Team:Aachen/Project/FRET Reporter
From 2014.igem.org
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=The REACh Construct= | =The REACh Construct= | ||
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+ | == A Fluorescence Answer Faster Than Expression == | ||
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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. | ||
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+ | ''Add: [Fig. 6 Scheme of a traditional biosensor.]'' | ||
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+ | 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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+ | This approach has two advantages: | ||
+ | * When ''Pseudomonas aeruginosa'' is detected by our cells, the reporter protein is already expressed and only waits to be cleaved off the REACh quencher. The cleavage reaction catalyzed by the TEV protease is a faster process than expression and correct folding of GFP. This way we hope for '''an earlier response''' by our sensor cells. | ||
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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. | ||
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+ | ''Add: [Fig. 7 Diagram of the expression levels/signal strength over time]'' | ||
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+ | {{Team:Aachen/BlockSeparator}} | ||
==The FRET (Förster Resonance Energy Transfer) System== | ==The FRET (Förster Resonance Energy Transfer) System== | ||
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Though quite popular in molecular biology, the TEV protease is not avaiable as a BioBrick yet. Hence, the Aachen team introduces a protease with anti-self cleavage mutation S219V and codon optimized for ''E. coli'' '''to the Parts Registry this year.''' | Though quite popular in molecular biology, the TEV protease is not avaiable as a BioBrick yet. Hence, the Aachen team introduces a protease with anti-self cleavage mutation S219V and codon optimized for ''E. coli'' '''to the Parts Registry this year.''' | ||
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{{Team:Aachen/Footer}} | {{Team:Aachen/Footer}} |
Revision as of 15:00, 6 October 2014
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