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

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

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2 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

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SLIDE 1
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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. 2003 05/01;423(6935):33-41.

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SLIDE 4

PDZ Domain PDZ Ligand

+

  • + + +

Periplasm Inner Membrane Cytosol

Voltage Sensor Peptide Polyproline Linkers (GGGGS)3 Linkers Effectors

+ + +

(GGGGS)3 Linkers Effectors 4

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SLIDE 5

PDZ Domain PDZ Ligand

+

  • + + +

Periplasm Inner Membrane Cytosol

Voltage Sensor Peptide Polyproline Linkers (GGGGS)3 Linkers Effectors

+ + +

(GGGGS)3 Linkers Effectors 5

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SLIDE 6

+

  • Periplasm

Inner Membrane Cytosol

6

+ + + + + +

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SLIDE 7

+

  • 7

+ + + + + +

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SLIDE 8

8

+

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SLIDE 9

We have made:

  • BBa_K1092006: Voltage Sensor Peptide

Voltage Sensor Peptide RBS 9

180bp

Voltage Sensor Peptide

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SLIDE 10

We have made:

  • BBa_K1092008:

ssDsbA-PDZ Domain-Voltage Sensor Peptide

  • BBa_K1092024:

T7-RBS-ssDsbA-PDZ Domain-VS-RFP C terminus

10 Voltage Sensor Peptide PDZ Domain

Voltage Sensor Peptide PDZ Domain RFP C terminus RBS

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SLIDE 12

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Acenaphthene Pyrene Acenaphthylene Fluorene Anthracene Benzo[a]pyrene

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  • Found in car exhaust, tar, cooking fume...

Pictures from http://chemistry.about.com/od/factsstructures/ig/Chemical-Structures---B/Benzo-a-pyrene.htm

Benzo[a]pyrene (BaP)

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  • Benzo[a]pyrene Diol Epoxide

(Carcinogenic!)

Human Body

Pictures from http://en.wikipedia.org/wiki/Benzo(a)pyrene

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  • Enzymes: Laccase and dioxygenase

– Inspired by a fungal pathway[1]

  • 1. Hadibarata T, Kristanti RA. Identification of metabolites from benzo[a]pyrene oxidation by ligninolytic enzymes of

Polyporus sp. S133. J Environ Manage. 2012 11/30;111(0):115-9.

Laccase

Catechol 1,2-Dioxygenase

Benzo[a]pyrene BaP-1,6-quinone 1-Hydroxy-2-naphthoic acid

Dangerous Safe 16

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SLIDE 17
  • BBa_K1092005

– T7 – RBS – Laccase

Laccase RBS

pSB1C3 Laccase (1542bp) Plasmid Restriction Digestion Gel Photo 1.5kbp 1kbp 750bp 500bp 250bp 100bp

Laccase

Benzo[a]pyrene BaP-1,6-Quinone

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  • BBa_K1092003

– T7 – RBS - Dioxygenase

Catechol 1,2-Dioxygeanse RBS

pSB1C3 Dioxygen

  • ase

(936bp) Plasmid Restriction Digestion Gel Photo 1kbp 750bp 500bp 250bp 100bp

Catechol 1,2- Dioxygenase BaP-1,6-Quinone 1-Hydroxy-2-naphthoic acid

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SLIDE 19

W T a c c a s e 0.00 0.05 0.10 0.15 0.20

**

Control T7 – Laccase

OD389 Value [1]

n = 3 p ≤ 0.01

  • 1. Method from: Ramesh A, Archibong AE, Niaz MS. Ovarian susceptibility to benzo[a]pyrene: tissue burden of

metabolites and DNA adducts in F-344 rats. Journal of Toxicology and Environmental Health, Part A. 2010 10/28; 2013/10;73(23):1611-25.

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40 80 120 0.0 0.1 0.2 0.3

EV T7-Dioxygenase

Time / min

Normalized OD298

n = 3 p ≤ 0.05

  • 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 December 01;190(23):7595-600.

[1] 20 Control T7-Dioxygenase

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  • Repressor: QsrR

– Quinone-mediated transcriptional repressor[1]

  • 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 26;110(13):5010-5.

