Genetic connection of two contiguous bacterial artificial chromosomes using homologous recombination in Bacillus subtilis genome vector

Journal of Biotechnology 139 (2009) 211–213

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Genetic connection of two contiguous bacterial artificial chromosomes using homologous recombination in Bacillus subtilis genome vector Shinya Kaneko 1 , Takashi Takeuchi, Mitsuhiro Itaya ∗ Mitsubishi Kagaku Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194-8511, Japan

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Article history: Received 27 August 2008 Received in revised form 3 December 2008 Accepted 9 December 2008 Keywords: Genome vector Recombination Transformation Mouse genome

a b s t r a c t A Bacillus subtilis genome (BGM) vector system using homologous recombination was applied to connect two contiguous BAC clones covering the entire 355-kb transcription unit of the mouse jumonji genomic region. Results from the convenient genomic manipulation indicated that the BGM system facilitates the connection of DNAs from a BAC library without exchange and deletion of original sequence, which can expand large-sized DNA construction beyond BAC-building in Escherichia coli. © 2008 Elsevier B.V. All rights reserved.

The cloning of giant DNA is an emerging topic in molecular cloning and synthetic biology (Itaya et al., 2005, 2008; Smailus et al., 2007; Gibson et al., 2008). The handling of DNA comprised of several hundreds of kb has been difficult because of the fragility of large DNA molecules in solution. To avoid physical shearing in vitro, the large-sized DNA is isolated from cells embedded in agarose (Sambrook et al., 1989; Lartigue et al., 2007). Otherwise a second approach for construction of the large segment is to use smaller, less fragile sub-fragments of DNA and build larger sections within a host organism (Itaya et al., 2005, 2008; Gibson et al., 2008). The Bacillus subtilis genome (BGM) vector system that has been developed by our group can accommodate stable incorporation of foreign large-sized DNA into the genome of this widely used Gram-positive bacterium. Actually, it was previously showed that the 3500-kb cyanobacterium Synechocystis PCC6803 genome (Itaya et al., 2005) and the circular form of the 135-kb rice chloroplast organelle genome (Itaya et al., 2008) can be recombined into the BGM vector. Herein we report, using two contiguous BAC clones,

Abbreviations: BGM, Bacillus subtilis genome; BAC, bacterial artificial chromosome; bp, base pair(s); kb, kilo bases; Mb, mega bases; spc, spectinomycin-resistance gene; cI, cI repressor gene; bsr, blasticidin S-resistance gene; neo, neomycin phosphotransferase gene; NmR, neomycin resistant; NmS, neomycin sensitive; CHEF, contour-clamped homogeneous electric field gel electrophoresis. ∗ Corresponding author. Present address: Institute for Advanced Biosciences, Keio University, Nipponkoku, Tsuruoka-shi, Yamagata 997-0017, Japan. Tel.: +81 235 29 0526; fax: +81 235 29 0529. E-mail address: [email protected] (M. Itaya). 1 Present address: Graduate School of Bioscience and Biotechnology, Department of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama-shi Kanagawa 226-8501, Japan. 0168-1656/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2008.12.007

pKANEG (198 kb) and pKANEH (219 kb) (Incyte Genomics, Inc.; Fig. 1), the reconstruction for a 355-kb transcriptional unit of the mouse jumonji (jmj) gene whose product plays an important role in embryonic development (Takeuchi et al., 2006). Our report is a new advance for the BGM system that will enable assembly of large BACs in B. subtilis in ways previously only reported for Escherichia coli (Kotzamanis and Huxley, 2004), which also represent a significant advance for the handling of widely available BACs. We carefully prepared intact BACs from E. coli by ultracentrifugation as described by Kaneko et al. (2003). BEST6528, a new BGM vector for large BAC handling, contains a 100-kb spacer DNA and two cI repressor-gene cassettes flanked by the BAC sequence at the B. subtilis proB locus (Fig. 1). Because the cI gene products bind Pr promoter and repress the neomycin-resistance gene that is inserted into the other locus in the genome as designated Pr-neo (Kaneko et al., 2005), BEST6528 shows sensitive to neomycin. However replacement of both the cI-spc and cI-bsr cassettes with the integration of the pKANEG or the pKANEH by homologous recombination between BAC sequences resulted in the loss of the cI gene, which enable to select BGM strains carrying large BAC clone as colonies resistant to neomycin (5 ␮g/ml). Representative strains, BEST6547 (pKANEG) and BEST6548 (pKANEH), contained the expected insert fragments. This was confirmed by the observation of a given single fragment of the predicted size upon I-PpoI digestion (Fig. 2A) and by Southern blot analysis using EcoRI (Fig. 2B) as well as several other type-II restriction enzymes (data not shown). With the exception of I-PpoI (Promega, Madison, WI, USA), all enzymes were from Toyobo (Tokyo, Japan). The scheme for connecting the two BAC inserts in BGM is shown in Fig. 1. The two antibiotic marker cassettes cI-spc and cI-bsr were inserted at the far side of the mouse DNA insert of BEST6547

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S. Kaneko et al. / Journal of Biotechnology 139 (2009) 211–213

