A Synthetic Oscillatory Network of Transcriptional Regulators
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1 A Synthetic Oscillatory Network of Transcriptional Regulators Michael Elowitz & Stanislas Leibler Nature, 2000 Presented by Khaled A. Rahman
2 Background Genetic Networks Gene X Operator Operator Gene Y Operator Gene Z Regulates A group of Y genes Regulates A group of Z genes Khaled A. Rahman
3 Background Genetic Engineering 1- Design 2- Simulate 3- Implement & Test then Refine
4 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
5 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. - m i (t) = [mrna] - p i (t) = [repressor] - a = promoter rate ( - a 0 )without repressor - a 0 = 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
6 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
7 The Repressilator Modifications: - s (PLlac01, ) - 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
8 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
9 The Repressilator PLlac01 tetr-lite l PR laci-lite gfp-aav l ci-lite Khaled A. Rahman
10 The Repressilator - Products LacI tetr-lite PLlac01 TetR GFP l PR laci-lite CI gfp-aav GFP l ci-lite Khaled A. Rahman
11 The Repressilator - Synchronization LacI tetr-lite IPTG PLlac01 TetR l PR laci-lite gfp-aav l ci-lite Khaled A. Rahman
12 The Repressilator LacI PLlac01 tetr-lite TetR l PR laci-lite gfp-aav l ci-lite Khaled A. Rahman
13 The Repressilator LacI tetr-lite PLlac01 GFP l PR laci-lite CI gfp-aav GFP l ci-lite Khaled A. Rahman
14 The Repressilator PLlac01 tetr-lite TetR GFP l PR laci-lite CI gfp-aav GFP l ci-lite Khaled A. Rahman
15 The Repressilator LacI PLlac01 tetr-lite TetR l PR laci-lite gfp-aav l ci-lite Khaled A. Rahman
16 The Repressilator LacI tetr-lite PLlac01 GFP l PR laci-lite CI gfp-aav GFP l ci-lite Khaled A. Rahman
17 And so on.!!
18 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
19 Thank You Acknowledgement: The Fluorescence and The Bright field movies were provided by Dr Michael Elowitz and Dr Mike Surette.
A synthetic oscillatory network of transcriptional regulators
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