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

Naoto Ohtani, Miki Hasegawa, Mitsuru Sato, Masaru Tomita, Shinya Kaneko, Mitsuhiro Itaya

Research output: Contribution to journalArticle

11 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)867-876
Number of pages10
JournalBiotechnology Journal
Volume7
Issue number7
DOIs
Publication statusPublished - 2012 Jul

Fingerprint

Thermus
Bacterial Artificial Chromosomes
Base Composition
Bacillus subtilis
Genome
DNA
Clone Cells
Plasmids
Thermus thermophilus
Maintenance
Escherichia coli
Bacteria

Keywords

  • Bacillus subtilis
  • GC content
  • Genome synthesis
  • Genome vector
  • Megaplasmid

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Molecular Medicine

Cite this

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. / Ohtani, Naoto; Hasegawa, Miki; Sato, Mitsuru; Tomita, Masaru; Kaneko, Shinya; Itaya, Mitsuhiro.

In: Biotechnology Journal, Vol. 7, No. 7, 07.2012, p. 867-876.

Research output: Contribution to journalArticle

Ohtani, Naoto ; Hasegawa, Miki ; Sato, Mitsuru ; Tomita, Masaru ; Kaneko, Shinya ; Itaya, Mitsuhiro. / 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. In: Biotechnology Journal. 2012 ; Vol. 7, No. 7. pp. 867-876.
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