What about those hydrolases ?

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.

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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