Epitope Tagging Of The Chromosomal Gene In Yersinia Pestis By Recombineering Technique

Backgroud Yersinia pestis (Y. pestis) is the etiological agent of bubonic and pneumonic plague, which has claimed millions of lives in human history. At present, although plague is under the effective control, but the possibility of Y. pestis used as biological warfare or bioterrorism agent is still existent. The accumulation of genome sequence data from an increasing number of organisms is changing classical genetic approaches. Based on the recombineering technique, one-step procedures for disrupting and /or tagging genes directly in their natural chromosomal context play an important role in the study of gene function. To facilitate the functional analysis of chromosomal genes and their products in Yersinia pestis, we adapted the recombineering technique to epitope tagging of chromosomal genes of Y. pestis.METHODS The epitope tag was generated by primer annealing and then fused with an antibiotic resistance marker (Kn~R or Cm~R) by fusion PCR. The epitope-resistance cassette was inserted into pBluecript, resulted in the template plasmid. The tagging cassette for chromosomal gene was amplified by PCR with primers that carry extensions (as short as 40-50 nt) homologous to the last portion of the targeted gene and to a region downstream from it. Transformation of a strain expressing bacteriophage λ Red recombination system yields recombinants carrying the targeted gene fused to the epitope-encoding sequence. The resulting C-terminal-tagged protein can be identified by standard immunological techniques. In this study, we have added the sequences encoding the Myc and 6 × His epitopes to rpoS genes of Yersinia pestis. Epitope fusion proteins were detected in the bacteria growing in vitro. PCR and DNA sequencing were used to confirm the correct tagging event. The expression of the tagged protein was detected with Western blot by using monoclonal antibody to the 6 X His epitope. RESULTS The template plasmid containing fusion of Myc-His epitope and Kn~R gene was successfully constructed. The sigma factor gene, rpoS, was tagged with a MYC-HIS tag at the -COOH terminus. The expression of tagged rpoS gene was successfully detected indirectly by the antibody against His tag. CONCLUSION The ease of the manipulations, the sensitivity of the detection method,...