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Team:ETH Zurich/Experiments 3
From 2013.igem.org
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What about the hydolases ? How do they work and where do they come from ? Why do we use hydrolases ? | What about the hydolases ? How do they work and where do they come from ? Why do we use hydrolases ? | ||
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| - | <h1><b>Alkaline phosphatase</b> (PhoA)</h1> | + | <h1><b>Alkaline phosphatase</b>(PhoA)</h1> |
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[[File:PNPP_on_phoA_t7_strain.JPG|left|300px|thumb|<b>Fig.2: NPP on PhoA expressing colony</b>]] | [[File:PNPP_on_phoA_t7_strain.JPG|left|300px|thumb|<b>Fig.2: NPP on PhoA expressing colony</b>]] | ||
| + | Alkaline phosphatases are used as reporter enzymes in different assays such as Western Blotting and in situ hybridization[[Part:BBa_K1216001#References|<sup>[1]</sup>]]. Testing human blood for Alkaline Phosphatase levels is a routine test that can reveal different conditions[[Part:BBa_K1216001#References|<sup>[2]</sup>]]. | ||
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| + | Alkaline phosphatases cleave phosphate groups from organic compounds by hydrolysis while retaining stereochemistry[[Part:BBa_K1216001#References|<sup>[3]</sup>]]. | ||
| + | The explanation of the mechanism was found on the [http://chemwiki.ucdavis.edu/Organic_Chemistry/Organic_Chemistry_With_a_Biological_Emphasis/Chapter_10%3A_Phosphoryl_transfer_reactions/Section_10.3%3A_Hydrolysis_of__phosphates Chem Wiki ].<br> | ||
| + | Alkaline phosphatases, respectively their serum levels, are also related to several diseases e.g. metabolic myopathies and Paget Disease. [[Part:BBa_K1216001#References|<sup>[4]</sup>]] | ||
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<h1><b>Carboxyl esterase</b> (Aes)</h1> | <h1><b>Carboxyl esterase</b> (Aes)</h1> | ||
[[File:Magenta_butyrate_on_aes_t7_strain.JPG|left|248px|thumb|<b>Fig.1: Magenta butyrate on Aes expressing colony</b>]] | [[File:Magenta_butyrate_on_aes_t7_strain.JPG|left|248px|thumb|<b>Fig.1: Magenta butyrate on Aes expressing colony</b>]] | ||
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<h1><b>Glycoside hydrolase</b> (NagZ)</h1> | <h1><b>Glycoside hydrolase</b> (NagZ)</h1> | ||
[[File:XGlcNAc_on_nagZ_t7_strain.JPG|left|300px|thumb|<b>Fig.4: X-Glucnac on NagZ expressing colony</b>]] | [[File:XGlcNAc_on_nagZ_t7_strain.JPG|left|300px|thumb|<b>Fig.4: X-Glucnac on NagZ expressing colony</b>]] | ||
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<h1><b>Beta-glucuronidase</b> (GusA)</h1> | <h1><b>Beta-glucuronidase</b> (GusA)</h1> | ||
[[File:Salmon_glcUA_on_gusA_t7_strain.JPG |left|280px|thumb|<b>Fig.3 Magenta glucuronide on GusA expressing colony</b>]] | [[File:Salmon_glcUA_on_gusA_t7_strain.JPG |left|280px|thumb|<b>Fig.3 Magenta glucuronide on GusA expressing colony</b>]] | ||
Revision as of 13:08, 2 September 2013
Contents |
What about those hydrolases ?
We use hydrolases able to make a colorimetric response by converting a specific substrate. We will use 5 hydrolases : Beta-Galactosidase (lacZ),Beta-Glucuronidase (GusA); alkaline phosphatase (phoA);Glycoside hydrolase (NagZ) and the carboxyl esterase (aes).
On this page you will find all relevant informations about the hydrolases and enzyme substrate reaction based on papers and our results.
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 | 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 | 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 | ||
What about the hydolases ? How do they work and where do they come from ? Why do we use hydrolases ?
Beta-galactosidase (LacZ)
Alkaline phosphatase(PhoA)
Alkaline phosphatases are used as reporter enzymes in different assays such as Western Blotting and in situ hybridization[1]. Testing human blood for Alkaline Phosphatase levels is a routine test that can reveal different conditions[2].
Alkaline phosphatases cleave phosphate groups from organic compounds by hydrolysis while retaining stereochemistry[3].
The explanation of the mechanism was found on the [http://chemwiki.ucdavis.edu/Organic_Chemistry/Organic_Chemistry_With_a_Biological_Emphasis/Chapter_10%3A_Phosphoryl_transfer_reactions/Section_10.3%3A_Hydrolysis_of__phosphates Chem Wiki ].
Alkaline phosphatases, respectively their serum levels, are also related to several diseases e.g. metabolic myopathies and Paget Disease. [4]
Carboxyl esterase (Aes)
Glycoside hydrolase (NagZ)
Beta-glucuronidase (GusA)
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.
