ustc igem 2007 extensible logic circuit in bacteria aims how to implement elementary computations?...
TRANSCRIPT
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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
![Page 5: USTC iGEM 2007 Extensible Logic Circuit in Bacteria Aims How to implement elementary computations? How to form a more complex one?](https://reader036.vdocuments.site/reader036/viewer/2022070306/5519b8855503465b578b48b6/html5/thumbnails/5.jpg)
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 BacteriaUSTC 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. CurrOpin 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. CurrOpin 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 itscontrol., 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 BacteriaUSTC 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 operators400 candidates per operator11 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 operator3 repressors bind to 2 operators3x3 array for the demo system
9 Repressors vs. 4 Operators
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USTC iGEM 2007 Extensible Logic Circuit in BacteriaUSTC 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 BacteriaUSTC 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 Submitted247 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 sig
naling network• Do not interrupt each other• Provide supports for cis-ac
ting 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 Transistor1947
The First Integrated Circuit1958
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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