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Team:ETH Zurich/Experiments 3
From 2013.igem.org
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[[File:Expectedsubstrate.png|left|180px|thumb|<b>Figure 6.2: Expected colorimetric response</b>]] | [[File:Expectedsubstrate.png|left|180px|thumb|<b>Figure 6.2: Expected colorimetric response</b>]] | ||
<p>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.</p> | <p>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.</p> | ||
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Revision as of 12:26, 16 September 2013
Contents |
Enzyme-substrate reactions
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We have cloned fluorescent receiver systems as backup for our circuit in case the hydrolase reaction do not work properly.
The enzyme substrate reactions take less than 5 minutes and are visible by eye.
Our minesweeper is becoming better and better so keep on track for updates !
| 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.
