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. 2003 05/01;423(6935):33-41. 3

PDZ Domain PDZ Ligand

+

-

+ + +

Periplasm

Inner Membrane

Cytosol

Voltage Sensor Peptide

Polyproline Linkers

(GGGGS)3 Linkers

Effectors

+ + + (GGGGS)3 Linkers

Effectors

4

PDZ Domain PDZ Ligand

+

-

+ + +

Periplasm

Inner Membrane

Cytosol

Voltage Sensor Peptide

Polyproline Linkers

(GGGGS)3 Linkers

Effectors

+ + + (GGGGS)3 Linkers

Effectors

5

+

-

Periplasm

Inner Membrane

Cytosol

6

+ + +

+ + +

+

-

7

+ + +

+ + +

8

+

-

We have made:

• BBa_K1092006: Voltage Sensor Peptide

Voltage Sensor Peptide RBS

9

180bp

Voltage Sensor Peptide

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

11

12

Acenaphthene Pyrene

Acenaphthylene Fluorene

Anthracene Benzo[a]pyrene

13

• 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)

14

• Benzo[a]pyrene Diol Epoxide

(Carcinogenic!)

Human Body

Pictures from http://en.wikipedia.org/wiki/Benzo(a)pyrene 15

• 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

• 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

17

• 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

18

WT

T7-Laccase

0.00

0.05

0.10

0.15

0.20

**

OD

398

Control T7 – Laccase

OD

38

9 V

alu

e [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. 19

0 40 80 1200.0

0.1

0.2

0.3EV

T7-Dioxygenase

Time / min

No

rmali

zed

OD

298

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

• 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.

Cy

s-5

QsrR

QsrR Binding Site Downstream Genes (Regulated by QsrR)

Quinone

Cy

s-5

QsrR

mRNA

21

BaP-1,6-quinone

• 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

22

QsrR Binding Site RBS PDZ

Domain Voltage Sensor

Peptide Dioxygenase

23

RBS PDZ Ligand

Voltage Sensor Peptide

Laccase

Laccase

Catechol 1,2-Dioxygenase

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

+

-

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. 24

+ + +

+ + +

Laccase Dioxygenase

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. 25

+

-

Laccase Dioxygenase

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

QsrR Binding Site RBS PDZ

Domain Voltage Sensor

Peptide Dioxygenase

RBS PDZ Ligand

Voltage Sensor Peptide

Laccase

27

28

• 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). 29

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

30

- +

• BiFC

• Target protein to the corresponding membrane (e.g. ER, Golgi)

• The system works on its own base on the native membrane potential

31

RBS PDZ domain PDZ ligand Voltage sensor peptide

Voltage sensor peptide

RFP-N terminus RFP-C terminus

Terminator

Promoter

Signaling peptide

32

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

33

+

+

-

Voltage Switch

Reversible BiFC

DegradeCellular Content

Bacterial Screen

Biosafety Control

Study Protein

Complements

Much More

34

35

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 36

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

37

Interesting 30%

Useful 31%

Interesting and Useful

23%

Neither good or bad 11%

Dangerous 5%

38

0%

10%

20%

30%

40%

50%

60%

70%

iGEMonly

SyntheticBiology

only

Both Neither

Pe

rce

nta

ge

of

Re

spo

nd

en

ts

High School Students (Hong Kong) High School Students (Macau)

University Students (Hong Kong) University Students (International)

39

3.71 3.84

0

1

2

3

4

5

Believers Non Believers

3.37 3.62

0

1

2

3

4

5

Believers Non Believers

Me

an

De

gre

e o

f A

cce

pta

bil

ity

40

41

42

43

iGEM only 11%

Synthetic Biology

9%

Both 66%

Neither 14%

44

Yes 95%

No 5%

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. 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

47

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

49