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 Z Gene Y Promoter Operator Promoter Operator Regulates Regulates A group of Z A group of Y genes 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 - i,j = “successive” genes in the chain. the parameters to be adjusted to - m i (t) = [mRNA] produce sustained oscillations. - p i (t) = [repressor] The model was solved twice - a = promoter rate ( - a 0 )without repressor - a 0 = leakiness term in saturating repressor (with similar parameters): - - b = ratio of protein decay rate to mRNA decay using deterministic continuous rate approximation – using - n = Hill coefficient of the repressor stochastic discrete approximation (the model was modified). Mike Cantor, Stanford Medical Informatics

The Repressilator - Design Deterministic solution Stochastic solution sustained limit-cycle oscillations stable steady state 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 tetR-lite Hybrid Promoter P L lac01 lacI-lite gfp-aav Hybrid Promoter Promoter l P R P L tet01 l cI-lite Hybrid Promoter P L tet01 Khaled A. Rahman

The Repressilator - Products LacI tetR-lite Hybrid Promoter P L lac01 GFP TetR lacI-lite gfp-aav Hybrid Promoter Promoter l P R P L tet01 GFP CI l cI-lite Hybrid Promoter P L tet01 Khaled A. Rahman

The Repressilator - Synchronization LacI tetR-lite Hybrid Promoter IPTG P L lac01 TetR lacI-lite gfp-aav Hybrid Promoter Promoter l P R P L tet01 l cI-lite Hybrid Promoter P L tet01 Khaled A. Rahman

The Repressilator LacI tetR-lite Hybrid Promoter P L lac01 TetR lacI-lite gfp-aav Hybrid Promoter Promoter l P R P L tet01 l cI-lite Hybrid Promoter P L tet01 Khaled A. Rahman

The Repressilator LacI tetR-lite Hybrid Promoter P L lac01 GFP lacI-lite gfp-aav Hybrid Promoter Promoter l P R P L tet01 GFP CI l cI-lite Hybrid Promoter P L tet01 Khaled A. Rahman

The Repressilator tetR-lite Hybrid Promoter P L lac01 GFP TetR lacI-lite gfp-aav Hybrid Promoter Promoter l P R P L tet01 GFP CI l cI-lite Hybrid Promoter P L tet01 Khaled A. Rahman

The Repressilator LacI tetR-lite Hybrid Promoter P L lac01 TetR lacI-lite gfp-aav Hybrid Promoter Promoter l P R P L tet01 l cI-lite Hybrid Promoter P L tet01 Khaled A. Rahman

The Repressilator LacI tetR-lite Hybrid Promoter P L lac01 GFP lacI-lite gfp-aav Hybrid Promoter Promoter l P R P L tet01 GFP CI l cI-lite Hybrid Promoter P L tet01 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.