Team:Aachen/Project/FRET Reporter
From 2014.igem.org
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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. | ||
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==REACh proteins - dark quenchers of GFP== | ==REACh proteins - dark quenchers of GFP== | ||
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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. | 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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==Producing a GFP_Reach Fusion Protein== | ==Producing a GFP_Reach Fusion Protein== | ||
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 was 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. | 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 was 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. | ||
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==Cutting the fusion protein with the TEV Protease== | ==Cutting the fusion protein with the TEV Protease== | ||
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- | = A Fluorescence Answer Faster Than Expression = | + | == A Fluorescence Answer Faster Than Expression == |
Biosensors often work with a system that is comprised of a reported gene under the control of a promoter that is induced directly by the chemical that the sensor is supposed to detect. In the case of our 2D biosensor for ''Pseudomonas aeruginosa'', the expression our reporter gene, GFP, would be directly induced by the quorum sensing molecules of the bacterium. However, transcription, translation, folding and post-translational modifications take their time. Since our goal is to detect the pathogen as fast as possible, we wanted to use a system that gives a fluorescent answer fast than just expressing the fluorescent protein. | Biosensors often work with a system that is comprised of a reported gene under the control of a promoter that is induced directly by the chemical that the sensor is supposed to detect. In the case of our 2D biosensor for ''Pseudomonas aeruginosa'', the expression our reporter gene, GFP, would be directly induced by the quorum sensing molecules of the bacterium. However, transcription, translation, folding and post-translational modifications take their time. Since our goal is to detect the pathogen as fast as possible, we wanted to use a system that gives a fluorescent answer fast than just expressing the fluorescent protein. | ||
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* While a certain concentration of homoserine lactone will produce the same number of gene read-outs, one TEV protease can cleave many GFP_REACh constructs. Through the cleavage step we therefore introduce an amplification step into our system. With the TEV protease, we will be able to produce a much stronger signal in a short time interval. | * While a certain concentration of homoserine lactone will produce the same number of gene read-outs, one TEV protease can cleave many GFP_REACh constructs. Through the cleavage step we therefore introduce an amplification step into our system. With the TEV protease, we will be able to produce a much stronger signal in a short time interval. | ||
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{{Team:Aachen/Footer}} | {{Team:Aachen/Footer}} |
Revision as of 12:42, 6 October 2014
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