Team:Aachen/Project/FRET Reporter
From 2014.igem.org
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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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+ | ''Add: [Fig. 6 Scheme of a traditional biosensor.]'' | ||
Instead of the traditional approach, we '''constitutively express our reporter gene in its quenched form'''. As GFP_REACh fusion protein, fluorescence is suppressed. Our biosensor gives a response when homoserine lactones of ''Pseudomonas aeruginosa'' are taken up by our sensor cells where the '''autoinducer activates the expression of the TEV protease''' by binding to the LasI promoter in front of the protease gene. | Instead of the traditional approach, we '''constitutively express our reporter gene in its quenched form'''. As GFP_REACh fusion protein, fluorescence is suppressed. Our biosensor gives a response when homoserine lactones of ''Pseudomonas aeruginosa'' are taken up by our sensor cells where the '''autoinducer activates the expression of the TEV protease''' by binding to the LasI promoter in front of the protease gene. | ||
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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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+ | ''Add: [Fig. 7 Diagram of the expression levels/signal strength over time]'' | ||
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Revision as of 14:20, 6 October 2014
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