Recombinational Cloning of Large DNA Fragments of Bacterial Chromosome by Plasmid Rescue

Abstract:

[Obiective]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. [Conclusion] this recombinational cloning strategy provides us a new method to modify large DNA fragment of bacteria.