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 acetyl esterase, 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 (acetyl esterase) 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.

Acetyl esterase

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). According to Kobayashi et al. (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.
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.

Alkaline phosphatase

Alkaline phosphatase is a non-specific periplasmic phosphomonoesterase that catalyzes production of free inorganic phosphate and phosphoryl transfer reaction to various alcohols. In Escherichia coli, this hydrolase is involved in the acquisition of phosphate from esters when free inorganic phosphate is scarce (Kim). It has been found that Escherichia coli alkaline phosphatase also catalyzes the oxidation of phosphite to phosphate and molecular H2 (Yang).
This hydrolase is a homodimeric metalloenzyme with two Zn2+ and on Mg2+ forming the metal triplet at each active site, which is considered to be Asp101-Ser102-Ala103. It has been shown that with p-nitrophenyl phosphate (pNPP) as a substrate, this enzyme has a specific activity of 1020 µmol pNPP h-1 mg-1 at pH 8; and its kcat and Km are 13.6 s-1 and 7.4 µM, respectively (Kim).
In Colisweeper, the alkaline phosphatase used for colorimetric enzyme-substrate reaction is natively expressed in the Escherichia coli strain used. However, we made use of the Citrobacter phoA gene ([http://parts.igem.org/Part:BBa_K1216001 K1216001]) for a kinetics assay of this enzyme. Additionally, we have added an improved version of this part to the registry ([http://parts.igem.org/Part:BBa_K1216005 K1216005]), with TEV and HIS-tags added to enable protein purification.

β-Galactosidase (LacZ)

β-Glucuronidase (gusA)

The enzyme β-glucuronidase catalyzes the hydrolysis of D-glucuronic acids which are conjugated through a β-O-glycosidic linkage to an aglycone. The action of bacterial β-glucuronidase in the gastrointestinal tract is an important component in the enterohepatic circulation of many hydrophobic xenobiotics and endogenous waste compounds, which are conjugated to D-glucuronic acid during the main detoxification pathway of vertebrates, and are then excreted in the bile or urine (Russ). The reduction of these beta-d-glucuronides by β-glucuronidase activity frees aglycone residues with protective effects, such as lignans, flavonoids, ceramide and glycyrrhetinic acid (Beaud), enabling their absorption and enterohepatic circulation.
β-Glucuronidase is commonly used as a reporter system for various experimental measurements, and has been favoured as a reporter gene in plants. In contrast to β-galactosidase, which is commonly used a reporter protein, β-glucuronidase has a key advantage in its robustness and its absence in many organisms other than vertebrates and their attendant microflora, rendering the possibility of accurate measurements of β-glucuronidase activity. Lower and higher plants, most bacteria, fungi and many insects are largely lacking activity of this enzyme (Jeff).
Our part [http://parts.igem.org/Part:BBa_K1216000 K1216000] encodes the Bacillus subtilis β-Glucuronidase, a 68 kDa tetrameric and intracellular enzyme with the Escherichia coli homolog name as uidA.

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 (Cheng). The enzyme is active against the beta-1,4-glycosidic bonds and releases beta-N-acetylglucosamine (Yem). Inhibitors of NagZ are N-acetylglucosamine and N-acetylmuramic acid at high concentrations, or N-acetylglucosaminolactone (Yem). Using p-nitrophenyl-beta-N-acetylglucosaminide as a substrate, a kinetic assay by Vötsch and Templin (Vötsch) 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.

References

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