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.
Bacillus subtilis Marburg 168, a Gram-positive spore-forming bacterium, is used as a platform for giant DNA synthesis1,2,3. The DNA synthesis is achieved by the connection of overlapping DNA molecules, called domino, at the cloning locus prepared in the proB gene locus of the B. subtilis genome, as illustrated in Fig. 1. The domino method has been used for the synthesis of various genomes, such as those of mouse mitochondria (16.7 kbp)2, lambda phage (48.5 kbp)4, rice chloroplast (135 kbp)2, and those of Synechocystis spp PCC6803 (3542 kbp)1. The domino method is based on the inherent natural transformation (TF), which is accurate enough to incorporate DNA3. Domino connection through TF requires liquid genomic DNA biochemically isolated from donor B. subtilis. As shown in Fig. 1, the liquid DNA is added to recipient competent cells. However, TF efficiency dramatically decreases with the increase in the size of the liquid DNA to >100 kbp5,6,7,8. Accordingly, our latest TF-based protocol restricts one domino size to ≤100 kb8.
We developed a conjugal transfer (CT) system for transmission of the B. subtilis genome to eliminate the TF-associated size restriction9. The CT enables the transfer of antibiotic resistance markers from the donor B. subtilis genome, distant from the origin of transfer oriT110 at the xkdE locus, to the recipient B. subtilis genome by mating within 4 hours at 30 °C9. Large DNA inserted in the B. subtilis genome should be similarly transferable with the help of a Type IV secretion system called T4SS, which is encoded and supplied by the plasmid pLS20hyg9. Orientation of the oriT110 from BEST3909 and in Table 1 was used throughout the study.
Methods and Results
As illustrated in Fig. 2, the CT was used to connect two dominos (196 kbp and 171 kbp with a 22 kbp overlap) covering the mouse jmj region to yield the 345 kbp product efficiently and accurately.
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 added9, 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 hygromycin9. Donors are illustrated in Fig. 2 and intermediate strains are listed in Table 1. Mating starts by simple mixing of the stationary donor culture (50 μL) and stationary recipient culture (50 μL) in 2.0 mL fresh LB in a 15 mL-culture tube in the presence of DNase I (final concentration, 3.4 μg per mL). 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). As listed in Table 1, the domino connection by CT yielded 23 transcipients (171 kbp-carrier as donor) and 33 transcipients (196 kbp-carrier as donor) per mL. Representatives of the 23 and 33 transcipients were analyzed by Pulsed Field Gel Electrophoresis (PFGE) as shown in Fig. 2. The production of 345 kbp inserts of the transcipients was confirmed. Sequence comparison of the insert from the donor (171 kbp and 196 kbp kbp) and the transcipient (345 kbp) showed no nucleotide alterations, indicating that the domino connection was achieved accurately.
Next, we investigated whether the CT allows the transmission of large DNA fragments of >200 kb, which was difficult by TF as shown in Fig. 1. We selected four strains carrying synthesized DNA of 509, 586, 683, and 875 kbp, originated from the Cyanobacterium Synechocystis spp. PCC6803 genome1 as shown in Fig. 3. The original strains shown in Fig. 3 and listed in Table 1 were converted to CT donors by the same method described in Fig. 2. Mating with the common recipient BEST386, which was previously described9 and shown in Fig. 3, and selection of transcipients on LB plates supplemented with spectinomycin (Spc,150 μg per mL) and tetracycline (Tc, 10 μg per mL) yielded transcipients (per mL), 125, 86, 140 and 3, as summarized in Table 1. Representatives of each transcipient were analyzed by PFGE, as shown in Fig. 3, which demonstrated the transfer of undisrupted DNA of up to 875 kbp. Sequence comparison of the inserts from donors and transcipients revealed no nucleotide alterations. The efficiency of CT from donors of mouse domino strains to the recipient BEST386 was assessed, and the transcipients were scored as (per mL), 176 (196 kbp), 175 (171 kbp), and 146 (345 kbp), as summarized in Table 1. It seems likely that CT for B. subtilis proceeded in a largely size-independent manner, consistent with the Hfr frequency for F-mediated Eschericha coli, which is constant throughout the movement of the whole genome10,11. The reason for the decrease in the number of transcipients for BEST7834 (875 kbp) remains unclear. A slower growth rate of the donor BEST7834 (875 kbp) (our unpublished observations) may have affected the efficiency of CT.
The TF-mediated connection of one domino DNA of 100 kbp requires at least 2 weeks, including the preparation of high quality genomic DNA for TF and selection, followed by screening by antibiotic resistance, and confirmation of insert structures by PFGE. More than 40 weeks was required for the synthesis of 875 kbp DNA by TF1. One domino connection by CT is expected to require 1 week including the introduction of two functional elements and the selection of transcipients. Considering that CT-based DNA connection as domino donor and/or recipient can be achieved for up to 683 kbp, the time required for the connection of larger DNA fragments should be dramatically reduced. It should be addressed that the lack of nucleotide alterations caused by CT could minimize the screening for correct transcipients. In addition to the currently established genome synthesis methods using yeast12,13, the CT should be a valuable additional tool to accurately edit and synthesize large DNA by B. subtilis. The synthesis of existing DNA, even that with a high GC content of up to 70% from Thermus thermophilus8, chloroplast DNA carrying a long 25-kbp inverse repeat2, and mouse genomes with abundant small repeats7,14,15 has been demonstrated. In addition to existing DNA, preparation of long DNA de novo can be achieved by our method called OGAB16,17. Since few nucleotide changes occur during TF1,16,18, CT can be used as a daily tool to synthesize DNA fragments of >1000 kbp using long DNA fragments as initial dominos. B. subtilis allows the stable synthesis of DNA as large as 3500 kbp1 as well as the synthesis of several mouse genome segments;7,14,15 therefore, it might be suitable as a workhorse for the Human Genome Wright project19.
Lastly, the accurate transfer of 875 kbp DNA inserted in the genome is, to the best of our knowledge, the first and largest exogenous DNA horizontally transferred between Bacillus species. The present results and experimental tools might shed light on studies about horizontal DNA transfer1,3.
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This work was supported by Research funds from the Yamagata prefectural government and the City of Tsuruoka.
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