Proposed new biobrick designs for soluble methane monooxygenase expression in E. coli
The 2014 Braunschweig and the 2021 Wagneningen teams both worked on engineering E. coli to express soluble methane monooxygenase. By looking at the biobricks that was designed by both teams, we thought to alter the Braunschweig sMMO design by two ways. The first was to codon optimize the mmo genes since the original design contained the original nucleotide sequences from M. capsulatus. The second change was to replace the IPTG inducible promoter with arabinose inducible promoter. We realize that these two changes can be considered minor, but we believe that these simple changes can potentially increase the expression level of the sMMO enzyme. We hope future teams (including our own!) will be able to try out our new design.
Proposed a different to detect the activity of soluble methane monooxygenase using fluorogenic coumarin substrates
A key assay in our project is determining whether the E. coli expressed sMMO is functional. The traditional and more direct method involves direct detection and quantification of methane and/or methanol level. This method has a disadvantage in that any methanol or methane in the liquid culture can not be easily detected. Therefore, we designed an assay that indirectly measures sMMO activity by the addition of coumarin into the solution. The oxidation of coumarin into 7-hydroxycoumarin by the E. coli expressed sMMO would have been easily detected using a fluorometer. Although we were not able to directly test our assay, we believe this is an affordable alternative way to measure sMMO activity. We hope future teams can try out our assay.
Proposed a fluorometer design that is affordable
As part of the measurement assay, we came up with a 3D model of an affordable fluorometer that would be specific in detecting the fluorescent 7-hydroxycoumarin produced by the oxidation of coumarin by sMMO.