2 Jiang Y, Lee A, Chen J, Ruta V, Cadene M, Chait BT, et al. X-ray - PowerPoint PPT Presentation

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Jiang Y, Lee A, Chen J, Ruta V, Cadene M, Chait BT, et al. X-ray structure of a voltage-dependent K+ channel. Nature. 3 2003 05/01;423(6935):33-41.

PDZ Domain PDZ Ligand + Periplasm (GGGGS)3 Linkers Voltage Sensor + + + + + + Peptide Inner Polyproline Membrane Linkers - (GGGGS) 3 Linkers Effectors Effectors Cytosol 4

PDZ Domain PDZ Ligand + Periplasm (GGGGS)3 Linkers Voltage Sensor + + + + + + Peptide Inner Polyproline Membrane Linkers - (GGGGS) 3 Linkers Effectors Effectors Cytosol 5

+ Periplasm + + + + + + Inner Membrane - Cytosol 6

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- + 8

We have made: • BBa_K1092006: Voltage Sensor Peptide Voltage Sensor Peptide RBS Voltage Sensor Peptide 180bp 9

We have made: • BBa_K1092008: ssDsbA-PDZ Domain-Voltage Sensor Peptide PDZ Domain Voltage Sensor Peptide • BBa_K1092024: T7-RBS-ssDsbA-PDZ Domain-VS-RFP C terminus RBS PDZ Domain Voltage Sensor Peptide RFP C terminus 10

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Acenaphthene Pyrene Acenaphthylene Fluorene Anthracene Benzo[ a ]pyrene 13

• Found in car exhaust, tar, cooking fume... Benzo[ a ]pyrene (BaP) 14 Pictures from http://chemistry.about.com/od/factsstructures/ig/Chemical-Structures---B/Benzo-a-pyrene.htm

Human Body • Benzo[ a ]pyrene Diol Epoxide (Carcinogenic!) 15 Pictures from http://en.wikipedia.org/wiki/Benzo(a)pyrene

• Enzymes: Laccase and dioxygenase – Inspired by a fungal pathway [1] Dangerous Safe Catechol 1,2-Dioxygenase Laccase Benzo[ a ]pyrene BaP-1,6-quinone 1-Hydroxy-2-naphthoic acid 1. Hadibarata T, Kristanti RA. Identification of metabolites from benzo[ a ]pyrene oxidation by ligninolytic enzymes of 16 Polyporus sp. S133. J Environ Manage. 2012 11/30;111(0):115-9.

Plasmid Restriction Digestion Gel Photo • BBa_K1092005 – T7 – RBS – Laccase pSB1C3 Laccase RBS Laccase 1.5kbp (1542bp) 1kbp 750bp Laccase 500bp 250bp Benzo[ a ]pyrene BaP-1,6-Quinone 100bp 17

Plasmid Restriction Digestion Gel Photo • BBa_K1092003 – T7 – RBS - Dioxygenase pSB1C3 RBS Catechol 1,2-Dioxygeanse Dioxygen 1kbp -ase Catechol 1,2- 750bp (936bp) Dioxygenase 500bp 250bp 1-Hydroxy-2-naphthoic acid BaP-1,6-Quinone 100bp 18

0.20 OD 389 Value [1] 0.15 p ≤ 0.01 n = 3 ** 0.10 0.05 0.00 Control T T7 – Laccase e s W a c c 1. Method from: Ramesh A, Archibong AE, Niaz MS. Ovarian susceptibility to benzo[ a ]pyrene: tissue burden of a metabolites and DNA adducts in F-344 rats. Journal of Toxicology and Environmental Health, Part A. 2010 10/28; 19 2013/10;73(23):1611-25.

[1] 0.3 Normalized OD 298 Control EV T7-Dioxygenase T7-Dioxygenase 0.2 n = 3 p ≤ 0.05 0.1 0.0 0 40 80 120 Time / min 1. Method from: Huang Y, Xun R, Chen G, Xun L. Maintenance role of a glutathionyl-hydroquinone lyase (PcpF) in pentachlorophenol degradation by Sphingobium chlorophenolicum ATCC 39723. Journal of Bacteriology. 2008 20 December 01;190(23):7595-600.

• Repressor: QsrR – Quinone-mediated transcriptional repressor [1] Cys-5 Cys-5 Quinone BaP-1,6-quinone QsrR QsrR Downstream Genes (Regulated by QsrR) QsrR Binding Site mRNA 1. Ji Q, Zhang L, Jones MB, Sun F, Deng X, Liang H, et al. Molecular mechanism of quinone signaling mediated through S- quinonization of a YodB family repressor QsrR. Proceedings of the National Academy of Sciences. 2013 March 21 26;110(13):5010-5.

