Team:ETH Zurich/Experiments 3

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<h1>Enzyme-substrate reactions</h1>
<h1>Enzyme-substrate reactions</h1>
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<p>As our reporter system, we use orthogonal hydrolase enzymes that react with specific substrates to give a chromogenic product. The color produced from the enzyme-substrate reaction gives the cue for the player to carry out the next logical move. The player adds a multi-substrate solution on to colonies on the agar mine-field. In case the player is certain of a colony to be a mine colony, another second specific substrate is added. The substrates react with the enzymes to give the color in couple of 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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[[File:2013-09-16 09.30 (1).png|450px|right|thumb|<b>Figure 1: Colorimetric output</b> This image shows liquid cultures of our mutant ''Escherichia coli'' strain expressing the enzymes PhoA, NagZ, LacZ, GusA or Aes to convert specific chromogenic substrates into the color outputs needed for our project. There is a possibility to create more colors, since our hydrolases have a large spectrum of substrates to convert to give different colorimetric outputs. As an example of multi-substrate conversion, there is the LacZ which can convert X-Gal into a blue output and Green-β-D-Gal into a green output.]]
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[[File:2013-09-16 09.30 (1).png|450px|left|thumb|<b>Figure 1: The colorimetric output</b> Our hydrolases can convert specific substrates to an colorimetric output visible by eye. The colors on the pictures are the colors we use for our project. We could create more colors because the hydrolases have a large spectrum of substrates to convert which gives different colorimetric outputs.Here we see the 5 hydrolases (annotated in bold) we use with a specific substrate (annotated in normal weight under the bold hydrolases). As an example of mulit substrate conversion we have lacZ which can convert X-Gal to a blue output and beta-green-X-Gal to a green output]]
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<p>The picture to the left shows different colors produced by the hydrolase-substrate reactions. Each hydrolase was tested with the corresponding substrate in liquid cultures. The hydrolases are ''lacZ'','' nagZ'' , ''aes'', ''gusA'' and ''phoA''. Multiple substrates were tested for the hydrolases as well.</p>
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The following table lists each of the specific hydrolase-substrate reactions with the corresponding color produced from the reaction.
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<p align="justify">To generate visible output by adding substrates to colonies in Colisweeper, we made use of orthogonal enzyme-substrate reactions. A set of chromogenic substrates was chosen to produce different colors depending on the abundant hydrolases and thereby to uncover the identity of each colony to the player.<br>
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Chromogenic substrates incorporate a chromophore whose absorbance properties change after the enzyme reaction, and the color signal produced then is directly related to the enzyme-catalyzed reaction.<br>
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Indican is a chromogenic glycoside hydrolase substrate which belongs to a family of natural glycosides found in plants. Cleavage of the glycosidic bond forms an unstable hydroxyindole intermediate, which dimerizes by oxidation to form indigo as a blue precipitate:<br>
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Numerous enzyme substrates have been designed following this natural product example, giving rise to many colored phenols that are used to detect enzyme activities.<br><br>
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In Colisweeper, the set of enzyme-substrate pairs are chosen as illustrated in the following table:</p>
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Revision as of 13:03, 3 October 2013

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Contents

Enzyme-substrate reactions

Figure 1: Colorimetric output This image shows liquid cultures of our mutant Escherichia coli strain expressing the enzymes PhoA, NagZ, LacZ, GusA or Aes to convert specific chromogenic substrates into the color outputs needed for our project. There is a possibility to create more colors, since our hydrolases have a large spectrum of substrates to convert to give different colorimetric outputs. As an example of multi-substrate conversion, there is the LacZ which can convert X-Gal into a blue output and Green-β-D-Gal into a green output.

To generate visible output by adding substrates to colonies in Colisweeper, we made use of orthogonal enzyme-substrate reactions. A set of chromogenic substrates was chosen to produce different colors depending on the abundant hydrolases and thereby to uncover the identity of each colony to the player.
Chromogenic substrates incorporate a chromophore whose absorbance properties change after the enzyme reaction, and the color signal produced then is directly related to the enzyme-catalyzed reaction.
Indican is a chromogenic glycoside hydrolase substrate which belongs to a family of natural glycosides found in plants. Cleavage of the glycosidic bond forms an unstable hydroxyindole intermediate, which dimerizes by oxidation to form indigo as a blue precipitate:
Numerous enzyme substrates have been designed following this natural product example, giving rise to many colored phenols that are used to detect enzyme activities.

In Colisweeper, the set of enzyme-substrate pairs are chosen as illustrated in the following table:


Hydrolase Complementary substrate / IUPAC name Visible color Concentration[M]
LacZ Beta-Galactosidase X-Gal 5-Bromo-4chloro-3-indolyl-beta-galactopyranoside Blue
Blau.png
LacZ Beta-Galactosidase Green-beta-D-Gal N-Methyl-3-indolyl-beta-D_galactopyranoside Green
Grün.png
GusA Beta-glucuronidase Magenta glucuronide 6-chloro-3-indolyl-beta-D-glucuronide-cycloheylammonium salt Salmon
Rot.png
PhoA Alkaline phosphatase pNPP 4-Nitrophenylphosphatedi(tris) salt Yellow
Gelb.png
Aes Acetyl esterase Magenta butyrate 5-bromo-6-chloro-3-indoxyl butyrate Magenta
Lila.png
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

Acetyl esterase(Aes)

Chemical conversion

Aeshydro.png

Colorimetric response in liquid culture

Colorimetric response on LB-Agar

Beta-glucuronidase(GusA)

Chemical conversion

GusAhydro.png

Colorimetric response in liquid culture

Colorimetric response on LB-Agar

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.



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