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 !

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