Fig 5.

Allelic replacement flowchart for replacing the yfg allele with a kanamycin resistance cassette (kanA). A nonreplicative vector is forced to recombine (thick X) with the chromosome by selection on erythromycin (Ery) and kanamycin (Kan). The recombination can occur only via L or R, which are homologous to the regions adjacent to yfg in the chromosome (recombination with L is shown). Once the vector has been integrated into the chromosome, then FOA is used to counterselect the presence of the backbone vector, while kanamycin is used to select for the recombination that results in a substitution of yfg by kanA (solid black double-headed arrow). The dotted double-headed arrow indicates an alternative recombination which can happen if there is no selection with kanamycin and which will result in a wild-type strain. Vector DNA (thick black lines) and chromosomal DNA (thin black lines) are indicated. yfg is the gene that is substituted by kanA. kanA is the kanamycin resistance marker. L and R are ∼1 kb of sequence to the left and right of yfg, respectively. ermC is the erythromycin resistance marker. PpyrFE is pyrF and pyrE from B. subtilis with a Pspac constitutive promoter. The chromosomal copies of the pyrF and/or pyrE genes that have been inactivated (pyrFE-mut) (see the text for further information) are indicated.