Gene knockout primers have 20- to 30-nucleotide ends for priming upstream (P1) and downstream (P2) of the FRT sites flanking the kanamycin resistance gene in pKD13 and 50-nucleotide ends homologous to upstream (H1) and downstream (H2) chromosomal sequences for targeting the gene deletion. H1 includes the gene B (target) initiation codon. H2 includes codons for the six C-terminal residues, the stop codon, and 29 nucleotides downstream. The same primer design with respect to gene B was used to target deletions regardless of whether gene B lies in an operon with genes A and C, as shown, or in different chromosomal arrangements. Novel junctions created between the resistance cassette and neighboring upstream (gene A) and downstream (gene C) sequences were verified by PCR with kanamycin (k1 or k2) and locus-specific (U or D) primers. Structures created after excision of the resistance gene are verified by PCR with neighboring gene-specific primers and by direct DNA sequencing of the region encompassing the H1-P1-FRT-P2-H2 scar to verify correct ones, as described elsewhere (Datsenko and Wanner, 2000). SD, Shine–Dalgarno ribosome binding sequence. (Baba et al., 2004).
Of 4288 genes targeted, deletions were obtained for 3985 ORFs. Based on finding mutants with the predicted structure, these 3985 genes are likely nonessential, while the 303 genes (including 37 genes of unknown function), for which no mutants were found, are candidates for essential genes. (Baba et al., 2004)