Fig. 4

Gene knockout of P. aeruginosa via homology recombination. Primer 1 and 2, universal pEX18 forward and reverse primers flanking the multiple cloning sites, respectively. Primers 3 and 4, specific to genomic DNA sequences flanking the sequences cloned into the pEX18Tc vector. Primers 1 and 2 were used to screen E. coli DH5α transformants containing the deletion allele plasmid. Primers 1 and 4 or primers 2 and 3 were used to screen merodiploids in which the deletion construct was integrated into the chromosome. Primers 3 and 4 were used to screen deletion mutants following secondary recombination