Plasmid Restriction Digestion Gel Photo • Biobricks – BBa_K1092001: T7 – RBS – QsrR pSB1C3 RBS QsrR 1kbp – BBa_K1092014: Constitutive Promoter (BBa_J23100) – RBS – 750bp QsrR Bind Site – Dioxygenase 500bp QsrR (339bp) 250bp 100bp QsrR Binding RBS Catechol 1,2- Site Dioxygenase 22

PDZ Voltage Sensor RBS Laccase Ligand Peptide PDZ Voltage Sensor QsrR Binding Site RBS Dioxygenase Domain Peptide Catechol 1,2-Dioxygenase Laccase Benzo[ a ]pyrene BaP-1,6-quinone 1-Hydroxy-2-naphthoic acid 23

+ + + + + + + - Laccase Dioxygenase Arai R, Ueda H, Kitayama A, Kamiya N, Nagamune T. Design of the linkers which effectively separate domains of a 24 bifunctional fusion protein. Protein Engineering. 2001 August 01;14(8):529-32.

- + Dioxygenase Laccase Arai R, Ueda H, Kitayama A, Kamiya N, Nagamune T. Design of the linkers which effectively separate domains of a 25 bifunctional fusion protein. Protein Engineering. 2001 August 01;14(8):529-32.

BBa_K1092000 QsrR BBa_K1092001 T7-RBS-QsrR BBa_K1092002 Dioxygenase BBa_K1092003 T7-RBS-Dioxygenase BBa_K1092004 Laccase BBa_K1092005 T7-RBS-Laccase BBa_K1092006 Voltage Sensor Peptide BBa_K1092007 ssDsbA-PDZ Ligand-Voltage Sensor Peptide BBa_K1092008 ssDsbA-PDZ Domain-Voltage Sensor Peptide BBa_K1092013 T7-RBS-Voltage Sensor Peptide BBa_K1092014 T7-QsrR Binding Site-RBS-Dioxygenase BBa_K1092024 pSB1C3-T7-RBS-ssDsbA-PDZ Domain-Voltage Switch-RFP C terminus BBa_K1092105 RFP N terminus BBa_K1092106 RFP C terminus 26

PDZ Voltage Sensor RBS Laccase Ligand Peptide PDZ Voltage Sensor QsrR Binding Site RBS Dioxygenase Domain Peptide 27

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• Biomolecular Fluorescent Complementation (BiFC). Fluorescent proteins were splited at loop • regions at DNA level, and coexpressed. A few fluorescent signals are found. • Fluorescent signals are enhanced when linked to interacting proteins pair. Used widely to study protein-protein • interaction. No documented reversible BiFC up until • now. 29 Kodama, Y. & Hu, C.-D. BioTechniques. 53, 285 – 98 (2012).

Promoter RBS PDZ domain RFP-N terminus Voltage PDZ ligand Voltage RFP-C terminus sensor sensor Terminator peptide peptide + - 1 s 1 s 1 s 30

• BiFC • Target protein to the corresponding membrane (e.g. ER, Golgi) • The system works on its own base on the native membrane potential Signaling peptide Promoter RBS PDZ domain RFP-N terminus Voltage PDZ ligand Voltage RFP-C terminus sensor sensor Terminator peptide peptide 31

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+ - + Adapted from: Huang, L. J. et al. The Journal of cell biology 145, 951 – 9 (1999). 33

Reversible BiFC Degrade Much Cellular More Content Voltage Switch Study Bacterial Protein Screen Complements Biosafety Control 34

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CUHK Human Practice International Local Undergraduate Macau Others* High School Students (60 students) (110 students) (135+234students) (110 students) Lab Workshop Survey Lab Workshop Poster Exhibition Synthetic Biology Synthetic Biology Survey Talk Talk Survey Survey Open Day Others* = Australia, Austria, Belgium, Canada, China, Denmark, England, Finland, France, Germany, Hong Kong, India, Indonesia, South 36 Korea, Malaysia, Mexico, Russia, Singapore, South Africa, Taiwan, Thailand, The Netherlands, Ukraine, USA, Vietnam

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Dangerous 5% Neither good or bad Interesting 11% 30% Interesting and Useful 23% Useful 31% 38

High School Students (Hong Kong) High School Students (Macau) University Students (Hong Kong) University Students (International) 70% 60% Percentage of Respondents 50% 40% 30% 20% 10% 0% iGEM Synthetic Both Neither only Biology only 39

5 5 Mean Degree of Acceptability 3.84 4 4 3.71 3.62 3.37 3 3 2 2 1 1 0 0 Believers Non Believers Believers Non Believers 40

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iGEM only 11% Neither Synthetic 14% Biology 9% Both 66% 44

No 5% Yes 95% 45

• Comics convey message about iGEM and synthetic biology well • Views on synthetic biology directly relates to religion’s view • Many agree that synthetic biology will be an integral part of knowledge-based economy • Future Plan: – Wider outreach and full detailed comics in English and Chinese languages 46

Research Group of Prof. School of Life Sciences, CUHK • • CHAN King Ming School of Biomedical • Research Group of Prof. Engineering, CUHK • CHAN Ting Fung University Safety Office, • Research Group of Prof. NGO CUHK • Chi Ki, Jacky Shanghai Jiao Tong • Prof. KONG Siu Kai University • Prof. GE Wei • Prof. JIANG Liwen • Jacky Loo • Qin Hao • Sunny • Nelson So • 47