Team:Manchester/Description/mechanism1
Mechanism 1
Cell Free System
How does it works?
Numerous methylotrophic yeasts are able to utilize primary alcohols as a sole source of energy and carbon [1]. This involves the function of specific enzymes called Alcohol Oxidases (AOx). AOx (AOX; Alcohol:O2 oxidoreductase, EC 1.1.3.13) is implicated in the methanol oxidation pathway in yeasts, however it can also oxidise other short-chain alcohols, such as ethanol [2].
Based on this system we have engineered Escherichia coli to express AOx from Pichia pastoris that will then be used in a cell-free colorimetric system. AOx is hence a new BioBrick we have characterised (BBa_K2092000). This Cell-free analysis is achieved without the use of living cells. Instead, all components needed to catalyse the alcohols are provided in solution for their use in vitro.
Our mechanism consists of two catalytic reaction steps. Firstly, in the presence of oxygen, ethanol is oxidised by AOx to acetaldehyde (ethanal), producing Hydrogen peroxide (H2O2) as a detectable by-product. In the second step, H2O2 is then used by Horseradish peroxidase (HRP) as a redox substrate to oxidise different chromagens and generate a colour change, where (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) is the most sensitive one [3], changing from colourless to green.
Different concentrations of ethanol will produce different concentrations of H2O2 in the first step of the total reaction. HRP will oxidise ABTS according to the varying amounts of H2O2 produced and therefore oxidising more or less dye. This will result in a gradient of colour intensity correlated to the amount of ethanol.
