br />
Biochemists
Bio-Memory<br />
Project Ideas<br />
Oscillator<br />
• Feasibility<br />
• Originality of Design<br />
• BioBrick Availability<br />
• Future Impact<br />
Imperial College<br />
Bio-Clock
Our Definition<br />
What is an Oscillator?<br />
Device producing a periodic variation in time<br />
of a measurable quantity, e.g. amplitude.<br />
Engineering Biology<br />
Imperial College
Testing/Validation<br />
The Engineering Approach<br />
Specifications<br />
Standard<br />
Engineering<br />
Certified<br />
Practice<br />
Implementation Modelling<br />
Imperial College<br />
Design
Fluorescence<br />
Main challenges of<br />
past oscillators:<br />
• Unstable<br />
• Noisy<br />
• Inflexible<br />
Repressilator<br />
Time (min.)<br />
Figure Reference : Michael B. Elowitz & Stanislas Leibler Nature 2000<br />
The Main Challenges<br />
Imperial College<br />
Requirement for a<br />
Our Specifications:<br />
typical engineering<br />
oscillator<br />
•<br />
Sustained<br />
Stability: >10<br />
Oscillations<br />
periods<br />
• High SNR: Signal High to Noise Ratio<br />
• Controllable Flexibility: Controllable Oscillations<br />
Amplitude and Frequency<br />
• Standardized Device for<br />
• Modular Design<br />
Easy Connectivity<br />
• Easy Connectivity
Our Initial Design Ideas<br />
Based on<br />
• Large populations of molecules to reduce<br />
influence of noise<br />
• Oscillations due to population dynamics<br />
• A well characterized model<br />
Molecular Predator - Prey<br />
Imperial College
d<br />
dt<br />
d<br />
dt<br />
X<br />
Y<br />
Population<br />
Size<br />
The Lotka-Volterra Model<br />
aX<br />
Prey Growth<br />
cXY dY<br />
Imperial College<br />
Prey bXY Killing<br />
by Predator<br />
Predator Growth Predator Death<br />
X<br />
Y<br />
: Prey<br />
: Predator<br />
Time
Graph of<br />
Prey vs. Time<br />
Small<br />
Amplitude<br />
Large<br />
Amplitude<br />
Typical LV Simulations<br />
Low Frequency High Frequency<br />
prey prey<br />
prey prey<br />
Imperial College<br />
time time<br />
time<br />
time
A<br />
B<br />
d<br />
dt<br />
d<br />
dt<br />
Population<br />
Size<br />
A<br />
B<br />
Required Biochemical Properties<br />
Self promoted<br />
Prey Growth<br />
expression of A<br />
A<br />
Imperial College<br />
Degradation<br />
Prey Killing<br />
by of Predator A by B<br />
Expression of B<br />
Degradation<br />
Predator promoted Growth by Predator Death<br />
of B<br />
AB interaction<br />
B A<br />
B<br />
B<br />
A<br />
Time
Prey Generator<br />
Cell<br />
Self promoted<br />
expression of A<br />
Degradation of B<br />
A<br />
B<br />
Molecular System<br />
A<br />
B<br />
Imperial College<br />
A<br />
• Cell-cell communication<br />
Degradation<br />
• Constraint: Chemostat of A by B<br />
A•<br />
Flexibility: B Ratio of populations<br />
Predator<br />
Generator Cell<br />
Expression of B<br />
promoted by AB<br />
interaction
+<br />
LuxR<br />
LuxI<br />
pLux<br />
Quorum sensing/quenching<br />
Vibrio fischeri<br />
Forms a complex with<br />
<strong>AHL</strong> to activate pLux<br />
pLux Promoter<br />
<strong>AHL</strong><br />
BioBricks available<br />
BBa_C0062 BBa_C0061<br />
pLux<br />
BBa_R0062<br />
LuxR<br />
pTet pLux<br />
BBa_F2620<br />
Imperial College<br />
BBa_C0160<br />
Bacillus<br />
aiiA<br />
Makes <strong>AHL</strong><br />
Degrades <strong>AHL</strong><br />
<strong>AHL</strong>->Pops Receiver
Required<br />
Dynamic<br />
Useful<br />
