A QUICK AND PRECISION METHOD TO CONSTRUCT ESCHERICHIA COLI HISTIDINE AUXOTROPH

Red in vivo recombination is a new kind of genetic engineering technique based on homologous recombination. In this work, plasmid pKD46 which expresses Red recombination proteins is transferred into Escherichia coli strain DH5α.The kanamycine resistant gene is generated by PCR by using primers with homology to hisDCB gene of E.coli chromosome. Thus, the hisDCB gene was replaced with kanamycine resistant gene by the plasmid recombination system, then the resistant gene was eliminated by a helper plasmid encoding the FLP recombinase. At last, a E.coli histidine auxotroph which is sensitive to kanamycine was got. The results indicate that Red in vivo recombination is a convenient method to construct auxotrophs.