Construction of the Escherichia coli DL11w-3 Strain with spoT relA Double Deletions

Stringent response, a form of cellular reprograming when encountering environmental stress, has been link to several physiological changes in Escherichia coli cells. The effector molecules that activate the stringent response, (p)ppGpp, are made by relA and spoT. These two genes regulate the accumulation of ppGpp through synthesis and hydrolysis. In this study, we utilized the lambda red recombinase system, a site-specific mutagenesis method, to knock out spoT in E. coli strain JW2755, which already had a deletion in relA. The JW2755 strain was first transformed with the plasmid, pCP20, to remove the kanamycin resistance, then again transformed with the plasmid pkD46 to introduce the lambda red recombinase system. The PCR product with sequence homology to the spoT gene and a kanamycin resistance marker was then transformed into the JW2755 strain. Evidence suggests that all transformations were successful. The final strains containing relA and spoT deletions were checked with PCR, and confirmed to have the correct recombination.