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 is becoming 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 is becoming better and better so keep on track for updates !</p>
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Revision as of 12:24, 16 September 2013

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Contents

Enzyme-substrate reactions

2013-09-16 09.30 (1).png

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 is becoming 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 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

LacZhydro.png

Colorimetric response in liquid culture

Colorimetric response on LB-Agar

Fig.5: X-Gal on LacZ expressing colony
Fig.6: Beta-green-X-gal on LacZ expressing colony


Alkaline phosphatase(PhoA)

Chemical conversion

PhoAhydro.png

Colorimetric response in liquid culture

Colorimetric response on LB-Agar

Fig.2: NPP on PhoA expressing colony


Acetyl esterase(Aes)

Chemical conversion

Aeshydro.png

Colorimetric response in liquid culture

Colorimetric response on LB-Agar

Fig.1: Magenta butyrate on Aes expressing colony


Beta-glucuronidase(GusA)

Chemical conversion

GusAhydro.png

Colorimetric response in liquid culture

Colorimetric response on LB-Agar

Fig.3 Magenta glucuronide on GusA expressing colony


β-N-Acetylglucosaminidase(NagZ)

Chemical conversion

NagZhydro.png

Colorimetric response in liquid culture

Colorimetric response on LB-Agar

Do the substrates and enzymes cross-react ?

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.


Figure 6.1: Enzyme-substrate test matrix