Enzymatic colourimetric assays are used to determine the concentration of a chemical in a solution by the conversion of a chromogen substrate into a coloured product. We have introduced a plasmid expressing recombinant Alcohol Oxidase 1 (AOx) from Pichia pastoris into Escherichia coli (E.coli) BL21 (DE3) strain that will then be used in the cell-free colorimetric system. This method involves the usage of AOx to oxidise ethanol, producing hydrogen peroxide (H₂O₂) as a by-product. H₂O₂ is used as an oxidising agent by horseradish peroxidase (HRP) to convert ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) to produce the colour change ^[1].

The alc gene expression system is one of the most reliable chemically inducible gene switches for use in plants and fungus. While the alc gene switch has been shown to function in a variety of dicotyledonous plants including tobacco ^[2], Arabidopsis ^[3], poplar ^[4], potato ^[5], oilseed rape ^[6], and tomato ^[7], it has not been reported to be functional in monocotyledonous plants. This system relies on the ability of AlcR, an alcohol-activated transcription factor, to bind to its target alcA promoter (PalcA). Based on this, we have engineered E. coli K-12 derivative DH5α and BL21 to induce expression of chromoproteins when AlcR binds to the native PalcA and variant of PalcA ( PalcA(var)) in the presence of ethanol ^[8].