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

USTC iGEM 2007 Extensible Logic Circuit in Bacteria

Aims

• How to implement elementary computations?

• How to form a more complex one?

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

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

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.

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

USTC iGEM 2007 Extensible Logic Circuit in Bacteria

Repression Model

Bintu, L. et al. Transcriptional regulation by the numbers: models. CurrOpin Genet Dev (2005)

USTC iGEM 2007 Extensible Logic Circuit in Bacteria

Repression Model

Bintu, L. et al. Transcriptional regulation by the numbers: models. CurrOpin Genet Dev (2005)

USTC iGEM 2007 Extensible Logic Circuit in Bacteria

Simulation and Score Function

USTC iGEM 2007 Extensible Logic Circuit in Bacteria

Simulation and Score Function

USTC iGEM 2007 Extensible Logic Circuit in Bacteria

Simulation and Score Function

USTC iGEM 2007 Extensible Logic Circuit in Bacteria

Cis-acting Logic Promoters

NOT Gate

USTC iGEM 2007 Extensible Logic Circuit in Bacteria

Cis-acting Logic Promoters

NOR Gate

USTC iGEM 2007 Extensible Logic Circuit in Bacteria

Cis-acting Logic Promoters

NAND Gate

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

USTC iGEM 2007 Extensible Logic Circuit in Bacteria

Effect of Operator Position

USTC iGEM 2007 Extensible Logic Circuit in Bacteria

Effect of Operator Composition

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)

USTC iGEM 2007 Extensible Logic Circuit in Bacteria

Hybrid Operator

USTC iGEM 2007 Extensible Logic Circuit in Bacteria

Dual-Repressed Operator

USTC iGEM 2007 Extensible Logic Circuit in Bacteria

Suggested Patterns

USTC iGEM 2007 Extensible Logic Circuit in Bacteria

Suggested Patterns

USTC iGEM 2007 Extensible Logic Circuit in Bacteria

Suggested Patterns

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

USTC iGEM 2007 Extensible Logic Circuit in Bacteria

Repressor-Operator Recognition

USTC iGEM 2007 Extensible Logic Circuit in Bacteria

Repressor-Operator Pairs

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

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

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

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

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

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

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

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

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

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

USTC iGEM 2007 Extensible Logic Circuit in Bacteria

A Demo: Diagram

USTC iGEM 2007 Extensible Logic Circuit in Bacteria

A Demo: Signaling Pathway

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

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

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

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

USTC iGEM 2007 Extensible Logic Circuit in Bacteria

Further More

The First Transistor1947

The First Integrated Circuit1958

USTC iGEM 2007 Extensible Logic Circuit in Bacteria

Further More

?

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

USTC iGEM 2007 Extensible Logic Circuit in Bacteria

Acknowledgments

We are sponsored by:

Univ. of Sci. and Tech. of China

HHTech Co. Ltd.

NNSFC