Point Mutations In The E. Coli Genome Engineering Method Mediated Recombination

Recombineering (Red/ET, recombination mediated genetic engineering) is a DNA cloning and modification technique that makes use of recombination between short oligonucleotides in Escherichia coli.Theoretically, recombineering mediated manipulations is simple, convenient and highly efficient for bacterial genomic DNA or DNA fragments of any size.In this paper, a novel recombineering method was presented to achieve the E. coli gene point mutation mediated by recombinant engineering. The first step is recombineering mediated chromosome integration of two I-SceI restriction sites (S),-homology arms and kanamycin resistance gene (neo) gene cassette. Homology arm the containing S-neo-S gene cassette was obtained by PCR.The gene cassette also contains the homologous with the genomic sequence and be introduced mutation point. After chlor-tetracycline (cTc) induced expression of I-SceI, I-SceI cleavage creates a double strand break on the genome. Further genome homologous recombination occurs under pressure to survive and in vivo recombinase, thus removing the kan resistance gene the any redundant sequence designated mutant strains. The addition, based on the restructuring the the I-SceI enzyme systems engineering, two-step recombination efficiency is80%.The method was used for lacZ gene and nanA gene knock out, as well as for lacZ gene and nanA gene point mutations. The first step efficiency reachs80%, while the wfficiency of secon drecombination is high up to90%.