A Synthetic Oscillatory Network of Transcriptional Regulators - - PowerPoint PPT Presentation

A Synthetic Oscillatory Network of Transcriptional Regulators

Michael Elowitz & Stanislas Leibler

Nature, 2000

Presented by Khaled A. Rahman

Background

Genetic Networks Gene X

Promoter Operator

Gene Y

Promoter Operator

Gene Z

Promoter Operator Regulates A group of Y genes Regulates A 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 transcriptional loop). Natural oscillators (cell cycle, circadian clock) Composition:

+

Black boxes are T1 terminators

Michael B. Elowitz & Stanislas Leibler

The Repressilator - Design

A mathematical model of transcription regulation. Aim of the model: determine the parameters to be adjusted to produce sustained oscillations. The model was solved twice (with similar parameters): - using deterministic continuous approximation – using stochastic discrete approximation (the model was modified).

• 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 decay

rate

• n = Hill coefficient of the repressor

Mike Cantor, Stanford Medical Informatics

The Repressilator - Design

Deterministic solution

stable steady state

Stochastic solution

sustained 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-lite

Promoter l PR

tetR-lite

Hybrid Promoter PLlac01

l cI-lite

Hybrid Promoter PLtet01

gfp-aav

Hybrid Promoter PLtet01

Khaled A. Rahman

The Repressilator - Products

lacI-lite

Promoter l PR

tetR-lite

Hybrid Promoter PLlac01

l cI-lite

Hybrid Promoter PLtet01

gfp-aav

Hybrid Promoter PLtet01

LacI CI TetR

GFP GFP

Khaled A. Rahman

The Repressilator - Synchronization

lacI-lite

Promoter l PR

tetR-lite

Hybrid Promoter PLlac01

l cI-lite

Hybrid Promoter PLtet01

gfp-aav

Hybrid Promoter PLtet01

LacI TetR

IPTG

Khaled A. Rahman

The Repressilator

lacI-lite

Promoter l PR

tetR-lite

Hybrid Promoter PLlac01

l cI-lite

Hybrid Promoter PLtet01

gfp-aav

Hybrid Promoter PLtet01

LacI TetR

Khaled A. Rahman

The Repressilator

lacI-lite

Promoter l PR

tetR-lite

Hybrid Promoter PLlac01

l cI-lite

Hybrid Promoter PLtet01

gfp-aav

Hybrid Promoter PLtet01

LacI CI

GFP GFP

Khaled A. Rahman

The Repressilator

lacI-lite

Promoter l PR

tetR-lite

Hybrid Promoter PLlac01

l cI-lite

Hybrid Promoter PLtet01

gfp-aav

Hybrid Promoter PLtet01

CI

GFP GFP

TetR

Khaled A. Rahman

The Repressilator

lacI-lite

Promoter l PR

tetR-lite

Hybrid Promoter PLlac01

l cI-lite

Hybrid Promoter PLtet01

gfp-aav

Hybrid Promoter PLtet01

TetR LacI

Khaled A. Rahman

The Repressilator

lacI-lite

Promoter l PR

tetR-lite

Hybrid Promoter PLlac01

l cI-lite

Hybrid Promoter PLtet01

gfp-aav

Hybrid Promoter PLtet01

LacI CI

GFP GFP

Khaled A. Rahman

And so on….!!

Conclusion

“Self Control is the quality that distinguishes the fittest to survive”

• 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 by Dr Michael Elowitz and Dr Mike Surette.