Optimization of Electroporation Method and Promoter Evaluation for Type-1 Methanotroph, Methylotuvimicrobium alcaliphilum Provisionally Accepted

Shubhasish Goswami¹ Deepika Awasthi^1* Steven Singer¹ Blake Simmons¹

• ¹Berkeley Lab (DOE), United States

Methanotrophic bacteria are promising hosts for methane bioconversion to biochemicals or bioproducts. However, due to limitations associated with long genetic manipulation timelines and, lack of choice in genetic tools required for strain engineering, methanotrophs are currently not employed for bioconversion technologies. In this study, a rapid and reproducible electroporation protocol is developed for type 1 methanotroph, Methylotuvimicrobium alcaliphilum using common laboratory solutions, analyzing optimal electroshock voltages and post-shock cell recovery time. Successful reproducibility of the developed method was achieved when different replicative plasmids were assessed on lab adapted Vs wild-type M. alcaliphilum strains (DASS Vs DSM19304). Overall, a ~ 3-fold decrease in time is reported with use of electroporation protocol developed here, compared to conjugation, which is the traditionally employed approach. Additionally, an inducible (3-methyl benzoate) and a constitutive (sucrose phosphate synthase) promoter is characterized for their strength in driving gene expression.