Cys-5 QsrR

QsrR Binding Site

Downstream Genes (Regulated by QsrR) Quinone Cys-5 QsrR

mRNA

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BaP-1,6-quinone

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SLIDE 22
  • Biobricks

– BBa_K1092001: T7–RBS–QsrR – BBa_K1092014: Constitutive Promoter (BBa_J23100)–RBS– QsrR Bind Site–Dioxygenase

Catechol 1,2- Dioxygenase QsrR Binding Site RBS QsrR RBS

pSB1C3 QsrR (339bp)

Plasmid Restriction Digestion Gel Photo

1kbp 750bp 500bp 250bp 100bp

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QsrR Binding Site RBS PDZ Domain Voltage Sensor Peptide Dioxygenase

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RBS PDZ Ligand Voltage Sensor Peptide Laccase

Laccase

Catechol 1,2-Dioxygenase

Benzo[a]pyrene BaP-1,6-quinone 1-Hydroxy-2-naphthoic acid

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SLIDE 24

+

  • Arai R, Ueda H, Kitayama A, Kamiya N, Nagamune T. Design of the linkers which effectively separate domains of a

bifunctional fusion protein. Protein Engineering. 2001 August 01;14(8):529-32.

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+ + + + + +

Laccase Dioxygenase

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

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+

  • Laccase

Dioxygenase

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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

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SLIDE 27

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

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SLIDE 28

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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.

Kodama, Y. & Hu, C.-D. BioTechniques. 53, 285–98 (2012).

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Promoter RBS PDZ domain PDZ ligand Voltage sensor peptide Voltage sensor peptide RFP-N terminus RFP-C terminus Terminator

1 s 1 s 1 s

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  • +
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  • BiFC
  • Target protein to the corresponding membrane

(e.g. ER, Golgi)

  • The system works on its own base on the native

membrane potential

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RBS PDZ domain PDZ ligand Voltage sensor peptide Voltage sensor peptide RFP-N terminus RFP-C terminus Terminator Promoter Signaling peptide

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

33 +

+

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SLIDE 34

Voltage Switch

Reversible BiFC Degrade Cellular Content

Bacterial Screen Biosafety Control

Study Protein Complements

Much More

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Others* = Australia, Austria, Belgium, Canada, China, Denmark, England, Finland, France, Germany, Hong Kong, India, Indonesia, South Korea, Malaysia, Mexico, Russia, Singapore, South Africa, Taiwan, Thailand, The Netherlands, Ukraine, USA, Vietnam

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CUHK Human Practice International Macau (60 students) Lab Workshop Synthetic Biology Talk Survey Others* (110 students) Survey Local High School (135+234students) Lab Workshop Synthetic Biology Talk Survey Open Day Undergraduate Students (110 students) Poster Exhibition Survey

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Interesting 30% Useful 31% Interesting and Useful 23%

Neither good

  • r bad

11% Dangerous 5%

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0% 10% 20% 30% 40% 50% 60% 70%

iGEM

  • nly

Synthetic Biology

  • nly

Both Neither

Percentage of Respondents High School Students (Hong Kong) High School Students (Macau) University Students (Hong Kong) University Students (International)

39

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

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iGEM only 11% Synthetic Biology 9%

Both 66%

Neither 14%

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Yes 95%

No 5% 45

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  • 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

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  • Research Group of Prof.

CHAN King Ming

  • Research Group of Prof.

CHAN Ting Fung

  • Research Group of Prof. NGO

Chi Ki, Jacky

  • Prof. KONG Siu Kai
  • Prof. GE Wei
  • Prof. JIANG Liwen
  • Jacky Loo
  • Qin Hao
  • Sunny
  • Nelson So
  • School of Life Sciences, CUHK
  • School of Biomedical

Engineering, CUHK

  • University Safety Office,

CUHK

  • Shanghai Jiao Tong

University

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Arai R, Ueda H, Kitayama A, Kamiya N, Nagamune T. Design of the linkers which effectively separate domains of a bifunctional fusion protein. Protein Engineering. 2001 August 01;14(8):529-32. Hadibarata T, Kristanti RA. Identification of metabolites from benzo[a]pyrene oxidation by ligninolytic enzymes of Polyporus sp. S133. J Environ Manage. 2012 11/30;111(0):115-9. 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 December 01;190(23):7595-600.

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 26;110(13):5010-5. Jiang Y, Lee A, Chen J, Ruta V, Cadene M, Chait BT, et al. X-ray structure of a voltage- dependent K+ channel. Nature. 2003 05/01;423(6935):33-41. Kodama Y and Hu CD. Bimolecular fluorescence complementation (BiFC): A 5-year update and future perspectives. BioTechniques. 2012 November; 53(5): 285-298. Ramesh A, Archibong AE, Niaz MS. Ovarian susceptibility to benzo[a]pyrene: tissue burden of metabolites and DNA adducts in F-344 rats. Journal of Toxicology and Environmental Health, Part A. 2010 10/28; 2013/10;73(23):1611-25.

48

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