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O-Key Outline The Project 1. Design & Construction of the - - PowerPoint PPT Presentation
O-Key Outline The Project 1. Design & Construction of the - - PowerPoint PPT Presentation
AegiSafe O-Key Outline The Project 1. Design & Construction of the O-Key System 2. Regulation of the metabolism networks by O-Key System 3. BioBricks submitted to the Registry 4. Potential Combination of O-Key System with other SynBio
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Outline
The Project
- 1. Design & Construction of the O-Key
System
- 2. Regulation of the metabolism networks
by O-Key System
- 3. BioBricks submitted to the Registry
- 4. Potential Combination of O-Key System
with other SynBio technologies
Human Practice
- 1. Collaboration
- 2. SynBio Museum & Lectures
- 3. Biosafety Handbook
- 4. HP Modeling
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Project Part 1 Design & Construction of the O-Key system
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AUG
UCCUCC AGGAGG UCCUCC AGGCGG
ΔG
UCCUCC AUGCGG
C U
Shine-Dalgarno (SD), RBS Anti-Shine-Dalgarno (ASD)
50S subunit 30S subunit
↑
mRNA
Project
Design & Construction of the O-Key system
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AUG
AUGCGG Modified SD Modified ASD UACGCC
50S subunit 30S subunit mRNA
O-Key System
O-Key O-Lock Project
Design & Construction of the O-Key system
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3
O-Lock (O-RBS) N-16S
2 1 4
N-RBS O-Key (O-16S)
Project
O-Key: an orthogonal system
Design & Construction of the O-Key system
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1
A U G 50s subunit A G G A G G 30s subunit U C C U C C
Native RBS Native ASD Sequence
O-Lock (O-RBS)
N-16S N-RBS
O - Key (O-16S)
Project
Design & Construction of the O-Key system
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2
A U G A G G A G G
Native RBS
30s subunit C A A G G C
O-Key
O-Lock (O-RBS)
N-16S N-RBS
O - Key (O-16S)
Project
Design & Construction of the O-Key system
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A U G G U U C C G
Orthogonal RBS
30s subunit U C C U C C
Native ASD Sequence
O-Lock (O-RBS)
N-16S N-RBS
O- Key (O-16S)
3 Project
Design & Construction of the O-Key system
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4
A U G 50s subunit G U U C C G 30s subunit C A A G G C
Orthogonal RBS Orthogonal ASD Sequence
O-Lock (O-RBS)
N-16S N-RBS
O- Key (O-16S)
Project
Design & Construction of the O-Key system
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𝐹 = 𝑙 ∙ 𝑛 ∙ 𝑆 ∙ exp(−𝛾∆𝐻𝑢𝑝𝑢)
- k: system parameter
- m: the number of mRNA transcript
- R: the number of ribosomes
- β: the apparent Boltzmann constant
- ΔG: the Gibbs free energy change
ΔGtot = ΔGmRNA:rRNA + ΔGstart + ΔGspacing −ΔGstandby − ΔGmRNA
Howard M Salis, Ethan A Mirsky, Christopher A Voigt. Automated dedsign of synthetic ribosome binding sites to control protein expression. Nature biotechnology 27, 946-950(2009).
