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Team:ETH Zurich/Experiments 7
From 2013.igem.org
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<h1>What about those <i>hydrolases</i> ?</h1> | <h1>What about those <i>hydrolases</i> ?</h1> | ||
| - | <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 ''Escherichia coli''. These hydrolases include the ''Citrobacter'' alkaline phospohatase, the ''Bacillus subtilis'' β-glucuronidase and the ''Escherichia coli'' acetylesterase, N- | + | <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 ''Escherichia coli''. These hydrolases include the ''Citrobacter'' alkaline phospohatase, the ''Bacillus subtilis'' β-glucuronidase and the ''Escherichia coli'' acetylesterase, β-N-Acetylglucosaminidase and β-galactosidase. In order to prevent background expression of these native hydrolases in ''Escherchia coli'', we use a triple knockout strain that has three hydrolase genes knocked out: ''gusA'' (β-glucuronidase), ''aes'' (acetylesterase) and ''nagZ'' (β-N-Acetylglucosaminidase). Addition of a multi-substrate mix by the player leads to an enzyme-susbtrate reaction which specifically cleaves the chromogenic substrates, thereby producing a visible color output. <br> |
</p> | </p> | ||
<br clear="all"/> | <br clear="all"/> | ||
<h1>Alkaline phosphatase</h1> | <h1>Alkaline phosphatase</h1> | ||
| + | <p align="justify"></p> | ||
<h1>β-Glucuronidase</h1> | <h1>β-Glucuronidase</h1> | ||
| + | <p align="justify"></p> | ||
<h1>Acetylesterase</h1> | <h1>Acetylesterase</h1> | ||
| - | <h1>N- | + | <p align="justify"></p> |
| + | <h1>β-N-Acetylglucosaminidase (<i>nagZ</i>)</h1> | ||
| + | <p align="justify">β-N-Acetylglucosaminidase is a cytoplasmatic hydrolase which is involved in the formation of monosaccharides from the released disaccharides during the murein tripeptide recycling pathway of <i>Escherichia coli</i>. | ||
| + | |||
| + | </p> | ||
<h1>β-Galactosidase</h1> | <h1>β-Galactosidase</h1> | ||
| + | <p align="justify"></p> | ||
<br clear="all"/> | <br clear="all"/> | ||
{{:Team:ETH_Zurich/templates/footer}} | {{:Team:ETH_Zurich/templates/footer}} | ||
Revision as of 17:19, 16 September 2013
Contents |
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 Escherichia coli. These hydrolases include the Citrobacter alkaline phospohatase, the Bacillus subtilis β-glucuronidase and the Escherichia coli acetylesterase, β-N-Acetylglucosaminidase and β-galactosidase. In order to prevent background expression of these native hydrolases in Escherchia coli, we use a triple knockout strain that has three hydrolase genes knocked out: gusA (β-glucuronidase), aes (acetylesterase) and nagZ (β-N-Acetylglucosaminidase). Addition of a multi-substrate mix by the player leads to an enzyme-susbtrate reaction which specifically cleaves the chromogenic substrates, thereby producing a visible color output.
Alkaline phosphatase
β-Glucuronidase
Acetylesterase
β-N-Acetylglucosaminidase (nagZ)
β-N-Acetylglucosaminidase is a cytoplasmatic hydrolase which is involved in the formation of monosaccharides from the released disaccharides during the murein tripeptide recycling pathway of Escherichia coli.
β-Galactosidase
