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iGEM in Tsinghua

iGEM in Tsinghua. The Department of Biological Sciences and Biotechnology, Tsinghua University, China 2007.11.3. Why do we come for iGEM?. We have a common dream: To build BioMachines as powerful as electronic and mechanical machines.

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iGEM in Tsinghua

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  1. iGEM in Tsinghua The Department of Biological Sciences and Biotechnology, Tsinghua University, China 2007.11.3

  2. Why do we come for iGEM? We have a common dream: To build BioMachines as powerful as electronic and mechanical machines.

  3. Even the simplest machine has two parts: • Power generator -The original force to drive the system. • Wire -information propagation

  4. What did we make? • An oscillator: RAP (AC power) Recoverable Auto-Pulse generator (RAP) by using a negative feedback loop. • A communicator: CelCuit (wire) Cell-cell communication system by using conjugating DNA as protein carrier.

  5. Part 1. RAP • Oscillator is a basic element in engineering. - It’s importance in electronic engineering is well known. - It’s also widely required for genetic machine and natural organism (circadian rhythm).

  6. Only one successful genetic oscillator published up to now. (Nature 403:335-338, 2000)

  7. We use another strategy • Negative feedback loop consisting of two components.

  8. Principle so easy!Never constructed…So, why not try it?

  9. T7 RNA polymerase (T7 RNAP) is used as the driver and Lac operon repressor (LacI) is used as the brake. • Both protein are tagged with Umd to implement fast degradation.

  10. Implementation Amplifier: Repressor:

  11. Modeling • We model the mRNA levels and protein levels respectively. (In the following description, we use lower-case of the first letter to indicate mRNA levels whereas up-case to indicate protein levels. )

  12. mRNA level is governed by: 1. spontaneous degradation (minus) 2. transcription (plus) Binding of proteins to DNA according to M-C equation First order reaction

  13. M-C equation Protein concentration Occupation ratio Hill coefficient Dissociation constant

  14. Protein levels are governed by: 1. spontaneous degradation (minus); 2. translation (plus) First order reaction

  15. Stochastic Model • To make our model more realistic, we modify our model to a stochastic one. In this model, all the parameters, except copy numbers of plasmids, are assumed to obey to a norm distribution and a ‘sigma’ value (which indicates the peak width) is set for each parameter.

  16. Parameter Estimation • Copy number of the repressor plasmid: n_lac_plasmid=30 copy • Copy number of the amplifier plasmid: n_t7_plasmid=5 copy

  17. Transcription rate of T7 polymerase: r_transcribe_T7=300 mRNA/min • Transcription rate of host polymerase: r_transcribe_P=30 mRNA/min Transcription rate of host polymerase is adapted form (Michael B. Elowitz, Nature, 2000 ) Transcription rate of T7 polymerase is about 10-20 folds of host polymerase.

  18. Translation rate: r_translate=5.0 proteins/min Adapted from (Michael B. Elowitz Nature, 2000 )

  19. Modified from (Michael B. Elowitz, Nature, 2000 ) • Half life time of mRNA: τ(mRNA)=3min • Half life time of LacI: τ(LacI)=10min • Half life time of T7: τ(T7)=10min • Half life time of EGFP: τ(EGFP)=40min

  20. Dissociation constant of LacI: K(LacI)=20 dimers per cell • Dissociation constant of T7 polymerase: K(T7)=20 monomers per cell K(LacI) is adapted from (Michael B. Elowitz Nature, 2000 ) According to (William R. McClure, PNAS, 1980), the Kd of T7 binding to double strand DNA is comparable to that of LacI.

