Cloning and physical map construction of a large plasmid pBMB165 in Bacillus thuringiensis

Abstract:

The largest detected plasmid pBMB165 from Bacillus thuringiensis subsp. tenebrionis strain YBT-1765 (H8ab) was cloned and its physical map was analyzed. For the cloning, two BAC libraries were constructed with their plasmid DNA and genomic DNA, respectively. The plasmid DNA BAC library was obtained by partially digesting plasmid DNA with BamHI and then cloning to pBeloBAC11 vector, whereas the genomic DNA BAC library was done with HindIII partial digestion. With the chromosome walking strategy, the plasmid BAC library was initially screened by the primers designed according the sequence coding replication protein Rep165 on a previously identified 3.6kb DNA fragment (pBMB165-F4A). Finally, 5 clones covering the most of plasmid pBMB165 were obtained. When screening the genomic DNA BAC library, 8 clones covering whole plasmid pBMB165 were isolated. By restriction analysis of these 13 BAC clones, the physical map and the linear linkage map of plasmid pBMB165 were constructed and the size of pBMB165 was calculated to be 82kb. Based on the DNA sequence of the BAC insertion ends and a previously published 20kb fragment on recombinant plasmid pBMB165A2, there were redundant transposable elements appeared on this large plasmid. This study provided a novel way to clone large plasmid from B. thuringiensis, to draw the physical map by construction of BAC library, and to dissolve the problem in cloning large plasmid from B. thuringiensis.