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A Synthetic Oscillatory Network of Transcriptional Regulators Michael Elowitz & Stanislas Leibler Nature, 2000 Presented by Khaled A. Rahman
A Synthetic Oscillatory Network ofTranscriptional Regulators
Michael Elowitz & Stanislas LeiblerNature, 2000
Presented by Khaled A. Rahman
Background
Genetic Networks
Gene XPromoter Operator
Gene YPromoter Operator
Gene ZPromoter Operator
RegulatesA group of Y
genes
RegulatesA group of Z
genes
Khaled A. Rahman
Background
Genetic Engineering
1- Design
2- Simulate
3- Implement & Test
then …… Refine
http://www.accessexcellence.org/AB/GG/plasmid.html
The Repressilator
Definition (A 3-element negative feedback transcriptionalloop).
Natural oscillators (cell cycle, circadian clock)
Composition:
+
Black boxes are T1 terminators
Michael B. Elowitz & Stanislas Leibler
The Repressilator - Design
A mathematical model oftranscription regulation.
Aim of the model: determinethe parameters to be adjusted toproduce sustained oscillations.
The model was solved twice(with similar parameters): -using deterministic continuousapproximation – usingstochastic discreteapproximation (the model wasmodified).
- i,j = “successive” genes in the chain.- mi(t) = [mRNA]- pi(t) = [repressor]- a = promoter rate ( - a0)without repressor- a0 = leakiness term in saturating repressor- b = ratio of protein decay rate to mRNA decayrate- n = Hill coefficient of the repressor
Mike Cantor, Stanford Medical Informatics
The Repressilator - Design
Deterministic solution
stable steady state
Stochastic solutionsustained limit-cycle oscillations
Oscillations in the levels of the three repressor proteins using numerical integration.
Conclusion: To obtain sustained oscillations we need
- strong promoters.
- tight transcription repression with low leakiness.
- comparable protein and mRNA decay rates
Michael B. Elowitz & Stanislas Leibler
The Repressilator
Modifications:
- Hybrid Promoters (PLlac01, PLtet01)
- Carboxy terminal tag (_ t1/2 60’ to 4’ and GFP t1/2 stable to 40’)
Output:
- Synchronization
- Periodic synthesis of GFP (150 minutes, 3x cell cycle time)
- The state of the network is transmitted to the siblings
- Average decorrelation time = 95 +/- 10 minutes
Michael B. Elowitz & Stanislas Leibler
The Repressilator
“ And what is the use of a book,”
thought Alice,
“ without pictures and conversations?”
Alice in Wonderland – Lewis Carroll
Michael B. Elowitz & Stanislas Leibler
The Repressilator
lacI-litePromoter
l PR
tetR-liteHybrid Promoter
PLlac01
l cI-liteHybrid Promoter
PLtet01
gfp-aavHybrid Promoter
PLtet01
Khaled A. Rahman
The Repressilator - Products
lacI-litePromoter
l PR
tetR-liteHybrid Promoter
PLlac01
l cI-liteHybrid Promoter
PLtet01
gfp-aavHybrid Promoter
PLtet01
LacI
CI
TetRGFP
GFP
Khaled A. Rahman
The Repressilator - Synchronization
lacI-litePromoter
l PR
tetR-liteHybrid Promoter
PLlac01
l cI-liteHybrid Promoter
PLtet01
gfp-aavHybrid Promoter
PLtet01
LacI
TetR
IPTG
Khaled A. Rahman
The Repressilator
lacI-litePromoter
l PR
tetR-liteHybrid Promoter
PLlac01
l cI-liteHybrid Promoter
PLtet01
gfp-aavHybrid Promoter
PLtet01
LacI
TetR
Khaled A. Rahman
The Repressilator
lacI-litePromoter
l PR
tetR-liteHybrid Promoter
PLlac01
l cI-liteHybrid Promoter
PLtet01
gfp-aavHybrid Promoter
PLtet01
LacI
CI
GFP
GFP
Khaled A. Rahman
The Repressilator
lacI-litePromoter
l PR
tetR-liteHybrid Promoter
PLlac01
l cI-liteHybrid Promoter
PLtet01
gfp-aavHybrid Promoter
PLtet01
CI
GFP
GFP
TetR
Khaled A. Rahman
The Repressilator
lacI-litePromoter
l PR
tetR-liteHybrid Promoter
PLlac01
l cI-liteHybrid Promoter
PLtet01
gfp-aavHybrid Promoter
PLtet01
TetR
LacI
Khaled A. Rahman
The Repressilator
lacI-litePromoter
l PR
tetR-liteHybrid Promoter
PLlac01
l cI-liteHybrid Promoter
PLtet01
gfp-aavHybrid Promoter
PLtet01
LacI
CI
GFP
GFP
Khaled A. Rahman
And so on….!!
Conclusion
“Self Control is the quality that distinguishes the fittest tosurvive”
- George Bernard Shaw
Noise from stochastic reactions and intrinsic complex dynamics.Positive feedback Control, potential input in the system.Applications of forward engineering:- Biotechnology- Experimental systems- Validation of models- Combined with reverse engineering to study natural networks
Thank You
Acknowledgement:The Fluorescence and The Bright field movies were provided byDr Michael Elowitz and Dr Mike Surette.
