View
3
Download
0
Category
Preview:
Citation preview
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
RFP
OD600
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0 2 4 6 8
time(h)
OD600
0uM
2.5uM
12.5uM
25uM
50uM
100uM
200uM
635(546)/OD600
0
100
200
300
400
500
600
700
800
900
1000
0 2 4 6 8
time(h)
635(546)/OD600 0uM
2.5uM
12.5uM
25uM
50uM
100uM
200uM
437(425)
0
100
200
300
400
500
600
700
800
0 2 4 6 8
time(h)
437(425)
0uM
2.5uM
12.5uM
25uM
50uM
100uM
200uM
635(546)/437(425)
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
0 2 4 6 8
time(h)
635(546)/437(425) 0uM
2.5uM
12.5uM
25uM
50uM
100uM
200uM
635(546)/437(425)
0
0.5
1
1.5
2
2.5
3
3.5
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
10
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
1961
1967
1973
1976
1984
1990
1993
1995
1998
2000
2007
2011
1961
1967
1973
1976
1984
1990
1993
1995
1998
2000
2007
2011
1961
1967
1973
1976
1984
1990
1993
1995
1998
2000
2007
2011
1961
1967
1973
1976
1984
1990
1993
1995
1998
2000
2007
2011
1961
1967
1973
1976
1984
1990
1993
1995
1998
2000
2007
2011
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.
1961
1967
1973
1976
1984
1990
1993
1995
1998
2000
2007
2011
Molecular evolution of rhodopsin family protein
by
Random and site-directed mutagenesis
1961
1967
1973
1976
1984
1990
1993
1995
1998
2000
2007
2011
1961
1967
1973
1976
1984
1990
1993
1995
1998
2000
2007
2011
1961
1967
1973
1976
1984
1990
1993
1995
1998
2000
2007
2011
1961
1967
1973
1976
1984
1990
1993
1995
1998
2000
2007
2011
1961
1967
1973
1976
1984
1990
1993
1995
1998
2000
2007
2011
1961
1967
1973
1976
1984
1990
1993
1995
1998
2000
2007
2011
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)
Recommended