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.

As shown in Figure 1, some of the hydrolases used in the Colisweeper reporter system can catalyze hydrolysis of various substrates, with different chromophores that give rise to a wide range of colors. This variety of substrates and color outputs enables change of positions and function of these enzymes. Other possible substrates that can be used for the enzymes of this system can be seen in the Hydrolases section. In Colisweeper, the set of enzyme-substrate pairs are chosen as illustrated in the following table:

Hydrolase		Complementary substrate / IUPAC name		Visible color
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	Salmon
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

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

Colorimetric response in liquid culture

Colorimetric response on LB-Agar

Acetyl esterase(Aes)

Chemical conversion

Colorimetric response in liquid culture

Colorimetric response on LB-Agar

Beta-glucuronidase(GusA)

Chemical conversion

Colorimetric response in liquid culture

Colorimetric response on LB-Agar

β-N-Acetylglucosaminidase(NagZ)

Chemical conversion

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.