BioBricks<br />
Final<br />
Construct<br />
Designing the Prey Generator<br />
LuxI<br />
C0061<br />
LuxR<br />
LuxR <strong>AHL</strong><br />
LuxR<br />
tetR pLux<br />
Imperial College<br />
Self promoted<br />
expression of A<br />
LuxR<br />
tetR pLux<br />
F2620<br />
<strong>AHL</strong><br />
LuxI<br />
LuxI<br />
<strong>AHL</strong>
Required<br />
Dynamic<br />
Useful<br />
BioBricks<br />
Final<br />
Construct<br />
Designing the Predator Generator<br />
Expression of B<br />
promoted by<br />
AB interaction<br />
pLux<br />
R0062<br />
<strong>AHL</strong><br />
Sensing<br />
LuxR<br />
C0062<br />
LuxR<br />
<strong>AHL</strong><br />
pLux<br />
<strong>AHL</strong><br />
Imperial College<br />
Degradation<br />
of A by B<br />
aiiA<br />
C0060<br />
LuxR<br />
<strong>AHL</strong><br />
LuxR aiiA<br />
<strong>AHL</strong><br />
Lactonase<br />
Degradation<br />
of B<br />
Natural<br />
degradation<br />
<strong>AHL</strong><br />
Killing
Prey Generator Cell<br />
pTet<br />
Lux<br />
R<br />
LuxR<br />
Lux<br />
R<br />
<strong>AHL</strong><br />
pLux<br />
<strong>AHL</strong><br />
System Overview<br />
LuxI<br />
LuxI<br />
pLux<br />
Imperial College<br />
Lux<br />
R<br />
<strong>AHL</strong><br />
LuxR<br />
<strong>AHL</strong><br />
Lux<br />
R<br />
Pool of <strong>AHL</strong> will<br />
oscillate<br />
<strong>AHL</strong><br />
Predator<br />
Generator Cell<br />
<strong>AHL</strong><br />
Lactonase<br />
aiiA<br />
<strong>AHL</strong>
LuxR<br />
tetR pLux<br />
LuxI<br />
Prey molecule generator<br />
pLux<br />
LuxR aiiA<br />
Predator molecule generator<br />
Full System set-up<br />
In-flow<br />
reserv oir<br />
Well mixed culture<br />
In chemostat<br />
Imperial College<br />
Wash-out<br />
Cell population<br />
Input<br />
signal<br />
Change in<br />
population ratio<br />
tim<br />
e<br />
[<strong>AHL</strong>] Output<br />
signal<br />
tim<br />
e
Start!<br />
Specifications Design Modelling Implementation Testing<br />
Specifications<br />
oscillator<br />
Based on<br />
Lotka-Volterra<br />
Based on<br />
Quorum Sensing<br />
Path to Our Goal<br />
Modelling<br />
Lotka-Volterra<br />
Modelling<br />
Full system<br />
Imperial College
d<br />
d<br />
d<br />
dt<br />
dt<br />
dt<br />
<strong>AHL</strong><br />
LuxR<br />
aiiA<br />
Modelling the Full System<br />
Self<br />
promoted<br />
expression of<br />
<strong>AHL</strong><br />
Expression<br />
of LuxR<br />
Expression<br />
of aiiA<br />
Imperial College<br />
Degradation<br />
of <strong>AHL</strong> by<br />
aiiA<br />
Degradation<br />
of <strong>AHL</strong><br />
Degradation<br />
of LuxR<br />
Degradation<br />
of aiiA
d [ <strong>AHL</strong>]<br />
<strong>AHL</strong><br />
dt<br />
d [ LuxR LuxR]<br />
dt<br />
d [ aiiA]<br />
aiiA<br />
dt<br />
Modelling the Full System<br />
Self<br />
promoted<br />
expression of<br />
<strong>AHL</strong><br />
Expression<br />
of LuxR<br />
Expression<br />
of aiiA<br />
Imperial College<br />
Degradation<br />
of <strong>AHL</strong> by<br />
aiiA<br />
Degradation<br />
of <strong>AHL</strong><br />
Degradation<br />
of LuxR<br />
Degradation<br />
of aiiA
d [ <strong>AHL</strong>]<br />
<strong>AHL</strong><br />
dt<br />
d [ LuxR LuxR]<br />
dt<br />
d [ aiiA]<br />
aiiA<br />
dt<br />
Modelling the Full System<br />
Gene Expression<br />
[ ][ ]<br />
[ ][ ]<br />
Production c <strong>AHL</strong> LuxRof<br />
c + <strong>AHL</strong> LuxR LuxR<br />
0<br />
a<br />
a<br />
0<br />
