Fig. 4

a Overview of spoIIE disruption by single crossover (Campbell-like integration) and b confirmation of spoIIE disruption by PCR amplification. a The constructed plasmid unable to replicate in the host organism is depicted. It contains a section of the target gene (between FRT sites) plus a selectable marker (Cm^R). Homologous recombination occurs between the gene fragment on the plasmid and the identical gene segment on the chromosome followed by genome integration of the entire plasmid. The target gene is ‘insertionally inactivated’, i.e. disrupted by splitting it into a truncated forward and rear end (with the homologous gene sequence present twice in the altered genome). b Amplification across each junction was done using one primer binding in the genome and one primer binding within the integrated plasmid-derived fragment. 5′ a, amplification across 5′ junction using primers spo5′fwd2 and CmF (1.9 kb product); 5′ b, amplification across 5′ junction using primers spo5′fwd3 and Cm_pMK_inner_fwd2 (1.2 kb product); 3′, amplification across 3′ junction using primers M13 fwd and spo3′rev2 (2.3 kb product); ΔE, spoIIE knockout strain; C, B. megaterium PHA05 wild type control; P, plasmid control (pFRT-Cm-spoIIE). For supplemental confirmation, the PCR product obtained with primers spo5′-fwd2 and CmF was sequenced. L, ladder λ PstI (PstI digested λ DNA)