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 P_LuxR 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 68 kDa tetrameric 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 51 kDa. See more details in the part registry for phoA [http://parts.igem.org/Part:BBa_K1216001 click here]

Acetyl esterase (aes)

This enzyme is a 36 kDa monomer that is present in the cytosol. It catalyses hydrolysis of p-nitrophenyl esters of fatty acids. The enzyme is involved in the regulation of the maltose operon. See more details in the part registry for aes [http://parts.igem.org/Part:BBa_K1216002 click here]

β-N-Acetylglucosaminidase (nagZ)

The β-N-Acetylglucosaminidase enzyme from Escherichia Coli encoded by the nagZ gene, is a cytoplasmatic hydrolase which is involved in the cell wall recycling pathway. This enzyme is also of therapeutic interest as it is involved in the hyperproduction of the AmpC β-lactamase in gram-negative bacteria [http://www.cazypedia.org/index.php/Glycoside_Hydrolase_Family_3 1]. See more details in the part registry for nagZ [http://parts.igem.org/Part:BBa_K1216003 click here]

β-Galactosidase (LacZ)

References

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