The Construction of E. coli DH5α △asd Deletion Mutant and the Evaluation on Its Potential on Gene Transforma-tion-expression System

Abstract:

E. coli DH5α △asd deletion mutant was constructed by using Red recombination system. First, the chloramphenicol resistance(cat)gene flanked by homology extensions of asd gene was amplified by PCR. The PCR products were electro-transformed into E. coli DH5α strain, with the help of Red recombinant system, the most part of asd gene was in vivo replaced by homology extensions connected with cat gene. E. coli DH5α (△asd::cat)deletion mutant with cat gene was selected by LB plate with DAP and chloramphenicol. The cat gene was then eliminated by using a helper plasmid, pCP20, en-coding the FLP recombinase; the mutant for the recombinant E. coli was named E. coli DH5α △asd which lost the capability of growth on LB plate. The deletion mutant recovered the capability of growth on LB plate when added with DAP component. The function of the asd deletion mutant also could be compensated by the plasmid expressing asd gene. There were no significant difference in the key charac-ters of growth speed, growth log phase, accepting different origin plasmids with high efficiency between E. coli DH5α and E. coli DH5α △asd mutant. Based on the DH5α △asd mutant, the chromosome-plasmid balanced-lethal system was set up successfully, which was stable for 50 generations of passage culture in vitro and expressed the FedF adhesin without antibiotic resistance gene.

Key words:E. coli DH5α, Host strain, Red recombination system, Asd gene, Mutant