Fig. 1.

Schematic work-flow of the knockout of Lon gene in E coli K-12 strain using a Crelox cassette. The Lon 5′ target region (U) and Lon 3′ target region (D) represent sequences (250 bp) upstream and downstream of the Lon ORF respectively. The 34 bp lox sequences (lox66 and lox71) are direct repeats located adjacent to the 250 bp sequence. It flanks the chromosomal Lon gene sequence to be deleted, the T7 promoter, Cre recombinase ORF, the T7 transcription terminator, a marker KanR ORF mediating kanamycin resistance. A double homologous recombination event replaced the Lon ORF in the genome with the crelox cassette. Growth of recombinant cells on IPTG induced the production of the Cre recombinase which recognized the two Lox sites and excised the inner sequence, leaving only one lox site in the genome