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Team:ETH Zurich/Experiments 3
From 2013.igem.org
Contents |
Enzyme-substrate reactions
As our reporter system, we use orthogonal hydrolase enzymes that react with specific substrates to give a chromogenic product. The color produced from the enzyme-substrate reaction gives the cue for the player to carry out the next logical move. The player adds a multi-substrate solution on to colonies on the agar mine-field. In case the player is certain of a colony to be a mine colony, another second specific substrate is added. The substrates react with the enzymes to give the color in couple of minutes and are visible by eye.
Our minesweeper is becoming better and better so keep on track for updates !
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In Figure 1 The hydrolases are given below and also.
| Hydrolase | Complementary substrate / IUPAC name | Visible color | Concentration[M] | Time(s)for colorimetric response | ||
|---|---|---|---|---|---|---|
| LacZ | Beta-Galactosidase | X-Gal | 5-Bromo-4chloro-3-indolyl-beta-galactopyranoside | Blue |
||
| LacZ | Beta-Galactosidase | Green-beta-D-Gal | N-Methyl-3-indolyl-beta-D_galactopyranoside | Green |
||
| GusA | Beta-glucuronidase | Magenta glucuronide | 6-chloro-3-indolyl-beta-D-glucuronide-cycloheylammonium salt | Red |
||
| PhoA | Alkaline phosphatase | pNPP | 4-Nitrophenylphosphatedi(tris) salt | Yellow |
||
| Aes | Acetyl esterase | Magenta butyrate | 5-bromo-6-chloro-3-indoxyl butyrate | Magenta |
||
| NagZ | Glycoside hydrolase | X-glucosaminide X-Glucnac | 5-bromo-4-chloro-3-indolyl-N-acetyl-beta-D-glucosaminide | Blue | ||
Beta-galactosidase(LacZ)
Chemical conversion
Colorimetric response in liquid culture
Colorimetric response on LB-Agar
Alkaline phosphatase(PhoA)
Chemical conversion
Colorimetric response in liquid culture
Colorimetric response on LB-Agar
Acetyl esterase(Aes)
Chemical conversion
Colorimetric response in liquid culture
Colorimetric response on LB-Agar
Beta-glucuronidase(GusA)
Chemical conversion
Colorimetric response in liquid culture
Colorimetric response on LB-Agar
β-N-Acetylglucosaminidase(NagZ)
Chemical conversion
Colorimetric response in liquid culture
Colorimetric response on LB-Agar
Do the substrates and enzymes cross-react ?
An enzyme-substrate test matrix (Figure 6) was established to test each substrate against each enzyme. The results were as expected (Figure 6.2) and no cross reaction is visible. The NagZ-X - glucosaminide X-Glunac reveal some difficulties in the liquid culture as well as on the agar plate.