Theoretical basis of Model: RBS calculator
Project
Design & Construction of the O-Key system
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50 100 150 200 250 300
Relative Fluorescence/OD (600nm)
Prediction of single O-Key system by modeling
N-16S O-16S N-RBS O-RBS
Project
Design & Construction of the O-Key system
N-16S~N-RBS N-16S~O-RBS O-16S~N-RBS O-16S~O-RBS
1 2 3 4
O-Lock (O-RBS)
N-16S N-RBS
O- Key (O-16S)
3 2 1 4
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1 3 4 Gene-circuit of the O-Key System
O-Lock (O-RBS)
N-16S N-RBS
O-Key (O-16S)
3 2 1 4
BBa_K821012 BBa_K82001 BBa_J04450
Project
Design & Construction of the O-Key system
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Group RFP-Plasmid O-16S- Plasmid Result 1 N-RBS N/A Expression 3 O-RBS Pbad+O-16S (No induced) N/A 3’ O-RBS N/A N/A 4 O-RBS Pbad+O-16S (induced) Expression
Part: BBa_K821001 BBa_K821012
Project
Design & Construction of the O-Key system
O-Lock (O-RBS)
N-16S N-RBS
O - Key (O-16S)
3 2 1 4
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50 100 150 200 250 300 350
Relative Fluorescence/OD(600nm)
N-16S~N-RBS N-16S~O-RBS O-16S~O-RBS
Result of Experiment
Project
Design & Construction of the O-Key system
1 3 4
O-Lock (O-RBS)
N-16S N-RBS
O - Key (O-16S)
3 1 4
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Project
Design & Construction of the O-Key system
BBa_K8210012
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Part: BBa_K821002
Project
Design & Construction of the O-Key system
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n-RBS
- 1-RBS
- 2-RBS
50 100 150 200 250 300 n-16s
- 1-16s
- 2-16s
Relative Fluorescence/OD(600nm)
Prediction of Two O-Key system by modeling
N- 16s O1- 16s
N- RBS O1- RBS
O2- 16s
O2- RBS
Project
Design & Construction of the O-Key system
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We have:
Designed & constructed the O-Key System Predicted the orthogonality using modeling Characterized the orthogonality in the wet lab Installed the O-Lock in front of the Ampr resistance gene Constructed three orthogonal BioBricks and sent to the Registry
Project
Design & Construction of the O-Key system
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Project Part 2 Regulation of the Metabolism Network by O-Key System
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Project
Regulation of the Metabolism Network
AND
O-Key O-Lock Cell native control transcription Protein
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N-mRNA to O-mRNA
Multiplex automated genome engineering(MAGE) Synthetize O-genomics
- r O-plasmids
RBS mutation in plasmid ……
Project
Regulation of the Metabolism Network
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Project
Regulation of the Metabolism Network
AND
O-Key O-Lock Manual control Cell native control transcription Protein
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Project
Regulation of the Metabolism Network
Manual Control
Temperature-inducible systems Constitutive promoter Chemical-inducible systems Quorum sensing systems
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F A B C D E H I
Open to Close
Single O-Key system
Project
Regulation of the Metabolism Network
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A B C D E H I
Project
Regulation of the Metabolism Network
Single O-Key system
Close to Open
F
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F A B C D E H I
Double O-Key system
Switch flux to only product C
Project
Regulation of the Metabolism Network
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F A B C D E H I
Double O-Key system
Switch flux only to right
Project
Regulation of the Metabolism Network
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F A B C D E H I
Switch flux from right to left
Double O-Key system
Project
Regulation of the Metabolism Network
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The metabolic pathway
- f the violacein
Project
Regulation of the Metabolism Network
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Experiment 1 Normal metabolic pathway of violacein The product is purple
Project
Regulation of the Metabolism Network
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Part: BBa_K821012
Project
Regulation of the Metabolism Network
The Product is green
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Project
Regulation of the Metabolism Network
Part: BBa_K821012
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Project
Regulation of the Metabolism Network
We have:
Successfully constructed the violacein pathway using yeast assembler Demonstrated the regulation role of O-Key System Designed various metabolism regulation modules
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Project Part 3 BioBricks submitted to the Registry
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Project
We have:
BioBricks
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Project
We have improved:
BioBricks
BBa_J04450: RFP Coding Device BBa_K821001:RFP with O-Key
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Project
We have reconstructed:
BioBricks
BBa_K274004, BBa_K274003: violacein operon
A L1 C L3 E L5 2 micromin URA3 Amp pUC VioA
T-P-N
vioB
T-P-N
vioC vioD L4 vioE
O-RBS
T-P-O T-P-N
T-P-N T-P-N
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Project
We have constructed:
BioBricks BBa_K821012: pBad+orthogonal 16S RBS, BBa_K821002: orthogonal 16S DNA sequence from rrnB
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Project Part 4 Potential Combination of O-Key System with other SynBio technologies
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Project
Potential Combination
Replacement of RBS in plasmid
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Assembling DNA constructs by Yeast
Project
Potential Combination
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Global introduction of O-Lock by MAGE
Project
Potential Combination
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Human Practice
Human Practice
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Collaboration
Peking University: lab visiting, discussion session SUST: Parts Registry software development Nanjing University: iGEM survey and questionnaire
Human Practice
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SynBio Museum of China Human Practice
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Human Practice
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SynBio Lecture for Nankai Student Human Practice
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Biosafety Handbook & Summary
- Operation Part
- Social Part
- SynBio Part
- Transcriptional Regulation
- RNA Process
- Translational Control
- Stability of mRNA
- DNA Rearrangement
Human Practice
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Human Practice
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Advisor
Professor Yingjin Yuan
Acknowledgement
The Key Laboratory of Systems Biongineering Tianjin University International Cooperation Office Tianjin University Jiankun Foundation
Supported by:
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