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| A Novel Temperatrue Sensitive Plasmid-based Method for Deletion of Chromosomal Genes |
1.Beijing Fisheries Research Institute, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100068, China
2.Beijing Institute of Biotechnology, Beijing 100071, China |
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Abstract A temperature sensitive plasmid-based procedure without linear DNA was developed for a rapid deletion of chromosomal target genes in Salmonella. To construct a recombinant vector, homologous regions and kana cassette with two FRT sites were amplified using corresponding templates and cloned into Ts-plasmid pHY304. The produced shuttle vector was then transformed into S.ty2. Under the pressure of temperature and antibiotic, homologous recombination occurred between the vector and genome of the host bacterium and the recombinants were selected by agar media with kanamycin. To delete kan resistant cassette, plasmid pCP20 was introduced into the recombinant. The markerless mutant strains were detected by genome PCR. The ssaV gene was successfully deleted by the modified method. A novel Ts-plasmid-based method has been established, which could be used for deletion of gene in other Gram-negative bacteria.
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Received: 14 December 2009
Published: 25 March 2010
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