Team:ETH Zurich/Experiments 3

From 2013.igem.org

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<h1><b>What about those <i>hydrolases</i> ?</b></h1>
<h1><b>What about those <i>hydrolases</i> ?</b></h1>
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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).<br><br> On this page you will find all relevant informations about the hydrolases and enzyme substrate reaction based on papers and our results.<br>
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We use hydrolases to realize a colorimetric response by conversion of a specific substrate. We will use five hydrolases : Beta-Galactosidase (lacZ),Beta-Glucuronidase (GusA); alkaline phosphatase (phoA);Glycoside hydrolase (NagZ) and the acetyl esterase (aes).<br><br> On this page you will find all relevant information on the hydrolases and enzyme substrate reactions based on papers and our results.<br>
<h1><b>Enzyme-substrate reactions</b></h1>
<h1><b>Enzyme-substrate reactions</b></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 is becoming better and better so keep on track for updates !</p>
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<table style="float:left;margin-top:10px">
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<tr>
<td>Aes</td>
<td>Aes</td>
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<td>Carboxyl esterase</td>
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<td>Acetyl esterase</td>
<td>Magenta butyrate</td>
<td>Magenta butyrate</td>
<td>5-bromo-6-chloro-3-indoxyl butyrate</td>
<td>5-bromo-6-chloro-3-indoxyl butyrate</td>
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<td> NagZ</td>
<td> NagZ</td>
<td>Glycoside hydrolase</td>
<td>Glycoside hydrolase</td>
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<td>X-glucosaminide X-Glunac</td>
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<td>X-glucosaminide X-Glucnac</td>
<td>5-bromo-4-chloro-3-indolyl-N-acetyl-beta-D-glucosaminide</td>
<td>5-bromo-4-chloro-3-indolyl-N-acetyl-beta-D-glucosaminide</td>
<td style="color:Lightblue">Blue</td>
<td style="color:Lightblue">Blue</td>
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What about the hydolases ? How do they work and where do they come from ? Why do we use hydrolases ?
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What about the hydolases? How do they work and where do they come from? Why do we use hydrolases?
<br><br>
<br><br>
<h1><b>Beta-galactosidase</b> (LacZ)</h1>
<h1><b>Beta-galactosidase</b> (LacZ)</h1>
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<h1><b>Carboxyl esterase</b> (Aes)</h1>
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<h1><b>Acetyl 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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Revision as of 07:34, 7 September 2013

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Contents

What about those hydrolases ?

We use hydrolases to realize a colorimetric response by conversion of a specific substrate. We will use five hydrolases : Beta-Galactosidase (lacZ),Beta-Glucuronidase (GusA); alkaline phosphatase (phoA);Glycoside hydrolase (NagZ) and the acetyl esterase (aes).

On this page you will find all relevant information on the hydrolases and enzyme substrate reactions 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 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





What about the hydolases? How do they work and where do they come from? Why do we use hydrolases?

Beta-galactosidase (LacZ)

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


Alkaline phosphatase(PhoA)

Fig.2: NPP on PhoA expressing colony


Acetyl esterase (Aes)

Fig.1: Magenta butyrate on Aes expressing colony


Glycoside hydrolase (NagZ)

Fig.4: X-Glucnac on NagZ expressing colony


Beta-glucuronidase (GusA)

Fig.3 Magenta glucuronide on GusA expressing colony




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