Team:Aachen/Project/FRET Reporter
From 2014.igem.org
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==The Reach proteins as quenchers for GFP== | ==The Reach proteins as quenchers for GFP== | ||
+ | In 2006, [http://www.pnas.org/content/103/11/4089.full Ganesan et al.] were the first to present a previously undescribed FRET acceptor, a non-fluorescent yellow fluorescent protein (YFP) mutant called REACh (for Resonance Energy-Accepting Chromoprotein). YFP can be used as a FRET acceptor in combination with GFP as the donor in FRET microscopy and miscellaneous assays in molecular biology. The ideal FRET couple should possess a large spectral overlap between donor emission and acceptor absorption but separated emission spectra to allow their selective imaging. | ||
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+ | To optimize the spectral overlap of this FRET pair, the group obtained a genetically modified YFP acceptor. Mutations of amino acid residues that stabilize the excited state of the chromophore in enhanced YFP (EYFP) resulted in a non-fluorescent chromoprotein. Two mutations, H148V and Y145W, reduced the fluorescence emission by 82 and 98 %, respectively. Ganesan et al. chose the Y145W mutant and the Y145W/H148V double mutant as FRET acceptors and named them REACh1 and REACh2, respectively. Both REACh1 and REACh2 act as dark quenchers of GFP. | ||
==Producing a GFP_Reach Fusion Protein== | ==Producing a GFP_Reach Fusion Protein== |
Revision as of 10:38, 6 October 2014
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