Abstract
Bacillus subtilis offers a platform for giant DNA synthesis, which is mediated by the connection of overlapping DNA segments called domino DNA, in the cloning locus of the host. The domino method was successfully used to produce DNA fragments as large as 3500 kbp. However, domino DNA is limited to <100 kbp because of size restrictions regarding the transformation (TF) of B. subtilis competent cells. A novel conjugal transfer (CT) method was designed to eliminate the TF size limit. The CT method enables rapid and efficient domino reactions in addition to the transfer of giant DNA molecules of up to 875 kbp to another B. subtilis genome within 4 hours. The combined use of the TF and CT should enable significantly rapid giant DNA production.
Highlights
Bacillus subtilis Marburg 168, a Gram-positive spore-forming bacterium, is used as a platform for giant DNA synthesis[1,2,3]
We developed a conjugal transfer (CT) system for transmission of the B. subtilis genome to eliminate the TF-associated size restriction[9]
The recipient B. subtilis that accepts the DNA transferred from the donor cell by CT is called the transcipient
Summary
The original domino strains, BEST6568 (171 kbp, SpcR) and BEST6571 (196 kbp, BSR), which are described in Fig. 2 and Table 1, were converted to CT donors. Two functional elements should be added[9], namely the sequence oriT110-stm unit, introduced by TF from BEST390 and selected with streptomycin, and pLS20hyg, transferred by CT from BEST23124 and selected with hygromycin[9]. The recipient B. subtilis that accepts the DNA transferred from the donor cell by CT is called the transcipient. After incubation at 30 °C for 4 hours, the transcipient was selected on LB plates supplemented with Blasticidin S (BS, 500 μg per mL) and Spectinomycin (Spc, 150 μg per mL). Genotypesb leuB8, arg-15, hsdRM pHY300PLK xkdE::oriT110-stm unit pLS20hyg xkdE::oriT110-stm unit, pLS20hyg, yjcI::ne-spc, pycA::eo-bsr
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