[ <strong>AHL</strong>]<br />
+ [ <strong>AHL</strong>]<br />
[ ][ ]<br />
[ ][ ]<br />
Production<br />
c <strong>AHL</strong> LuxR<br />
of<br />
c0<br />
+ <strong>AHL</strong> aiiA LuxR<br />
Imperial College<br />
Degradation<br />
of <strong>AHL</strong> by<br />
aiiA<br />
Degradation<br />
of <strong>AHL</strong><br />
Degradation<br />
of LuxR<br />
Degradation<br />
of aiiA
d [ <strong>AHL</strong>]<br />
<strong>AHL</strong><br />
dt<br />
d [ LuxR LuxR]<br />
dt<br />
d aiiA]<br />
aiiA<br />
dt<br />
Modelling the Full System<br />
Gene Expression<br />
[ ][ ]<br />
[ ][ ]<br />
Production c <strong>AHL</strong> LuxRof<br />
c + <strong>AHL</strong> LuxR LuxR<br />
[ [ ][ ]<br />
[ ][ ]<br />
0<br />
a<br />
a<br />
0<br />
[ <strong>AHL</strong>]<br />
+ [ <strong>AHL</strong>]<br />
Production<br />
c <strong>AHL</strong> LuxR<br />
of<br />
c0<br />
+ <strong>AHL</strong> aiiA LuxR<br />
b<br />
Imperial College<br />
Enzymatic Reaction<br />
[ aiiA][<br />
<strong>AHL</strong>]<br />
b + [ <strong>AHL</strong>]<br />
0<br />
Degradation<br />
of <strong>AHL</strong><br />
Degradation<br />
of LuxR<br />
Degradation<br />
of aiiA
d [ <strong>AHL</strong>]<br />
<strong>AHL</strong><br />
dt<br />
d [ LuxR]<br />
LuxR<br />
dt<br />
d aiiA]<br />
aiiA<br />
dt<br />
Modelling the Full System<br />
Gene Expression Enzymatic Reaction<br />
[ ][ ]<br />
[ ][ ]<br />
Production c <strong>AHL</strong> LuxRof<br />
c + <strong>AHL</strong> LuxR LuxR<br />
[ [ ][ ]<br />
[ ][ ]<br />
0<br />
a<br />
a<br />
0<br />
[ <strong>AHL</strong>]<br />
+ [ <strong>AHL</strong>]<br />
Production<br />
c <strong>AHL</strong> LuxR<br />
of<br />
c0<br />
+ <strong>AHL</strong> aiiA LuxR<br />
b<br />
Imperial College<br />
[ aiiA][<br />
<strong>AHL</strong>]<br />
b + [ <strong>AHL</strong>]<br />
0<br />
Degradation<br />
Degradation<br />
e[<br />
<strong>AHL</strong>]<br />
of <strong>AHL</strong><br />
d1[ LuxR]<br />
d [ aiiA]<br />
2
Graph of<br />
Prey vs.<br />
Time<br />
Small<br />
Amplitude<br />
Large<br />
Amplitude<br />
Full System Simulations<br />
Low Frequency High Frequency<br />
prey prey<br />
prey prey<br />
Imperial College<br />
time time<br />
time<br />
time
Typical System Behaviours<br />
Oscillations with limit cycles No oscillations<br />
Predator<br />
Prey<br />
Prey<br />
Time<br />
Imperial College<br />
Predator<br />
Prey<br />
Prey<br />
Time
Population<br />
dependent<br />
d [ <strong>AHL</strong>]<br />
<strong>AHL</strong><br />
dt<br />
d [ LuxR]<br />
LuxR<br />
dt<br />
d aiiA]<br />
aiiA<br />
dt<br />
a<br />
a<br />
0<br />
Modelling the Full System<br />
Gene Expression Enzymatic Reaction<br />
[ <strong>AHL</strong>]<br />
+ [ <strong>AHL</strong>]<br />
[ ][ ]<br />
[ ][ ]<br />
Production c <strong>AHL</strong> LuxRof<br />
c + <strong>AHL</strong> LuxR LuxR<br />
[ [ ][ ]<br />
[ ][ ]<br />
0<br />
Production<br />
c <strong>AHL</strong> LuxR<br />
of<br />
c0<br />
+ <strong>AHL</strong> aiiA LuxR<br />
Constant<br />
Imperial College<br />
b<br />
[ aiiA][<br />
<strong>AHL</strong>]<br />
b + [ <strong>AHL</strong>]<br />
0<br />
Wash-out<br />
related<br />
Degradation<br />
Degradation<br />
e[<br />
<strong>AHL</strong>]<br />
of <strong>AHL</strong><br />
d1[ LuxR]<br />
d [ aiiA]<br />
2
Start!<br />
Specifications Design Modelling Implementation Testing<br />
Specifications<br />
oscillator<br />
Specifications<br />
test constructs<br />
Based on<br />
Lotka-Volterra<br />
