Figure 1

Illustration of markerless knockout construction in B. smithii. a pNW33n-derived plasmid containing the 1,000 bp-flanking regions of a gene of interest (‘g.o.i.’) is introduced into the cell via electroporation, plated on LB2 containing chloramphenicol at 55°C and subjected to PCR analysis to check for single crossovers using primers ‘Fw-chrom’ and ‘Rv-plasmid’. Colonies containing single crossovers are transferred to plates without chloramphenicol, after which PCR screening is performed to check for double crossovers using primers ‘Fw-chrom’ and ‘Rv-chrom’. If the 2nd recombination occurs via the same flank as the 1st recombination, a wild-type genotype will be the result. If the 2nd recombination occurs via the other flank, this results in a knockout genotype. To delete the ldhL gene, only PCR screening was performed and no lacZ gene was present on the plasmid. For creating the sigF and pdhA mutants, the plasmid also contained the lacZ gene. In those cases, lacZ counter-selection was performed by plating an overnight culture on LB2 plates containing 100 µg/mL X-gal, resulting in toxic concentrations of the X-gal cleavage product in the presence of lacZ, resulting in small blue colonies still containing the plasmid (either replicating plasmid or inserted in the genome via single crossover) and white colonies that have cured the plasmid. g.o.i. gene of interest, cat chloramphenicol acetyltransferase, chrom. chromosome, us upstream, ds downstream, Cm chloramphenicol, primers are indicated with black hooks.