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Construction of E. coli strains for parallel screening. (A) Plasmid-based strategy for regulating expression of a target gene that has been deleted from the chromosome (bent lines). PC, PBAD, promoters of the araC and araBAD gene clusters, respectively; genX, any bacterial gene X. The plasmid origin of replication (ori) is denoted by the small shaded box and represents any plasmid origin (from low copy to high copy number). (B) Recombination using linear, double-stranded DNA molecules and l-redgam. In this example, the gene for nicotinamide adenine dinucleotide A replaces the mutant version on the chromosome. Blunt-ended lines, linear DNA; large X, l-red-mediated crossover between regions of homologous DNA. (C) Linear DNA recombination to replace the defective lprophage. att+ refers to a fragment of E. coli chromosomal DNA containing the bacteriophage lattachment site, attB. (D) Deletion of target genes on the chromosome using the lox2kan cassette. Colored boxes, target genes (murA, fabI, and metG); shaded boxes, neighboring genes that encode open reading frames; curvy lines, homologous DNA shared by the linear DNA substrate and the bacterial chromosome. P1 and P2 designate promoter elements flanking the target genes; specific promoters for each target are identified by name. Illustrations are not to scale. (E) Agarose gel showing PCR products generated from genomic DNA templates of various E. coli strains constructed with the bacteriophage recombination systems. Lanes 1 and 12: marker DNA (0.1 kb and 1 kb ladders, respectively). Lanes 2 and 3: removal of transposon Tn10 from chromosome of DY329; a multiplex PCR using primers within tetA and flanking nadA shows that the smaller internal band (0.95 kb) is lost when the wild-type nadA allele is restored (1.45 kb band, lane 3). The faint 0.85 kb band (lane 3) is a nonspecific PCR product typical of multiplex PCR. Lanes 4 and 5: removal of the lprophage; a multiplex PCR done with primers flanking and within the prophage shows the smaller internal band is lost after the wild-type attB allele is restored. For the metG deletion, PCR was done with an internal primer (directed against the kan coding sequence) and two primers flanking metG. Lanes 6–8: PCR products representing metG, replacement by lox2–kan cassette, and the PCR product after removal of the kanamycin marker by P1 Cre, respectively. Lanes 9–11: corresponding PCR products with primers flanking the fabI coding sequence.