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 Bacteria
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)
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)
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
PCR Construction
Solo-Repression Assay
77 Promoter Synthesized
~ 400 Quantitative Assays
Co-Repression Assay
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 its
control., 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 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
USTC iGEM 2007
5000 colonies screened
3 artificial operators
400 candidates per operator
11 novel artificial repressors
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
Sequence evaluation
Test the results in vivo
USTC iGEM 2007
Extensible Logic Circuit in Bacteria
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
USTC 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 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
USTC iGEM 2007
Extensible Logic Circuit in Bacteria
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
USTC iGEM 2007
Extensible Logic Circuit in Bacteria
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 cisacting logic gates
– DNA Recognition
– Dimerization
– Tetramerization
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 Transistor
1947
The First Integrated Circuit
1958
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
NNSFC
HHTech Co. Ltd.
USTC iGEM 2007
Extensible Logic Circuit in Bacteria