What about those hydrolases ?

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).

On this page you will find all relevant informations about the hydrolases and enzyme substrate reaction 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 become 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

Carboxyl 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