Overexpression of mrpigG in M. ruber M7. ( a ) Schematic representation of the homologous recombination strategy yielding mrpigG overexpression strains. ( b ) Construction of mrpigG overexpression construct by double-joint PCR. Lane 1, 5′ flanking region of mrpigG ; lane 2, G418 resistance cassette; lane 3, the trpC promoter; lane 4, mrpigG ORF regions plus 3′ flanking region of mrpigG ; lane 5, double-joint PCR product. ( c ) Confirmation of mrpigG homologous recombination events. Three primer pairs were used, and PCR amplifications showed distinct bands in different strains. Lane 1, the M7:: PtrpC-mrpigG strain; lane 2, the wild-type strain. 