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USTC iGEM 2007 Extensible Logic Circuit in Bacteria Aims How to implement elementary computations? How to form a more complex one?

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria 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

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria 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

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria 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.

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria USTC iGEM 2007 Extensible Logic Circuit in Bacteria

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria Repression Model Bintu, L. et al. Transcriptional regulation by the numbers: models. Curr Opin Genet Dev (2005)

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria Repression Model Bintu, L. et al. Transcriptional regulation by the numbers: models. Curr Opin Genet Dev (2005)

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria Simulation and Score Function

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria Simulation and Score Function

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria Simulation and Score Function

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria Cis-acting Logic Promoters NOT Gate

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria Cis-acting Logic Promoters NOR Gate

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria Cis-acting Logic Promoters NAND Gate

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria Constructions and Measurements Solo-Repression Assay Co-Repression Assay PCR Construction 77 Promoter Synthesized ~ 400 Quantitative Assays

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria Effect of Operator Position

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria Effect of Operator Composition

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria 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)

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria Hybrid Operator

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria Dual-Repressed Operator

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria Suggested Patterns

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria Suggested Patterns

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria Suggested Patterns

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria USTC iGEM 2007 Extensible Logic Circuit in Bacteria

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria Repressor-Operator Recognition

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria Repressor-Operator Pairs

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria Directed Evolution Select Target Sites Mutagenesis by PCR Screen on Plates Quality Control Quantitative Measurements Result Analysis

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria Directed Evolution Select Target Sites Mutagenesis by PCR Screen on Plates Quality Control Quantitative Measurements Result Analysis

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria 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

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria Directed Evolution Select Target Sites Mutagenesis by PCR Screen on Plates Quality Control Quantitative Measurements Result Analysis

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria Directed Evolution Select Target Sites Mutagenesis by PCR Screen on Plates Quality Control Quantitative Measurements Result Analysis

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria Directed Evolution Select Target Sites Mutagenesis by PCR Screen on Plates Quality Control Quantitative Measurements Result Analysis Repression Matrix Diagonal Repression Matrix

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria Repressor Evolution in Silico Selection of target ligand and variable positions Side chain conformation optimization Sequence evaluation Test the results in vivo

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria Repressor Evolution in Silico Selection of target ligand and variable positions Side chain conformation optimization Test the results in vivo Sequence evaluation

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria Diagonal Repression Matrix 6 repressors bind to only 1 operator 3 repressors bind to 2 operators 3x3 array for the demo system 9 Repressors vs. 4 Operators

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria USTC iGEM 2007 Extensible Logic Circuit in Bacteria

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria A Demo: Diagram

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria A Demo: Signaling Pathway

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria USTC iGEM 2007 Extensible Logic Circuit in Bacteria 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

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria Cis-acting Logic Gates 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 Promoters with Cis-acting Elements

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria Wires without Interference 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 Highly-Specific Artificial Repressor

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria 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

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria Further More The First Transistor 1947 The First Integrated Circuit 1958

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria Further More ?

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria 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

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USTC iGEM 2007 Extensible Logic Circuit in Bacteria Acknowledgments We are sponsored by: Univ. of Sci. and Tech. of China HHTech Co. Ltd. NNSFC

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