Fig. 3

Schematic representation of upp deletion. Flanking genomic regions A′ and B′ of the target gene were amplified and fused via SOE-PCR. The resulting deletion construct was cloned into vector pUCTV2. Bacillus megaterium was transformed with the resulting plasmid pUCTV2_Δupp and cultivated at 30°C. After two subsequent recombination events with each homologous fragment the deletion of upp was obtained. Incubation at the non-permissive temperature prevents replication of the plasmid containing the intact copy of the upp gene (ori ^ts, temperature-sensitive origin of replication; P _upp , promoter uracil phosphoribosyltransferase; tetR, tetracycline resistance gene; upp, uracil phosphoribosyltransferase gene).