  21. Simulation Results • Numerically simulated in MATLAB by using ODE toolbox.

  22. When Translation Rate Fluctuates

  23. What makes our RAP different? • RAP is more stable than the repressilator. It should be emphasized here that it is unnecessary for the driver to degrade faster than the brake τ(T7)=40min

  24. In a synchronized oscillator model proposed by coupling oscillator to quorum sensing system, RAP, which is of an relaxation type, converges more rapidly than repressilator, which is an phase oscillator. (PNAS 99(2):679-84, 2002 )

  25. Proceedings • We finished almost all the molecular cloning. • We confirmed that T7 RNAP we cloned is functional by luciferase test. • We have no time to put repressor and amplifier together before we leave for the Jamboree… 

  26. Parts Registered for RAP pACYC-Lux pEB-AclANcoI- pEB-SC101 pEB-T7X T7BLUE pT7Blue-XisT7X pEB-EGFPL pET15b-lacI(-) AAV-110 LLA-110 LVA-110 AAV-111 LLA-111 LVA-111 AAV-112 LLA-112 LVA-112

  27. Summary of RAP Q&A • What is RAP proposed to? -automatic oscillation • How is RAP constructed? -negative feedback loop by T7 RNAP and LacI • What are the advantages of RAP over existing oscillator? -stability -converge rapid when synchronized

  28. Part 2. CelCuit • Cell-cell communication is a very important issue in synthetic biology. • It is widely required for synchronized oscillator, populational computation, control of population size, artificial organogenesis and so on.

  29. Drawbacks of current cell-cell communication system-quorum sensing system (small diffusion molecule as messenger) 1. unrecoverable 2. difficult to achieve multiple independent signals Why are they so important?

  30. Focus shifts from small chemical to CONJUGATION • Two strategies have been tried to implement information transference by using conjugation.

  31. CelCuit: Using DNA to Carry Proteins Step 1 FLP recombinase SSB EGFP Step 2 SSB binding site Terminator

  32. Step 3 movie

  33. Which Executor? • The pool of executor is very large: transcription factors, two-hybrid system, … • In this case, we chose FLP recombinase for its high efficiency.

  34. Which Carrier (SSB)?

  35. We also tried other modes: - Lamda cI protein as the carrier - “relaxase-fusion” strategy: Mob-FLP fusion protein

  36. Implementation

  37. Proceedings • We finish all the molecular cloning of CelCuit except SSB part. • We confirm that the FLP recombinase we cloned is functional by using LacZ reporter. • Because the cloning of SSB is not finished yet, we are still not sure whether the protein transference during conjugation does occur.

  38. Parts Registered for CelCuit pEB-Lambda Amp- pBL-FLP pEB-C1 pEB-Mob pBL-Linker pBL-FLPMob pGEM-FLP pLZHinDIII2 pLZCI3 FLP-p15a pEASY-p15a pBL-Blunt pKD-pBL pKD-pBL-Lambda pEB-FLP pEB-Lambda

  39. We are all exciting for what we are doing. We think the CelCuit, once finished, will certainly revise the methodology of gene machine in population in many cases!

  40. Three Application Examples • Pattern Formation (Nature. 2005 Apr 28;434(7037):1130-4 ) Pattern formation by reciprocal interaction between nearby cells.

  41. Bacterial neural network (U.C Berkley’s dream several years ago) Neurons in one layer innervates those in their downstream layer in an all-connection mode.

  42. Cell Circuit (ambitious concept, isn’t it?) If we could set a cell array… Insulate cells which are near from each other.

  43. Summary of CelCuit Q&A • What is CelCuit proposed to? -transfer proteins between cells • How is CelCuit constructed? -fuse signal proteins (FLP, T7 RNAP…) to SSB • What are the advantages of CelCuit over existing communicator? -recoverable -large signal pool

  44. These two projects are done by: Dr. Xu Feng Prof. Chen Guoqiang Jiang Ming, Li Keyu, Dong Peng, Zhao Xinyu, Chen Chen, Shi Zhenyu, Liu Yexing, Zhou Lijun, Yu Zhou, Liu Zhe, Song Xindong

  45. Acknowledgement • We thank Dr. Xie Liping for her encouragement. • We thank the students who took practical synthetic biology summer course for their technique help. • This work is supported by the International Competition Funds of Tsinghua University.

  46. Thank iGEM for giving us such an unforgettable experience! Thank you for your attention!

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