RRC ID 61168
Author Ohtani N, Hasegawa M, Sato M, Tomita M, Kaneko S, Itaya M.
Title Serial assembly of Thermus megaplasmid DNA in the genome of Bacillus subtilis 168: a BAC-based domino method applied to DNA with a high GC content.
Journal Biotechnol J
Abstract Bacillus subtilis is the only bacterium-based host able to clone giant DNA above 1000 kbp. DNA previously handled by this host was limited to that with GC content similar to or lower than that of the B. subtilis genome. To expand the target DNA range to higher GC content, we tried to clone a pTT27 megaplasmid (257 kbp, 69% of G+C) from Thermus thermophilus. To facilitate the reconstruction process, we subcloned pTT27 in a bacterial artificial chromosome (BAC) vector of Escherichia coli. Owing to the ability of BAC to carry around 100 kbp DNA, only 4 clones were needed to cover the pTT27 and conduct step-by-step assembly in the B. subtilis genome. The full length of 257 kbp was reconstructed through 3 intermediary lengths (108, 153, and 226 kbp), despite an unexpected difficulty in the maintenance of DNA >200 kbp. Retrieval of these four pTT27 segments from the B. subtilis genome by genetic transfer to a plasmid pLS20 was attempted. A stable plasmid clone was obtained only for the 108 and 153 kbp intermediates. The B. subtilis genome was demonstrated to accommodate large DNA with a high GC content, but may be restricted to less than 200 kbp by unidentified mechanisms.
Volume 7(7)
Pages 867-76
Published 2012-7-1
DOI 10.1002/biot.201100396
PMID 22553167
MeSH Bacillus subtilis / genetics* Base Composition* Chromosomes, Artificial, Bacterial* Cloning, Molecular / methods DNA, Bacterial / chemistry DNA, Bacterial / genetics Escherichia coli / genetics Genome, Bacterial Plasmids / genetics* Thermus / genetics* Transformation, Bacterial*
IF 3.543
Prokaryotes B. subtilis MBS840 MBS841 MBS842 MBS843 MBS844 MBS845 MBS846 MBS847 MBS848 MBS849 MBS850 MBS851