Team:Aachen/Project/FRET Reporter
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However, fluorescence is not an essential requirement for FRET. This type of energy transfer can also be obersved between donors that are capable of other forms of radiation, such as phosphorescence, bioluminescence or chemiluminescence, and fit acceptors. Acceptor chromophores do not necessarily emit the energy in form of light, and can lead to quenching. Thus, this kind of acceptors are also referred to as dark quenchers. In our project, we use a FRET system with a dark quencher, namely our Reach construct. | However, fluorescence is not an essential requirement for FRET. This type of energy transfer can also be obersved between donors that are capable of other forms of radiation, such as phosphorescence, bioluminescence or chemiluminescence, and fit acceptors. Acceptor chromophores do not necessarily emit the energy in form of light, and can lead to quenching. Thus, this kind of acceptors are also referred to as dark quenchers. In our project, we use a FRET system with a dark quencher, namely our Reach construct. | ||
- | == | + | ==REACh proteins - dark quenchers of 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. | 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. | ||
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. | 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. | ||
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+ | In our project, we reproduced the REACh1 and REACh2 proteins by subjecting YFP to a QuikChange mutation. Subsequently, we fused each REACh protein with wild-type GFP. The protein complex is linked via a protease cleavage site. Therefore, when GFP is connected to either REACh quencher, GFP will absorb light but the energy will be transferred to REACh via FRET and then emitted in the form of heat. However, when the complex is cleaved by a protease, REACh will be separated from GFP. The latter will then be able to absorb and emit light as usual. | ||
==Producing a GFP_Reach Fusion Protein== | ==Producing a GFP_Reach Fusion Protein== |
Revision as of 10:51, 6 October 2014
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