Part:BBa_K3747003

Figure 1: The particulate methane monooxygenase mimic plasmid as described in Kim et al 2019. This high copy number plasmid harbors the pMMO active site mimic by scaffolding the substructures pMMO-B1 and pMMO-B2 on a human heavy-chain ferritin (huHF) scaffold. This is under control of a lac operator and T7 promotor.

Through Gibson assembly the plasmid as shown in Figure 1 is constructed. For this two Gblocks are ordered from IDT DNA, the first one carrying both huHF and pMMO-B1, and the second harboring pMMO-B2. Additionally an empty pET-28a(+) is used from the Wageningen collection. This is transformed into a E. coli BL21 strain, which is specialized for protein expression. (NEB).

After successful transformation the protein is tested in vitro by first purifying through His-tagging and afterwards running protein assays. The protein assays contain both the reductant duroquinone, that was obtained following the reducing protocol as shown in Kim et al 2019, although ambiguous -and the protein in a Tris buffered solution. As substrate ¼ atmosphere of methane is held in an anaerobic bottle with ¾ air and after 25 hours the methanol concentration is measured using GC. Here bottles without duroquinone, enzyme or methane are used as negative controls. The protein is also tested in vitro by growing BL21 in similar conditions to the enzyme assay on M9-glucose and measuring the methanol concentration every 24 hours.

references

Kim, Hyun Jin; Huh, June; Kwon, Young Wan; Park, Donghyun; Yu, Yeonhwa; Jang, Young Eun; Lee, Bo-Ram; Jo, Eunji; Lee, Eun Jung; Heo, Yunseok; Lee, Weontae; Lee, Jeewon (2019). Biological conversion of methane to methanol through genetic reassembly of native catalytic domains. Nature Catalysis, (), –. doi:10.1038/s41929-019-0255-1