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 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).

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