Team:ETH Zurich/Experiments 3
From 2013.igem.org
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)
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.