a synthetic oscillatory network of transcriptional...

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.