• heterologous expression
• Methylococcus capsulatus
• soluble methane monooxigenase

This thesis project comprises the preliminary steps for the development of a biosensor for the detection of CH4 gas using sMMO as a biorecognition element. First, the generation of an Escherichia coli strain expressing the sMMO enzyme was attempted. In this sense, the sMMO operon was amplified through PCR and integrated into the plasmidic expression vector pET 302NT/His by using the Gibson assembly approach. The resulting expression vector pET_sMMO was transformed by chemical transformation in E. coli DH5α strain, and then further characterized by PCR, restriction analysis and sequencing using the Nanopore technology. pET_sMMO together with the plasmid pBB541 which contains the genes codifying for the chaperonines GroEL and GroES known to improve the enzyme folding and thus the sMMO expression in E. coli, were sequentially and successfully transformed by electroporation into the expression vector E. coli BL21. In vitro studies directed to evaluation of the expression of sMMO in E. coli were directed using coumarin as a substrate, which once hydroxylate generates a fluorescent compound. Taken together, the results of this work represent a fundamental step in the set-up of a biosensing system for the detection of CH4 gas using M. capsulatus sMMO enzyme as biorecognition element.