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Team:ETH Zurich/Experiments 3
From 2013.igem.org
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| - | < | + | <h1>Enzyme-substrate reactions</h1> |
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<p>We have cloned fluorescent receiver systems as backup for our circuit in case the hydrolase reaction do not work properly.<br>The enzyme substrate reactions take less than 5 minutes and are visible by eye.<br><br> Our minesweeper become better and better so keep on track for updates !</p> | <p>We have cloned fluorescent receiver systems as backup for our circuit in case the hydrolase reaction do not work properly.<br>The enzyme substrate reactions take less than 5 minutes and are visible by eye.<br><br> Our minesweeper become better and better so keep on track for updates !</p> | ||
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<th colspan="2">Complementary substrate / IUPAC name</th> | <th colspan="2">Complementary substrate / IUPAC name</th> | ||
<th>Visible color</th> | <th>Visible color</th> | ||
| + | <th>Time(s)for colorimetric response</th> | ||
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| - | [[File:Magenta_butyrate_on_aes_t7_strain.JPG|left|148px|thumb|Fig.1: Magenta butyrate on Aes expressing colony]] | + | [[File:Magenta_butyrate_on_aes_t7_strain.JPG|left|148px|thumb|<b>Fig.1: Magenta butyrate on Aes expressing colony</b>]] |
| - | [[File:PNPP_on_phoA_t7_strain.JPG|left|200px|thumb|Fig.2: NPP on PhoA expressing colony]] | + | [[File:PNPP_on_phoA_t7_strain.JPG|left|200px|thumb|<b>Fig.2: NPP on PhoA expressing colony</b>]] |
| - | [[File:Salmon_glcUA_on_gusA_t7_strain.JPG |left|180px|thumb|Fig.3 Magenta glucuronide on GusA colony]] | + | [[File:Salmon_glcUA_on_gusA_t7_strain.JPG |left|180px|thumb|<b>Fig.3 Magenta glucuronide on GusA expressing colony</b>]] |
| - | [[File:XGlcNAc_on_nagZ_t7_strain.JPG|left|200px|thumb|Fig.4: X-Glucnac on NagZ colony]] | + | [[File:XGlcNAc_on_nagZ_t7_strain.JPG|left|200px|thumb|<b>Fig.4: X-Glucnac on NagZ expressing colony</b>]] |
| - | [[File:XGlcNAc_on_nagZ_t7_strain.JPG|left|200px|thumb|Fig.5: X-Gal on LacZ colony]] | + | [[File:XGlcNAc_on_nagZ_t7_strain.JPG|left|200px|thumb|<b>Fig.5: X-Gal on LacZ expressing colony</b>]] |
| - | [[File:XGlcNAc_on_nagZ_t7_strain.JPG|left|200px|thumb|Fig.5: Beta-green-X-gal on LacZ colony]] | + | [[File:XGlcNAc_on_nagZ_t7_strain.JPG|left|200px|thumb|<b>Fig.5: Beta-green-X-gal on LacZ expressing colony</b>]] |
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| - | [[File:Substratecrosstest.png|left|300px|thumb|Figure 6: Enzyme-substrate test matrix]] | + | [[File:Substratecrosstest.png|left|300px|thumb|<b>Figure 6.1: Enzyme-substrate test matrix</b>]] |
| - | [[File:Expectedsubstrate.png|left|280px|thumb|Figure | + | [[File:Expectedsubstrate.png|left|280px|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 | + | <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 16:01, 26 August 2013
Enzyme-substrate reactions
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 become better and better so keep on track for updates !
| Hydrolase | Complementary substrate / IUPAC name | Visible color | 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 | Carboxyl esterase | Magenta butyrate | 5-bromo-6-chloro-3-indoxyl butyrate | Magenta | |
| NagZ | Glycoside hydrolase | X-glucosaminide X-Glunac | 5-bromo-4-chloro-3-indolyl-N-acetyl-beta-D-glucosaminide | Blue | |
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.
