Fig. 1

pUCG3.8Bgl-pdu ldh promoter integration protocol. The ldh promoter is inserted between the 5′ and 3′ flanking regions on pUCG3.8Bgl-pdu. The 1st crossover event relies on homologous recombination of the plasmid with two locations on the chromosome: the start of the target gene and the 3′ flanking region. The first double crossover will delete the target gene. This event is selected on X-Glu kanamycin agar, which only permits growth of cells that have the integrated construct with kanamycin resistance and have lost the plasmid backbone encoding the Bgl enzyme. The 2nd crossover event relies on homologous recombination between the 5′ flanking region of the target gene and the duplicate region incorporated from the plasmid in the 1st crossover event. This second crossover will render the cells sensitive to kanamycin and can be identified by replica plating