Construction of Escherichia coli K-12 Strain Deficient in relA and spot Using the γ Red Site-specific Recombinase System

Previous studies have shown that an exposure to sub-inhibitory levels of kanamycin induces capsule synthesis which confers antibiotic resistance. To examine the role of spoT and relA genes in capsule synthesis and acquired physiological antibiotic resistance during sub-inhibitory kanamycin treatment, multiple studies were performed but showed inconclusive results. A potential explanation for the lack of success in the past was the use of genetic mutants containing a number of uncharacterized mutations, which resulted in limited comparability. In this study, the λ Red recombinase system has been utilized to construct an E. coli K-12 Δ relA Δ spoT mutant by disrupting the spoT gene in E. coli K-12 Δ relA strain, JW2755-3. A PCR fragment was generated using primers with homologous sequences found upstream and downstream of the spoT gene, and a template plasmid, pKD3, carrying chloramphenicol resistance gene that is flanked by FLP recognition target sites. This product was electroporated into the strain JW2755-3 expressing the λ Red recombinase system. Five chloramphenicolresistant transformants were selected and PCR-tested for the presence of new junctions and locus-specific fragments. Construct SL11W447-4 had the correct structure as evidenced by four PCR tests performed using various combinations of locusand cat -specific primers. This work provides a basis for future studies.