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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 | + | <p>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>β-Glucuronidase (GusA)</h1> | <h1>β-Glucuronidase (GusA)</h1> | ||
| - | <p | + | <p> |
| - | gusA encodes β-Glucuronidase, an intracellular enzyme that catalyzes the hydrolysis of β-D-glucuronides.This is a 68kDa tetramer enzyme with the E.coli homolog name as uidA. It is commonly used as a reporter system for various experimental measurements. See more details in the part registry for gusA[http://parts.igem.org/Part:BBa_K1216000 click here]</p> | + | gusA encodes β-Glucuronidase, an intracellular enzyme that catalyzes the hydrolysis of β-D-glucuronides.This is a 68kDa tetramer enzyme with the E.coli homolog name as uidA. It is commonly used as a reporter system for various experimental measurements. See more details in the part registry for gusA [http://parts.igem.org/Part:BBa_K1216000 click here]</p> |
<h1>Alkaline phosphatase (PhoA)</h1> | <h1>Alkaline phosphatase (PhoA)</h1> | ||
| - | <p | + | <p> |
| - | The alkaline phosphatase is a periplasmic homodimeric hydrolase. Each monomer contains 429 amino acids. This monomer is a dimer of 51kDa. See more details in the part registry for phoA[http://parts.igem.org/Part:BBa_K1216001 click here] </p> | + | The alkaline phosphatase is a periplasmic homodimeric hydrolase. Each monomer contains 429 amino acids. This monomer is a dimer of 51kDa. See more details in the part registry for phoA [http://parts.igem.org/Part:BBa_K1216001 click here] </p> |
<h1>Acetylesterase (Aes)</h1> | <h1>Acetylesterase (Aes)</h1> | ||
<h1>β-N-Acetylglucosaminidase (NagZ)</h1> | <h1>β-N-Acetylglucosaminidase (NagZ)</h1> | ||
| - | <p | + | <p> beta-N-Acetylglucosaminidase (nagZ) from Escherichia Coli |
nagZ encodes β-N-Acetylglucosaminidase, a cytoplasmatic hydrolase which is involved in the murein tripeptide recycling pathway of E.coli</p> | nagZ encodes β-N-Acetylglucosaminidase, a cytoplasmatic hydrolase which is involved in the murein tripeptide recycling pathway of E.coli</p> | ||
<h1>β-Galactosidase (LacZ)</h1> | <h1>β-Galactosidase (LacZ)</h1> | ||
| - | <p | + | <p></p> |
<br> | <br> | ||
Revision as of 08:30, 18 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.
β-Glucuronidase (GusA)
gusA encodes β-Glucuronidase, an intracellular enzyme that catalyzes the hydrolysis of β-D-glucuronides.This is a 68kDa tetramer enzyme with the E.coli homolog name as uidA. It is commonly used as a reporter system for various experimental measurements. See more details in the part registry for gusA [http://parts.igem.org/Part:BBa_K1216000 click here]
Alkaline phosphatase (PhoA)
The alkaline phosphatase is a periplasmic homodimeric hydrolase. Each monomer contains 429 amino acids. This monomer is a dimer of 51kDa. See more details in the part registry for phoA [http://parts.igem.org/Part:BBa_K1216001 click here]
Acetylesterase (Aes)
β-N-Acetylglucosaminidase (NagZ)
beta-N-Acetylglucosaminidase (nagZ) from Escherichia Coli nagZ encodes β-N-Acetylglucosaminidase, a cytoplasmatic hydrolase which is involved in the murein tripeptide recycling pathway of E.coli
β-Galactosidase (LacZ)
References
