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 technologies Human Practice 1. Collaboration 2. SynBio Museum & Lectures 3. Biosafety Handbook 4. HP Modeling

Project Part 1 Design & Construction of the O-Key system

Project Design & Construction of the O-Key system 50S subunit U C Shine-Dalgarno (SD), RBS mRNA AUG AUGCGG AGGCGG AGGAGG ΔG ↑ 30S subunit UCCUCC UCCUCC UCCUCC Anti-Shine-Dalgarno (ASD)

Project Design & Construction of the O-Key system O-Key 50S subunit System O-Lock Modified SD mRNA AUG AUGCGG 30S subunit UACGCC Modified ASD O-Key

Project Design & Construction of the O-Key system O-Key: an orthogonal system O-Lock N-RBS (O-RBS) 2 3 4 1 O-Key N-16S (O-16S)

Project Design & Construction of the O-Key system O-Lock N-RBS (O-RBS) 1 O - Key N-16S (O-16S) 50s subunit Native RBS A U G A G G A G G 30s subunit U C C U C C Native ASD Sequence

Project Design & Construction of the O-Key system O-Lock N-RBS (O-RBS) 2 O - Key N-16S (O-16S) Native RBS A U G A G G A G G 30s subunit C A A G G C O-Key

Project Design & Construction of the O-Key system O-Lock N-RBS (O-RBS) 3 O- Key N-16S (O-16S) Orthogonal RBS A U G G U U C C G 30s subunit U C C U C C Native ASD Sequence

Project Design & Construction of the O-Key system O-Lock N-RBS (O-RBS) 4 O- Key N-16S (O-16S) 50s subunit Orthogonal RBS A U G G U U C C G 30s subunit C A A G G C Orthogonal ASD Sequence

Project Design & Construction of the O-Key system Theoretical basis of Model: RBS calculator ΔG tot = ΔG mRNA:rRNA + ΔG start + ΔG spacing −ΔG standby − ΔG mRNA 𝐹 = 𝑙 ∙ 𝑛 ∙ 𝑆 ∙ exp(−𝛾∆𝐻 𝑢𝑝𝑢 ) • 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 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).

Project Design & Construction of the O-Key system Prediction of single O-Key O-Lock system by modeling N-RBS Relative Fluorescence/OD (600nm) (O-RBS) 3 2 1 4 1 300 O- Key N-16S (O-16S) 250 200 4  N-16S~N-RBS 150 2  N-16S~O-RBS 100  O-16S~N-RBS O-RBS 50 3  O-16S~O-RBS 0 N-RBS N-16S O-16S

Project Design & Construction of the O-Key system Gene-circuit of the O-Key System O-Lock N-RBS (O-RBS) 3 2 1 4 BBa_J04450 BBa_K82001 O-Key N-16S (O-16S) 3 4 1 BBa_K821012

Project Design & Construction of the O-Key system O-Lock N-RBS (O-RBS) 2 3 1 4 O - Key N-16S (O-16S) O-16S- Group RFP-Plasmid Result Plasmid Part: 1 N-RBS N/A Expression BBa_K821001 Pbad+O-16S 3 O-RBS (No N/A BBa_K821012 induced) 3’ O-RBS N/A N/A Pbad+O-16S 4 O-RBS Expression (induced)

Project Design & Construction of the O-Key system O-Lock N-RBS (O-RBS) 3 Result of Experiment 1 4 Relative Fluorescence/OD(600nm) O - Key N-16S 350 (O-16S) 300 250 N-16S~N-RBS 200 N-16S~O-RBS 150 O-16S~O-RBS 100 50 0 1 3 4

Project Design & Construction of the O-Key system BBa_K8210012

Project Design & Construction of the O-Key system Part: BBa_K821002

Project Design & Construction of the O-Key system N- O1- O2- 16s 16s 16s Prediction of Two O-Key system by modeling N- O1- O2- Relative Fluorescence/OD(600nm) RBS 300 RBS RBS 250 200 150 100 o2-RBS 50 o1-RBS 0 n-RBS n-16s o1-16s o2-16s

Project Design & Construction of the O-Key system 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 Part 2 Regulation of the Metabolism Network by O-Key System

Project Regulation of the Metabolism Network Cell native control transcription O-Key O-Lock AND Protein

Project Regulation of the Metabolism Network Synthetize O-genomics or O-plasmids RBS mutation in N-mRNA plasmid to O-mRNA Multiplex automated genome engineering(MAGE) ……

Project Regulation of the Metabolism Network Manual Cell native control control transcription O-Key O-Lock AND Protein

Project Regulation of the Metabolism Network Constitutive promoter Chemical-inducible systems Manual Temperature-inducible Control systems Quorum sensing systems

Project Regulation of the Metabolism Network Single O-Key system A B I H C D E F Open to Close

Project Regulation of the Metabolism Network Single O-Key system A B I H C D E F Close to Open

Project Regulation of the Metabolism Network Double O-Key system A B I H C D E F Switch flux to only product C

Project Regulation of the Metabolism Network Double O-Key system A B I H C D E F Switch flux only to right

Project Regulation of the Metabolism Network Double O-Key system A B I H C D E F Switch flux from right to left

Project Regulation of the Metabolism Network The metabolic pathway of the violacein

Project Regulation of the Metabolism Network Experiment 1 Normal metabolic pathway of violacein The product is purple

Project Regulation of the Metabolism Network Part: BBa_K821012 The Product is green

Project Regulation of the Metabolism Network Part: BBa_K821012

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 35

Project Part 3 BioBricks submitted to the Registry

Project BioBricks We have:

Project BioBricks We have improved: BBa_J04450: RFP Coding Device BBa_K821001:RFP with O-Key

Project BioBricks We have reconstructed: BBa_K274004, BBa_K274003: violacein operon VioA vioB vioC L4 vioD vioE T-P-N T-P-N T-P-N T-P-N L1 A L3 C L5 E 2 micromin URA3 Amp pUC T-P-N O-RBS T-P-O

Project BioBricks We have constructed: BBa_K821012: pBad+orthogonal 16S RBS, BBa_K821002: orthogonal 16S DNA sequence from rrnB

Project Part 4 Potential Combination of O-Key System with other SynBio technologies

Project Potential Combination Replacement of RBS in plasmid

Project Potential Combination Assembling DNA constructs by Yeast

Project Potential Combination Global introduction of O-Lock by MAGE

Human Practice Human Practice

Human Practice Collaboration Peking University: lab visiting, discussion session SUST: Parts Registry software development Nanjing University: iGEM survey and questionnaire

Human Practice SynBio Museum of China

Human Practice

Human Practice SynBio Lecture for Nankai Student

Human Practice Biosafety Handbook & Summary • Operation Part • Social Part • SynBio Part • Transcriptional Regulation • RNA Process • Translational Control • Stability of mRNA • DNA Rearrangement

Human Practice

Acknowledgement Advisor Professor Yingjin Yuan Supported by: The Key Laboratory of Systems Biongineering Tianjin University International Cooperation Office Tianjin University Jiankun Foundation