Fig. 1. Structure of genomic jmj and reconstruction in BGM. The genomic jmj region (380 kb) with exons (bold vertical lines) on top. Two BAC clones, pKANEG (198 kb) and pKANEH (219 kb), are shown in linear form with the BAC vector parts at both ends (open [L] and striped [R] arrows). Mouse DNA inserts were integrated into the BAC-specific BGM vector, BEST6528, in dotted square using the method of Kaneko et al. (2005). A neomycin-resistance gene (neo) regulated by a Pr promoter, boxed P:n, was integrated between the NotI sites of yvfC and yveP of the B. subtilis genome (Uotsu-Tomita et al., 2005). The I-PpoI recognition sequence is indicated by a bold vertical bar. BEST6547 and BEST6548 carrying pKANEG and pKANEH, respectively, yield BEST6568 and BEST6571 as a resulted of cI cassette insertion (cI-spc; solid circle or cI-bsr; open circle) using plasmid pJcS and pJcB separately. After joining of two BAC clones was performed by transformation of BEST6571 using purified genome of BEST6568, each cI cassette markers were excised by selection using cI and Pr-neo with original BAC clones.

(pKANEG) and BEST6548 (pKANEH). By means of this marker insertion, two plasmids, pJcS and pJcB, illustrated in Fig. 1, yielded BEST6568 and BEST6571. The two BAC-DNAs in the BGM vector were connected by genetic transformation of bsr-marked BEST6571, using purified genomic DNA from spc-marked BEST6568. Homologous recombination of the 60-kb overlapping region (horizontal line in the upper panel of Fig. 1) and the common sequence in the B. subtilis genome resulted in 18 colonies resistant to both spectinomycin (50 ␮g/ml) and blasticidin (500 ␮g/ml). The representative strain BEST6586 provided the expected 355-kb I-PpoI fragment (Fig. 2A). Southern blot analysis using EcoRI (Fig. 2B) identified the faithful genetic connection of the two individual inserts pKANEG and pKANEH. The two inserted markers linked with the cI gene of BEST6586 were excised from the genome as a result of simultaneous transformation using the two original BACs, pKANEG and pKANEH. Selection for marker-less BEST6613 was as follows. BEST6586 containing two cI cassettes and a Pr-neo in the genome shows sensitive to neomycin. Transformation using the original BACs leads to replacement of the cI markers from the BGM and activation of the Pr-neo gene, rendering the BGM selectable by neomycin (Uotsu-

Tomita et al., 2005). We found that 57 colonies selected on LB plates supplemented with neomycin were sensitive to spectinomycin and blasticidin. The insert-structure of the representative BEST6613 was confirmed by I-PpoI (Fig. 2C) and Southern blot analysis (data not shown). The mouse jmj DNA containing abundant short repetitive sequences which are especially included in the first large intron as described in public database (http://www.ncbi.nlm.nih.gov/ mapview/maps.cgi?org=mouse&chr=13&maps=cntg-r,bes,scan, ugMm,loc&VERBOSE=ON&query=Jmj&cmd=focus&fill=40&size= 40&compress=no) was stably maintained in BGM after many generations without antibiotics such as confirmed in Fig. 2. This appears consistent with the observation that DNA segments cloned in BGM remain stable and allow the insertion of even a 21-kb long-inverted repeat of a chloroplast genome (Itaya et al., 2008). Two contiguous BACs have been connected (Kotzamanis and Huxley, 2004) in the original E. coli host using the Red recombination system. Although the usefulness of BACs in mouse genetics (Yang et al., 1997) and in synthetic biology (Gibson et al., 2008) is clear, the E. coli BAC cloning system seems to have size limit

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We suggest that the recombined jmj genomic DNA will facilitate the elucidation of transcription mechanisms and the characteristics of particular genomes. For these aims the DNA in the BGM vector must be retrieved from the genome of B. subtilis. We applied several retrieval methods developed for BGM (Kaneko et al., 2005; Itaya and Tanaka, 1997). Preparation of the 355-kb insert as an I-PpoI fragment can be reproducibly performed as shown in Fig. 2C. After reconstructed transcription unit would be moreover modified by insertion of reporter gene, etc., the purified fragment in agarose plugs with agarases could be employed in further manipulations (Lartigue et al., 2007). Efforts using a combination of BAC and TAR systems using homologous recombination in Saccharomyces cerevisiae (Kouprina and Larionov, 2006) are underway in our laboratory to improve the BGM vector for a variety of applications. Acknowledgement We thank Dr. Uotsu and Ms. Akioka for technical assistance and Dr. Yanagawa of Keio University for helpful discussions and continuous encouragement. References

Fig. 2. Reconstruction and retrieving of the 355-kb segment. (A) Open arrowheads show the I-PpoI fragment from the indicated BGM strains. The location of the 4.2-Mb BGM vector linearized with I-PpoI is shown by a horizontal arrow on the left. Running condition: 5 V cm−1 , 45 s pulse time, and 19 h running time at 14 ◦ C. (B) Genomic Southern blot analysis for the indicated BGM strains. The genetic connection of two inserts is shown as the sum of the respective bands of BEST6586. Running condition: 3 V cm−1 , 18 s pulse time, and 16 h running time at 14 ◦ C. (C) The genome of B. subtilis (4.2 Mb) and the 355-kb segment with the BAC vector are indicated by closed and open arrowheads, respectively. Running condition: 5 V cm−1 , 45 s pulse time, and 20 h running time at 14 ◦ C.

for stable cloning up to 350 kb inserts at most (Sheng et al., 1995; Kouprina and Larionov, 2006). In contrast manipulations of BAC libraries based on the BGM system will provide the method for construction of far larger DNA than the current 355 kb.

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