Fig. 1
From: Efficient dual-negative selection for bacterial genome editing
An optimized method for genome editing in Salmonella enterica. a Map of suicide plasmid pFOK (aphA, aminoglycoside phosphotransferase gene conferring resistance to kanamycin; I-sceI gene encoding meganuclease; oriT, origin of conjugational transfer; PtetA, tetA promoter; R6K γ ori, pi-dependent origin of replication; sacB, levansucrase gene; tetR, tetracycline repressor gene; traJ, transcriptional activator for conjugational transfer genes; MCR, multi cloning region containing EcoRI and BamHI recognition sites). b Mechanisms of negative selection for SacB and I-SceI, c Efficiency of negative selection for various chromosomal loci (sitABCD deletion - orange, foxA deletion - yellow, ssrB point mutation – green, and phoQ chimeric insertion - magenta [30]) using either SacB or I-SceI, or a combination of both. Fifty colonies were screened for each mutation. d Schematic representation of the consecutive single crossover procedure. Recombination can occur in one of the two homologous sequences (routes 1 and 2). Only alternate single crossover events involving both homologous sequences lead to the desired mutation, while two consecutive single crossovers in the same regions lead to reversion to wild-type (WT) e Overview of the entire procedure. Ideally, each step can be completed in one working day. f Schematic representation of preferential recombination in the right flanking region. External primers 1 and 2 together with plasmid-specific primers oOPC-614 and oOPC-615 can be used to screen co-integrant clones to reveal such bias and to identify rare variants for promoting mutant generation in the second single crossover. g Recombination bias for foxA deletion. PCR results of ex-conjugant screening using external primer 1 (oOPC-396) / oOPC-614 or external primer 2 (oOPC-397) / oOPC-615. Rare ex-conjugants (clones 5, 10) with recombination in the non-preferred flanking region were used for subsequent counter-selection