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 language | English |
|---|---|
| Pages (from-to) | 867-876 |
| Number of pages | 10 |
| Journal | Biotechnology Journal |
| Volume | 7 |
| Issue number | 7 |
| DOIs | |
| Publication status | Published - 2012 Jul |
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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 journal › Article
}
TY - JOUR
T1 - Serial assembly of Thermus megaplasmid DNA in the genome of Bacillus subtilis 168
T2 - A BAC-based domino method applied to DNA with a high GC content
AU - Ohtani, Naoto
AU - Hasegawa, Miki
AU - Sato, Mitsuru
AU - Tomita, Masaru
AU - Kaneko, Shinya
AU - Itaya, Mitsuhiro
PY - 2012/7
Y1 - 2012/7
N2 - 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.
AB - 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.
KW - Bacillus subtilis
KW - GC content
KW - Genome synthesis
KW - Genome vector
KW - Megaplasmid
UR - http://www.scopus.com/inward/record.url?scp=84863313638&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84863313638&partnerID=8YFLogxK
U2 - 10.1002/biot.201100396
DO - 10.1002/biot.201100396
M3 - Article
C2 - 22553167
AN - SCOPUS:84863313638
VL - 7
SP - 867
EP - 876
JO - Biotechnology Journal
JF - Biotechnology Journal
SN - 1860-6768
IS - 7
ER -