Team:ETH Zurich/Experiments 3

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<h1>Enzyme-substrate reactions</h1>
<h1>Enzyme-substrate reactions</h1>

Revision as of 05:50, 27 August 2013

Header2.png
80px-Eth igem logo.png

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




Fig.1: Magenta butyrate on Aes expressing colony
Fig.2: NPP on PhoA expressing colony
Fig.3 Magenta glucuronide on GusA expressing colony
Fig.4: X-Glucnac on NagZ expressing colony
Fig.5: X-Gal on LacZ expressing colony
Fig.6: Beta-green-X-gal on LacZ expressing colony


Figure 6.1: Enzyme-substrate test matrix
Figure 6.2: Expected colorimetric response

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.


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