Project I

Synthetic Bile Acid receptor in E.coli

Project II

Proteorhodopsin in mitochondrial inner membrane

Project I Synthetic Bile Acid receptor in E.coli

Crystal structure of LXRb ligand-binding domain (1P8D) and LacI-DNA (1EFA). The pink

region is fused to green part of lacI protein to generate lacI-LXR chimeric protein

Amino acid sequence of LacI-LXR chimeric protein.

The DNA-binding domain of LacI is boxed.

pET28 origin: pBR322, which is from pMB1

Fig3. pSB2k3-BBa_J04450 and pET28a-lacI-LXR constructs. In the dual-plasmid

assay, four lac operons are present.

RFP

LacI LaXR

ori

ori

Kan

IPTG BA

In BL21(DE3) cells

containing PET28-LacI-LXR and pSB1A3-RFP plasmids,

concentration of 3 compounds will influence RFP expression:

IPTG

Kanmycin

Bile acid

Gene worknet

Kanamycin

Binding site

16S rRNA

RFP

OD600

0

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1

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time(h)

OD600

0uM

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635(546)/OD600

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437(425)

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437(425)

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635(546)/437(425)

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time(h)

635(546)/437(425) 0uM

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635(546)/437(425)

0

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0 50 100 150 200 250

Compound B（uM）

635(5

46)/

437(4

25)

0uM Kanamycin

1uM Kanamycin

10uM Kanamycin

100uM Kanamycin

635(546)/437(425)

0.01

0.1

1

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0 50 100 150 200 250

Compound B（uM）

63

5(5

46

)/4

37

(42

5

25uM IPTG

50uM IPTG

75uM IPTG

100uM IPTG

Project I

Conclusion: Synthetic bile acid receptor is successfully

constructed in E.coli.

Project II Proteorhodopsin in mitochondrial inner membrane

《山海经·海外北经》：“夸父与日逐走入日渴欲得饮饮于河渭河渭不足北饮大泽未至道渴而死弃其杖化为邓林”

《山海经·大荒北经》：“大荒之中有山名曰成都载天有人珥两黄蛇把两黄蛇名曰夸父后

土生信信生夸父夸父不量力欲追日景逮之于禺谷将饮河而不足也将走大泽未至死于此应龙已杀蚩尤又杀夸父乃去南方处之故南方多雨”

《列子·汤问》：“夸父不量力欲追日影逐之于隅谷之际渴欲得饮赴饮河渭河渭不足将走北饮大泽未至道渴而死弃其杖尸膏肉所浸生邓林邓林弥广数千里焉”

http://baike.baidu.com/view/33954.htm

A Man pursuing

the Sun

from East to West

stopped at last.

Realizing a 50-year old dream, a millennium young myth

1920-1992

1978 Nobel

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Journal of Molecular Biology

Volume 213, Issue 4, 20 June 1990, Pages 899-929

Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-

microscopy* R. Henderson, J.M. Baldwin, T.A. Ceska†, F. Zemlin, E. Beckmann, K.H. Downing MRC Laboratory of Molecular Biology, Hills Road Cambridge CB2 2QH, U.K.

Fritz-Haber-Institut der Max-Planck-Gesellschaft Faradayweg 4-6, D-1000 Berlin 33, F.R.G.

Cell and Molecular Biology Division Lawrence Berkeley Laboratory Berkeley, CA 94720, U.S.A.

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Molecular evolution of rhodopsin family protein

by

Random and site-directed mutagenesis

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A new Biobrick device:

Mitochondrial proteorhodopsin

(humanized genetic code bias)

Partsregistry submission：

BBa_K603000 (pSB1AC3)

BBa_K603001 (pSB1C3)

Transient expression of mitochondrial proteorhodopsin and GFP

in human embryonic kidney cells HEK293

Light exposure increased level of transient GFP expression

in HEK293 cells co-transfected with mPR and GFP expression vectors

2% ATP is consumed by each heart beat, 90% ATP is from mitocondria.

Jacobus, W. E. (1985) Annu. Rev. Physiol. 47, 707-725

Project II

Conclusion:mPR(BBa_K603001)is a functional light-driven

proton pump in human mitochondria.

Acknowledgement:

Prof. Ching Song Doc. Huai Zhang Wen-qing Yao

BBa_K572005 Proteorhodopsin

S: Proteorhodopsin D: Proteorhodopsin, a photon dependent proton pump, is a

photoactive retinylidene protein. A transmembrane protein homologous to

bacteriorhodopsin in Archaea creates a proton gradient across the cell membrane in

many marine organisms. It has been demonstrated that photophosphorylation occurs

with the help of transmembrane ATP Synthase on expression of Proteorhodopsin gene

in Escherichia coli* *Martinez A, Bradley AS, Waldbauer JR, Summons RE, DeLong EF

(2007). "Proteorhodopsin photosystem gene expression enables photophosphorylation

in a heterologous host". PNAS 104 (13): 5590–5595. Bibcode 2007PNAS..104.5590M.

doi:10.1073/pnas.0611470104. PMC 1838496. PMID 17372221.

BBa_K572006 Proteorhodopsin Generator

S: Proteorhodopsin Generator D: To study the expression and function of

proteorhodopsin under various promoters

Designed by Avaneesh T Group: iGEM11_IIT_Madras (2011-09-18)

BBa_I711033 Proteorhodopsin (light-activated proton pump)

S: Proteorhodopsin (light-activated proton pump)

BBa_I711040 proteorhodopsin generator

S: proteorhodopsin generator

Designed by Ranjan Khan Group: iGEM07_Virginia (2007-10-09)