Figure 4. Schematic representation of allelic replacement by homologous recombination.

. (A) Generation of SCO strains. p2Nbk-duth was electroporated into WT competent M. smegmatis, and single-crossover (SCO) transformants were selected. (B) Merodiploid strains were constructed by electroporating the complementing plasmid (pGem-dut) into the SCO strains. (C) Generation of disrupted dut deletion mutant strain. The double crossover event may result either a disrupted dut deletion mutant strain (a), or a wild type strain (b). (D) Strategy for SCO and DCO screening. a) shows primers and expected PCR products for the knock-out (KO) allele while b) shows the same for the WT allele. Abbreviations: WT; wild type; SCO; single crossover; DCO; double cross over.