Objective       To establish the scarless knockout method in Escherichia coli (E. coli) based on the Red recombination system

and study the effect of hns gene on biofilm formation of E. coli.        Methods       E. coli MG1655 was used as the target strain and transformed with plasmid pKD46 by electroporation. The chloramphenicol resistance gene was amplified using template plasmid pKD3

and then introduced into E. coli MG1655/pKD46. The strains expressing chloramphenicol resistance gene were selected with chloramphenicol agar plate. The chloramphenicol resistance gene was deleted using plasmid pCP20

and then the MG1655 hns gene knockout (△hns) strain was constructed. The effect of hns gene on biofilm formation of E. coli △hns strain was analyzed with 96-well crystal violet staining. The content of extracellular polymeric substance (EPS) was determined by phenol-sulfuric acid testing.        Results       The 434 bp product was amplified from MG1655 △hns strain after the chloramphenicol resistance gene was deleted

and it was identified by sequencing

indicating that the MG1655 △hns strain was successfully constructed. The biofilm formation and EPS production of MG1655 △hns strain were significantly lower than that of MG1655 strain (P0.01).        Conclusion       MG1655 △hns strain is successfully constructed by Red recombination technology. The hns gene plays an important regulatory role in the biofilm formation of E. coli.