Team:ETH Zurich/Experiments 7

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What about those hydrolases ?

Colisweeper depends largely on processing of molecular signals and generation of visible and distinguishable outputs accordingly. Next to our PLuxR promoter mutations, which detect ranges of OHHL concentrations, we made use of a reporter system which gives colorimetric responses and additionally provides that the visibility of output is only to be triggered by the player of Colisweeper: A set of orthogonal hydrolases which specifically cleave the chromogenic hydrolase substrates added by the player.
These hydrolases include the Citrobacter alkaline phospohatase, the Bacillus subtilis β-glucuronidase and the Escherichia coli acetylesterase, N-acetyl-β-glucosaminidase and β-galactosidase. To ensure specific enzyme-substrate reactions, we used a triple deletion Escherichia coli strain, lacking aes (acetylesterase), gusA (β-glucuronidase) and nagZ (N-acetyl-β-glucosaminidase).


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

Fig.2: NPP on PhoA expressing colony


Acetyl esterase(Aes)

Chemical conversion

Aeshydro.png

Colorimetric response in liquid culture

Colorimetric response on LB-Agar

Fig.1: Magenta butyrate on Aes expressing colony


Beta-glucuronidase(GusA)

Chemical conversion

GusAhydro.png

Colorimetric response in liquid culture

Colorimetric response on LB-Agar

Fig.3 Magenta glucuronide on GusA expressing colony


β-N-Acetylglucosaminidase(NagZ)

Chemical conversion

NagZhydro.png

Colorimetric response in liquid culture

Colorimetric response on LB-Agar