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Aims

Aims. How to implement elementary computations? How to form a more complex one?. Concrete Example. Gates: NAND, NOR, NOT gates, 2 levels Wires: 3 wires, which can cross and branch off I/O: 2 inputs and 2 outputs. Concrete Example. Gates: NAND, NOR, NOT gates, 2 levels

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Aims

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  1. Aims • How to implement elementary computations? • How to form a more complex one?

  2. Concrete Example • Gates: NAND, NOR, NOT gates, 2 levels • Wires: 3 wires, which can cross and branch off • I/O: 2 inputs and 2 outputs

  3. Concrete Example • Gates: NAND, NOR, NOT gates, 2 levels • Wires: 3 wires, which can cross and branch off • I/O: 2 inputs and 2 outputs

  4. Objectives • Transcriptional Regulation can be utilized to implement NAND, NOR, NOT gates in E.coli. • Transcriptional Factors can transmit message from one component to another.

  5. Extensible Logic Circuit in Bacteria USTC iGEM 2007

  6. Repression Model Bintu, L. et al. Transcriptional regulation by the numbers: models. Curr Opin Genet Dev (2005)

  7. Repression Model Bintu, L. et al. Transcriptional regulation by the numbers: models. Curr Opin Genet Dev (2005)

  8. Simulation and Score Function

  9. Simulation and Score Function

  10. Simulation and Score Function

  11. Cis-acting Logic Promoters NOT Gate

  12. Cis-acting Logic Promoters NOR Gate

  13. Cis-acting Logic Promoters NAND Gate

  14. Constructions and Measurements PCR Construction Solo-Repression Assay Co-Repression Assay 77 Promoter Synthesized ~ 400 Quantitative Assays

  15. Effect of Operator Position

  16. Effect of Operator Composition

  17. DNA-Looping [1] Müller, J., et al. Repression of lac promoter as a function of distance, phase and quality of an auxiliary lac operator. J. Mol. Bio. (1996) [2] Saiz, L. and Vilar, J. M. G. DNA looping: the consequences and its control., Curr Opin Struct Biol (2006)

  18. Hybrid Operator

  19. Dual-Repressed Operator

  20. Suggested Patterns

  21. Suggested Patterns

  22. Suggested Patterns

  23. Extensible Logic Circuit in Bacteria USTC iGEM 2007

  24. Repressor-Operator Recognition

  25. Repressor-Operator Pairs

  26. Directed Evolution • Select Target Sites • Mutagenesis by PCR • Screen on Plates • Quality Control • Quantitative Measurements • Result Analysis

  27. Directed Evolution • Select Target Sites • Mutagenesis by PCR • Screen on Plates • Quality Control • Quantitative Measurements • Result Analysis

  28. Directed Evolution • Select Target Sites • Mutagenesis by PCR • Screen on Plates • Quality Control • Quantitative Measurements • Result Analysis 5000 colonies screened 3 artificial operators 400 candidates per operator 11 novel artificial repressors

  29. Directed Evolution • Select Target Sites • Mutagenesis by PCR • Screen on Plates • Quality Control • Quantitative Measurements • Result Analysis

  30. Directed Evolution • Select Target Sites • Mutagenesis by PCR • Screen on Plates • Quality Control • Quantitative Measurements • Result Analysis

  31. Directed Evolution • Select Target Sites • Mutagenesis by PCR • Screen on Plates • Quality Control • Quantitative Measurements • Result Analysis Repression Matrix Diagonal Repression Matrix

  32. Repressor Evolution in Silico Side chain conformation optimization Sequence evaluation Test the results in vivo Selection of target ligand and variable positions

  33. Repressor Evolution in Silico Side chain conformation optimization Selection of target ligand and variable positions Sequence evaluation Test the results in vivo

  34. Diagonal Repression Matrix 9 Repressors vs. 4 Operators 6 repressors bind to only 1 operator 3 repressors bind to 2 operators 3x3 array for the demo system

  35. Extensible Logic Circuit in Bacteria USTC iGEM 2007

  36. A Demo: Diagram

  37. A Demo: Signaling Pathway

  38. What we have done: Patterns for NAND, NOR, NOT gates Highly-specific artificial repressors A demonstration system 123 Parts Submitted 247 Part Sequences 77 Synthesized Promoters 11 Novel Artificial Repressors ~ 350 New Strains ~ 130 DNA Strands Sequenced > 5000 Colonies Screened ~ 400 Quantitative Assays Extensible Logic Circuit in Bacteria USTC iGEM 2007

  39. Cis-acting Logic Gates Promoters with Cis-acting Elements • Work in vivo • Can be systematically constructed • Small in scale • About 2.0nm in width • 20 - 70nm in length • Can be cascaded to implement complex combinational logic computation

  40. Wires without Interference Highly-Specific Artificial Repressor • The number can grow • Do not interrupt natural signaling network • Do not interrupt each other • Provide supports for cis-acting logic gates • DNA Recognition • Dimerization • Tetramerization

  41. What We Plan To Do • Further Optimization • Size of the Wires • Response Time • More Input Signals • Better NOR pattern • Conductance Adjusting • Using different RBS • Using different operators

  42. Further More The First Transistor 1947 The First Integrated Circuit 1958

  43. Further More ?

  44. USTC iGEM 2007 • Graduates • Zhan Jian • Ding Bo • Ma Rui • Ma Xiaoyu • Undergrads • Liu Ziqing • Su Xiaofeng • Zhao Yun • Advisors • Prof. HY Liu • Prof. JR Wu • Prof. ZH Hou

  45. Acknowledgments We are sponsored by: Univ. of Sci. and Tech. of China NNSFC HHTech Co. Ltd.

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