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 68kDa tetramer enzyme with the E.coli homolog name as uidA. It is commonly used as a reporter system for various experimental measurements. <a href="url">http://parts.igem.org/Part:BBa_K1216000</a>

Alkaline phosphatase (PhoA)

The alkaline phosphatase is a periplasmic homodimeric hydrolase. Each monomer contains 429 amino acids.

Acetylesterase (Aes)

β-N-Acetylglucosaminidase (NagZ)

nagZ encodes β-N-Acetylglucosaminidase, a cytoplasmatic hydrolase which is involved in the murein tripeptide recycling pathway of E.coli

β-Galactosidase (LacZ)

References

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