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)

The Escherichia coli acetyl esterase is encoded by the aes gene and has been identified as a member of the hormone-sensitive lipase family (1). Acetyl esterase interferes with the expression of the maltose system by counteracting maltose sensitivity (2). This cytosolic enzyme is a 36 kDa monomer and catalyzes hydrolysis of short chain fatty esters with acyl chain lengths of up to eight carbons (1). It is composed of 319 amino acid residues (3) and its catalytic triad is composed of Ser165, Asp262 and His292 (4). Various substrates can used for chromogenic enzyme assays with this enzyme, e.g. p-nitrophenyl acetate (2), p-nitrophenyl butanoate (5) and p-nitrophenyl butyrate (6). Kinetic constants for the Escherichia coli acetyl esterase are K_m 170 ± 41 µM and k_cat 29 ± 3.0 s^-1 when using p-nitrophenyl butyrate as a substrate (6).
In Colisweeper, 5-Bromo-6-Chloro-3-indoxyl butyrate is used in the multi-mix substrate. After hydrolysis of this substrate, an indigo analog precipitates, which absorbs at 565 nm (visible as magenta color).
This enzyme's coding region has been added to the registry by our team as [http://parts.igem.org/Part:BBa_K1216002 K1216002]. We used this part in our final circuit and have made this our favorite part.

β-N-Acetylglucosaminidase (nagZ)

The β-N-Acetylglucosaminidase enzyme is a cytoplasmic hydrolase which is involved in the Escherichia coli cell wall recycling pathway. Its natural substrates are muropeptides and anhydro-muropeptides, which are released into the cytosol from cell wall murein (7). The enzyme is active against the beta-1,4-glycosidic bonds and releases beta-N-acetylglucosamine (8). Inhibitors of NagZ are N-acetylglucosamine and N-acetylmuramic acid at high concentrations, or N-acetylglucosaminolactone. Using p-nitrophenyl-beta-N-acetylglucosaminide as a substrate, a kinetic assay by Cheng et al. (8) showed a K_m 310 µM and V_max 13.3 µmol/min mg protein at 25°C.
We have added the coding region for β-N-Acetylglucosaminidase to the registry as [http://parts.igem.org/Part:BBa_K1216003 K1216003] and used this part for Colissweeper's final circuit in the mine cells.

β-Galactosidase (LacZ)

References

(1) Kanaya S, Koyanagi T, Kanaya E, Biochem. J., 332, 75-80 (1998).
(2) Peist R, Koch A, Bolek P, Sewitz S, Kolbus T, Boos J, J. Bacteriol., 179, 7679-7686 (1997).
(3) Blattner FR, Plunkett III G, Bloch CA, Perna NT, Burna V, Riley M, Collado-Vides J, Glasner JD, Rode CK, Mayhew GF, Gregor J, Davis NW, Kirkpatrick HA, Goeden MA, Rose DJ, Mau B, Shao Y, Science, 277, 1453-1461(1997).
(4) Haruki M, Oohashi Y, Mizuguchi S, Matsuo Y, Morikawa M, Kanaya S, FEBS Lett., 454, 262-266 (1999).
(5) Farias T, Mandrich L, Rossi M, Manco G, Protein Pept. Lett., 14, 65-69 (2007).
(6) Kobayashi R, Hirano N, Kanaya S, Haruki M, Biosci. Biotechnol. Biochem., 76 (11), 2082-2088 (2012).

(7) Cheng Q, Li H, Merdek K, Park JT, J. Bacteriol., 182, 4836-4840 (2000).
(8) Yem DW, Wu HC, J. Bacteriol., 125, 324-331 (1976).
(9) Vötsch W, Templin MF, J. Biol. Chem., 275, 39032-39038 (2000).