University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine E. coli Automatic Directed Evolution Machine a Universal Framework for Evolutionary Approaches in Synthetic Biology University of Science and Technology of China
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University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
E. coli Automatic Directed Evolution Machine
a Universal Framework for Evolutionary Approaches in Synthetic Biology
University of Science and Technology of China
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
The power of evolution
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
Biology Engineering
Evolutionary
Algorithm
Directed
Evolution
E. coli Automatic Directed Evolution Machine
Our inspiration
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
The Goal
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
The Goal
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
E.ADEM Framework
Change
Act On
PoPS
Score
PoPS
Score
Kill
PoPSVariation PoPS Selection
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
Demand Analysis
Variaton = C Score
Death = Population [ ( . )]BasalC Score Avg Score
1 2Death Population ( . ) ( )C Avg Score C Score
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
The Prototype
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
pCon 0.15 luxI pCon 0.70 luxR
tetR
AHL
luxR
luxI tetR
pLux-Tet
Self-Adaptive Controller
luxR-AHL
PoPS
PoPS
1 2Death Population ( . ) ( )C Avg Score C Score
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
pCon 0.15 luxI pCon 0.70 luxR
tetR
AHL
luxR
luxI tetR
pLux-Tet
Self-Adaptive Controller
luxR-AHL
PoPS
PoPS
1 2Death Population ( . ) ( )C Avg Score C Score
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
pCon 0.15 luxI pCon 0.70 luxR
tetR
AHL
luxR
luxI tetR
pLux-Tet
Self-Adaptive Controller
luxR-AHL
PoPS
PoPS
1 2Death Population ( . ) ( )C Avg Score C Score
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
pCon 0.15 luxI pCon 0.70 luxR
tetR
AHL
luxR
luxI tetR
pLux-Tet
Self-Adaptive Controller
luxR-AHL
PoPS
PoPS
1 2Death Population ( . ) ( )C Avg Score C Score
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
pCon 0.15 luxI pCon 0.70 luxR
tetR
AHL
luxR
pCon X
VR X
mRFP luxI tetR
pLux-Tet ccdB
Selection FunctionScoring Function
Self-Adaptive ControllerReporter
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
Selection function
tetR
pLux-Tet ccdB
luxR-AHL
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
tetR
pLux-Tet ccdB
luxR-AHL
AHL
High AHL & Low tetR
tetR
pLux-Tet ccdB
luxR-AHL
AHL
High AHL & High tetR
tetR
pLux-Tet ccdB
AHL
Low AHL & High tetR
luxR
tetR
pLux-Tet ccdB
luxR
AHL
Low AHL & Low tetR
Selection function
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
pCon X
VR X
mRFP luxI tetR
tetRAHL
High score E.coli
tetRAHLLow score E.coli
pLux-Tet ccdB
pCon X
VR X
mRFP luxI tetRpLux-Tet ccdB
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
pCon X
VR X
mRFP luxI tetR
tetRAHL
High score E.coli
tetRAHLLow score E.coli
pLux-Tet ccdB
pCon X
VR X
mRFP luxI tetRpLux-Tet ccdB
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
pCon X
VR X
mRFP luxI tetR
tetR
AHL
tetRAHLLow score E.coli
High score E.coli
pLux-Tet ccdB
pCon X
VR X
mRFP luxI tetR
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
Measurement and Modeling
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
• Basic measurement strategy
B0034 GFP B0015
PoPS
Use spectrfluorophotometer to measure fluorescence intensity
Measurement and Modeling
Reporter
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
• Previous work: lacZα-ccdB
• Did not work very well. Still working…
• Different versions of ccdB we have tried
ccdB
lacZα-ccdB
B0031
B0031
B0015
B0015
ccdB-LVA
lacZα-ccdB-LVA
B0031
B0031
B0015
B0015
ccdB
lacZα-ccdB
B0034
B0034
B0015
B0015
ccdB-LVA
lacZα-ccdB-LVA
B0034
B0034
B0015
B0015
ccdB parts
You L, Cox RS 3rd, Weiss R, Arnold FH. Programmed population control by cell-cell communication and regulated killing. Nature. 2004 Apr 22;428(6985):868-71.
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
We estimate the values of k3, rAHLin, C1, C2, C3 and C4, using the data from our measurements. The simulation results as follows. k3=0.8min-1; rAHL=0.001min-1; C1=1.5*10-10; C2=1*1010; C3=8*10-7; C4=5.8*1011
ModelingpLux-Tet
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
The dots in the graph represent our experiment data and the curve and surface represent our simulation results.
Simulation results
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
★ Submit over 160 BioBrick parts to the registry
★ Characterize most of the parts
★ Comprehensive measurement data for whole system modeling
★ Introduce evolutionary algorithm into synthetic biology
★ A modular framework for automatic in vivodirected evolution
Summary
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
Future Plan
Short-term plan:
Use GFP to check the output of the self-adaptive controller
Try alternative proposal of the selection function, such as antisense RNA
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
pCon 0.15 luxI pCon 0.70 luxR
tetR
AHL
luxR
pCon X
VR X
mRFP luxI tetR
pLux-Tet ccdB
Selection FunctionScoring Function
Self-Adaptive ControllerReporter
Quorum-sensing I
Quorum-sensing II
luxR-AHL
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
pCon 0.15 luxI pCon 0.70 luxR
tetR
AHL
luxR
pCon X
VR X
mRFP luxI tetR
pLux-Tet ccdB
Selection FunctionScoring Function
Self-Adaptive ControllerReporter
Quorum-sensing I
Quorum-sensing II
luxR-AHL
PoPS
GFP
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
Future Plan
Short-term plan:
Use GFP to check the output of the self-adaptive controller
Try alternative proposal of the selection function, such as antisense RNA
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
Future Plan
Long-term plan:
E.ADEM as collaborative project in OpenWetWare
Realize the scoring functions for transcription repressor, sensor, logic gate, enzyme and binding partner
Develop variation functions for site-specific mutagenesis and recombination
Fine-tune the parameters in the self-adaptive controller
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
• Sponsors
• iGEM community
• Teaching Affairs Office, USTC
• Graduate School, USTC
• Foreign Affairs Office, USTC
• School of Life Sciences, USTC
Acknowledgments
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
Tsien Hsue-shen (钱学森)1911.12.11-2009.10.31
Father of Space Tech in China
Bei Shi-zhang (贝时璋)1903.10.10-2009.10.29
Father of Biophysics in China
In Memory of Our Great Masters
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
Thank You
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine
Supplementary Slides:
University of Science and Technology of China - iGEM 2009 - E.coli Automatic Directed Evolution Machine