Freiburg07/report light sensor
From 2007.igem.org
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Towards optical signal processing in E. coli
iGEM team Freiburg
Introduction
Phytochrome A (PhyA) is a member of the phytochrome family of red/far-red photoreceptors. These photoreceptors are involved in a wide range of developmental responses in plants. Fhy1 is a protein which binds PhyA under light conditions, enable us to use this protein-protein interaction. In plants Fhy1 is responsable for nuclear accumulation of PhyA in case of light exposure. In our case it wasn´t neccessary to use the full Fhy1 protein, that is why we restriced to the PhyA binding domain of Fhy1. We choose two different sizes of the Fhy1 fragment, a small version and a big one. Our goal was to show the interaction of both by performing a FRET, fusing CFP to PhyA and YFP to Fhy1.
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Materials and Methods
Cloning of constructs:
PhyA Insert was gained by PCR from plasmid D153ah-phyA using 5´ (5´-TATCGACGAA TTCGCGGCCG CTTCTAGAAA GAGGAGAAAT
TAACTATGTC AGGCTCTAGG CCGAC-3´) and 3´ (5´- AACGATCACT AGTTCCGCCG TGATGGTGAT GGTGGCCTCC GTTATCGAGT TCCACCTCCT TA-3´) primers.
Fhy1 Insert1 was gained by PCR from pGADT7 using 5´ (5´-TATCGACGCT AGCGATTACA TCTATGGGAC TCAGA-3´) and 3´
(5´-AACGATCGGC GCGCCCAGCA TTAGCGTTGA GAAGTAT-3´) primers.
Fhy1 Insert2 was gained by PCR from pGADT7 using 5´ (5´-TATCGACGCT AGCAGTAGTA ATGCTGCTAA GTTTGT-3`) and 3´
(5´-AACGATCGGC GCGCCCAGCA TTAGCGTTGA GAAGTAT-3´) primers.
Results
Discussion
References
Hiltbrunner A, Tscheuschler A, Viczian A, Kunkel T, Kircher S, Schäfer E. "FHY1 and FHL act together to mediate nuclear accumulation of the phytochrome A photoreceptor." Plant Cell Physiol. 2006 Aug;47(8):1023-34.
Hiltbrunner A, Viczian A, Bury E, Tscheuschler A, Kircher S, Toth R, Honsberger A, Nagy F, Fankhauser C, Schäfer E "Nuclear accumulation of the phytochrome A photoreceptor requires FHY1." Curr Biol. 2005 Dec 6;15(23):2125-30.
Gambetta GA, Lagarias JC "Genetic engineering of phytochrome biosynthesis in bacteria." Proc Natl Acad Sci U S A. 2001 Sep 11;98(19):10566-71.
