Team:ETH Zurich/Experiments 7

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<h1>What about those <i>hydrolases</i> ?</h1>
<h1>What about those <i>hydrolases</i> ?</h1>
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<p align="justify">Colisweeper depends largely on processing of molecular signals and generation of visible and distinguishable outputs accordingly. Next to our P<sub>LuxR</sub> promoter mutations, which detect ranges of OHHL concentrations, we made use of a reporter system which gives colorimetric responses and additionally provides that the visibility of output is only to be triggered by the player of Colisweeper: A set of orthogonal hydrolases which specifically cleave the chromogenic hydrolase substrates added by the player. <br>
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<p align="justify">Colisweeper depends largely on processing of molecular signals and generation of visible and distinguishable color outputs used as logic to play the game. Next to our P<sub>LuxR</sub> promoter mutations, which detect ranges of OHHL concentrations, we make use of a reporter system which gives colorimetric responses only to be triggered by the player. As our reporter system, we rely on a set of orthogonal hydrolase enzymes that are native to E.coli. In order to prevent background expression of these native hydrolases in E.coli, we use a triple knockout strain that have three hydrolase genes knocked out. Addition of a multi-substrate mix by the player leads to cleavage of the hydrolases which specifically cleave the chromogenic substrates, thereby producing a visible color output. <br>
These hydrolases include the ''Citrobacter'' alkaline phospohatase, the ''Bacillus subtilis'' β-glucuronidase and the ''Escherichia coli'' acetylesterase, N-acetyl-β-glucosaminidase and β-galactosidase. To ensure specific enzyme-substrate reactions, we used a triple deletion ''Escherichia coli'' strain, lacking ''aes'' (acetylesterase), ''gusA'' (β-glucuronidase) and ''nagZ'' (N-acetyl-β-glucosaminidase).</p>
These hydrolases include the ''Citrobacter'' alkaline phospohatase, the ''Bacillus subtilis'' β-glucuronidase and the ''Escherichia coli'' acetylesterase, N-acetyl-β-glucosaminidase and β-galactosidase. To ensure specific enzyme-substrate reactions, we used a triple deletion ''Escherichia coli'' strain, lacking ''aes'' (acetylesterase), ''gusA'' (β-glucuronidase) and ''nagZ'' (N-acetyl-β-glucosaminidase).</p>

Revision as of 10:24, 16 September 2013

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What about those hydrolases ?

Colisweeper depends largely on processing of molecular signals and generation of visible and distinguishable color outputs used as logic to play the game. Next to our PLuxR promoter mutations, which detect ranges of OHHL concentrations, we make use of a reporter system which gives colorimetric responses only to be triggered by the player. As our reporter system, we rely on a set of orthogonal hydrolase enzymes that are native to E.coli. In order to prevent background expression of these native hydrolases in E.coli, we use a triple knockout strain that have three hydrolase genes knocked out. Addition of a multi-substrate mix by the player leads to cleavage of the hydrolases which specifically cleave the chromogenic substrates, thereby producing a visible color output.
These hydrolases include the Citrobacter alkaline phospohatase, the Bacillus subtilis β-glucuronidase and the Escherichia coli acetylesterase, N-acetyl-β-glucosaminidase and β-galactosidase. To ensure specific enzyme-substrate reactions, we used a triple deletion Escherichia coli strain, lacking aes (acetylesterase), gusA (β-glucuronidase) and nagZ (N-acetyl-β-glucosaminidase).