title: [Recombinational cloning of large DNA fragments of bacterial chromosome by plasmid rescue].
abstract: OBJECTIVE
  To develop a new method to cline large DNA fragment by combining whole genome sequence information and the principle of plasmid rescue.
  
  
  METHODS
  First, we amplified a fragment downstream the region to be cloned and cloned the fragment into a suicide plasmid to construct targeting plasmid, which was then targeted into chromosome by homologous recombination. Then, genomic DNA was isolated, digested with appropriate enzyme, re-ligated and transformed into host bacteria to rescue the plasmid and the large DNA fragment. The rescued DNA fragment was sub-cloned into new plasmids for special purposes.
  
  
  RESULTS
  Using this method, we successfully cloned the 11kb virB operon of Brucella and constructed complementary plasmid. The transcription of the disrupted virB operon was restored with the complementary plasmid, validating the feasibility of the strategy.
  
  
  CONCLUSIONS
  This recombinational cloning strategy provides us a new method to modify large DNA fragment of bacteria.
authors.affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing 100071. wangyf1618@yahoo.com.cn
authors.forename: Yufei
authors.initials: Y
authors.lastname: Wang
authors.forename: Zeliang
authors.initials: Z
authors.lastname: Chen
authors.forename: Feng
authors.initials: F
authors.lastname: Qiao
authors.forename: Xinying
authors.initials: X
authors.lastname: Du
authors.forename: Leili
authors.initials: L
authors.lastname: Jia
authors.forename: Jing
authors.initials: J
authors.lastname: Yuan
authors.forename: Liuyu
authors.initials: L
authors.lastname: Huang
mesh_terms: D002876:Chromosomes, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics*; D004274:DNA, Recombinant / Q000235:genetics*; D009876:Operon / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D014158:Transcription, Genetic
chemical_list: D004269:DNA, Bacterial; D004274:DNA, Recombinant