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Team:ETH Zurich/Experiments 3
From 2013.igem.org
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<td>Magenta butyrate</td> | <td>Magenta butyrate</td> | ||
<td>5-bromo-6-chloro-3-indoxyl butyrate</td> | <td>5-bromo-6-chloro-3-indoxyl butyrate</td> | ||
| - | <td style="color:Magenta">Magenta[[Image:lila.png| | + | <td style="color:Magenta">Magenta[[Image:lila.png|100px|left|thumb|]]</td> |
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Revision as of 17:57, 25 September 2013
Contents |
Enzyme-substrate reactions
The enzyme substrate reactions take less than 5 minutes and are visible by eye.
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In Figure 1 you can see the different hydrolases we use.
| 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.
