[Recombinational cloning of large DNA fragments of bacterial chromosome by plasmid rescue].
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.
Wang
Yufei
Y
Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing 100071. wangyf1618@yahoo.com.cn
Chen
Zeliang
Z
Qiao
Feng
F
Du
Xinying
X
Jia
Leili
L
Yuan
Jing
J
Huang
Liuyu
L
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
D004269:DNA, Bacterial; D004274:DNA, Recombinant