Based on<br />
Quorum Sensing<br />
Design<br />
test constructs<br />
Path to Our Goal<br />
Modelling<br />
Lotka-Volterra<br />
Modelling<br />
Full system<br />
Modelling<br />
Test construct<br />
Imperial College<br />
Build<br />
test constructs<br />
Characterized<br />
test constructs
Prey Generator<br />
PoPs<br />
Prey Sensing<br />
Breaking Down the Complexity<br />
<strong>AHL</strong><br />
a<br />
a0<br />
Predator Sensing<br />
PoPs<br />
Imperial College<br />
Predator Generator<br />
<strong>AHL</strong><br />
c<br />
c0<br />
Predator Killing<br />
<strong>AHL</strong><br />
time<br />
b<br />
b0
Test part<br />
Predictive model<br />
transfer function<br />
Experimental data<br />
Characterization Predator Sensing<br />
[GFP]<br />
Imperial College<br />
Experimental Data<br />
Average with variance and curve fit<br />
[<strong>AHL</strong>]
Test part<br />
Predictive model<br />
transfer function<br />
Experimental data<br />
Fitting model to data<br />
Parameter extractions<br />
Characterization Predator Sensing<br />
[GFP]<br />
Average with variance and curve fitting<br />
Imperial College<br />
[<strong>AHL</strong>]
Registry<br />
Catalogue<br />
Parts<br />
Prey<br />
J37034<br />
RS+J37034<br />
Prey with<br />
Riboswitch<br />
J37023<br />
J37024<br />
AiiA Test<br />
Construct<br />
J37025<br />
Final predator<br />
J37033<br />
J37019<br />
Sensing<br />
predator<br />
J37031<br />
Sensing<br />
predator<br />
J37032<br />
+<br />
+<br />
+<br />
Implementation<br />
Assembly process<br />
+<br />
+<br />
+<br />
+<br />
Imperial College<br />
+<br />
+<br />
+<br />
+<br />
+<br />
+<br />
+
Testing/Validation<br />
On Our Experience<br />
Specifications<br />
Implementation Modelling<br />
Imperial College<br />
Design
Start!<br />
Specifications Design Modelling Implementation Testing<br />
Specifications<br />
oscillator<br />
Specifications<br />
test constructs<br />
Based on<br />
Lotka-Volterra<br />
Based on<br />
Quorum Sensing<br />
Design<br />
test constructs<br />
Path to Our Goal<br />
Modelling<br />
Lotka-Volterra<br />
Modelling<br />
Full system<br />
Modelling<br />
Test construct<br />
Modelling<br />
characterized sys<br />
Imperial College<br />
Build<br />
test constructs<br />
Build full<br />
system<br />
Characterized<br />
test constructs<br />
Characterized<br />
Full system<br />
Our Goal!
Final Prey<br />
J37015<br />
Sensing Prey<br />
T9002<br />
Cre/Lox<br />
J37027<br />
Functional Parts<br />
Sensing Predator<br />
J37016<br />
Intermediate Parts<br />
J37033<br />
J37034<br />
Contributions to the Registry<br />
Built Sequenced Tested Characterized Documented<br />
Built Sequenced Built Sequenced<br />
Imperial College<br />
J37019<br />
J37032<br />
J37023
Imperial College<br />
Our Wiki<br />
• Documentation<br />
• Communication<br />
• Organization<br />
http://openwetware.org/wiki/IGEM:IMPERIAL/<strong>2006</strong>
Acknowledgements:<br />
• Prof. Tony Cass<br />
• Dr. Anna Radomska<br />
• Dr. Rupert Fray<br />
• Dr. David Leak<br />
• Dr. Mauricio Barahona<br />
• Dr. Danny O'Hare<br />
• Dr. Geoff Baldwin<br />
• Susan E. Wryter<br />
• David Featherbe<br />
• Ciaran Mckeown<br />
• James Mansfield<br />
Thank You<br />
Imperial College<br />
Funding:<br />
• European Commission<br />
• Imperial College Deputy<br />
Rectors Fund<br />
• Faculty of Engineering<br />
• Faculty of Natural<br />
Sciences
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