Freiburg07/report light sensor
From 2007.igem.org
Contents |
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.
 |
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.
PhyA Insert was digested with EcoRI and SpeI, the CFP containing plasmid pAR200-cJunCFP as well. Fhy1 Insert1 was digested with AscI and NheI, the YFP containing plasmid pAR200-YFP-Dummy (for N-terminal fusion) and pAR200-cFOS-YFP (for C-terminal fusion) as well.
Insert and Vector fragments were ligated.
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.
