We aim to construct bacteria able to detect methanol in ethanol solutions. Our project focuses on using a methanol-dependent promoter and a gene regulated by it – methanol dehydrogenase. We would like to introduce bacteria, which –in presence of methanol – would produce a dye, for instance, GFP.
The main problem is to find a promoter that is insensible to ethanol. Unfortunately, the most suitable organism – Picha pastoris – has methanol-dependent promoters which are blocked in the presence of ethanol. Therefore, we have found a bacterium which is an obligate methylotroph – Methylobacterium organophilum. In theory, its methanol-dependent promoter should react only to methanol.
We would like to present two different approaches to our project. Depending on initial results, we will take different measures to reaching our goal. Being restricted by the possibility that the promoter may not act as we would like it to, we thought of another route.
The first method, based on methanol-dependent promoter is very clear and easy to achieve. Provided that this promoter is insensible to ethanol and that it works well in our final, transformed organism, we only have to measure the efficiency of dye production in different concentrations of alcohols.
The other approach focuses on the reaction catalyzed by methanol dehydrogenase. This enzyme, in the presence of NAD+, is able to alter methanol into formaldehyde. While detecting the methanol with simple measures is very hard, performing a colorimetric reaction with formaldehyde seems to be quite possible.
The bacterium, which we want to use as a host is E. coli and if it works, we will try to perform the transformation on Zymomonas mobilis, which ethanol resistance is much hiigher. Z. mobilis transformation is much harder, so firstly we will focus on more known organism, E.coli .
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