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An inducible recA expression Bacillus subtilis genome vector for stable manipulation of large DNA fragments.

Ogawa T, Iwata T, Kaneko S, Itaya M, Hirota J - BMC Genomics (2015)

Bottom Line: We developed a novel BGM vector with inducible recA expression system, iREX, which enables us to manipulate large DNA fragments more stably than the conventional BGM vector by suppressing undesirable recombination.In addition, we demonstrate that the iREX can be applied to handling the DNA, which has several homologous sequences, such as multiple-reporter expression cassettes.Thus, the iREX expands the utility of the BGM vector as a platform for engineering large DNA fragments.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Graduate School of Bioscience and Bioengineering, Tokyo Institute of Technology, Yokohama, 226-8501, Japan. togawa@bio.titech.ac.jp.

ABSTRACT

Background: The Bacillus subtilis genome (BGM) vector is a novel cloning system based on the natural competence that enables B. subtilis to import extracellular DNA fragments into the cell and incorporate the recombinogenic DNA into the genome vector by homologous recombination. The BGM vector system has several attractive properties, such as a megabase cloning capacity, stable propagation of cloned DNA inserts, and various modification strategies using RecA-mediated homologous recombination. However, the endogenous RecA activity may cause undesirable recombination, as has been observed in yeast artificial chromosome systems. In this study, we developed a novel BGM vector system of an inducible recA expression BGM vector (iREX), in which the expression of recA can be controlled by xylose in the medium.

Results: We constructed the iREX system by introducing the xylose-inducible recA expression cassette followed by the targeted deletion of the endogenous recA. Western blot analysis showed that the expression of recA was strictly controlled by xylose in the medium. In the absence of xylose, recA was not expressed in the iREX, and the RecA-mediated recombination reactions were greatly suppressed. By contrast, the addition of xylose successfully induced RecA expression, which enabled the iREX to exploit the same capacities of transformation and gene modifications observed with the conventional BGM vector. In addition, an evaluation of the stability of the cloned DNA insert demonstrated that the DNA fragments containing homologous sequences were more stably maintained in the iREX by suppressing undesirable homologous recombination.

Conclusions: We developed a novel BGM vector with inducible recA expression system, iREX, which enables us to manipulate large DNA fragments more stably than the conventional BGM vector by suppressing undesirable recombination. In addition, we demonstrate that the iREX can be applied to handling the DNA, which has several homologous sequences, such as multiple-reporter expression cassettes. Thus, the iREX expands the utility of the BGM vector as a platform for engineering large DNA fragments.

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Cloning of BAC1 into the iREX. (a) One-step cloning of BAC1 into the iREX. NmS, neomycin sensitive; NmR, neomycin resistant; SpcS, spectinomycin sensitive; SpcR, spectinomycin resistant; I, I-PpoI recognition sequence. (b) iREX/BAC1 was digested with I-PpoI followed by CHEF gel electrophoresis. The BAC1 insert is indicated as an open arrowhead, and the BGM vector is indicated as a closed arrowhead. A lambda DNA concatemer was used as a size marker in lane M. (c) Original BAC1 and genomic DNA of the iREX recombinant were digested with EcoRI or HindIII and hybridized with the original BAC1 clone as a probe. Band patterns identical to the original BAC1 clones were confirmed in the iREX recombinant, except for the bands derived from the BAC end sequences. The closed arrowhead indicates a BAC1-specific signal, and the open arrowheads indicate BGM-specific signals. In lane M, lambda/HindIII fragments were used as a size marker.
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Fig3: Cloning of BAC1 into the iREX. (a) One-step cloning of BAC1 into the iREX. NmS, neomycin sensitive; NmR, neomycin resistant; SpcS, spectinomycin sensitive; SpcR, spectinomycin resistant; I, I-PpoI recognition sequence. (b) iREX/BAC1 was digested with I-PpoI followed by CHEF gel electrophoresis. The BAC1 insert is indicated as an open arrowhead, and the BGM vector is indicated as a closed arrowhead. A lambda DNA concatemer was used as a size marker in lane M. (c) Original BAC1 and genomic DNA of the iREX recombinant were digested with EcoRI or HindIII and hybridized with the original BAC1 clone as a probe. Band patterns identical to the original BAC1 clones were confirmed in the iREX recombinant, except for the bands derived from the BAC end sequences. The closed arrowhead indicates a BAC1-specific signal, and the open arrowheads indicate BGM-specific signals. In lane M, lambda/HindIII fragments were used as a size marker.

Mentions: One of the most attractive properties of the BGM vector system is its capacity to clone very large DNA fragments. To examine this important feature in the iREX, we conducted one-step cloning of BAC DNA into the iREX. The BAC clone, designated BAC1, carried a 114 kb mouse genomic DNA fragment containing two class I odorant receptor genes [10]. We transformed the iREX with BAC1 to construct iREX/BAC1 (Figure 3a). Briefly, the iREX is resistant to spectinomycin and sensitive to neomycin because the CI repressor represses the Pr-neo cassette. Once the BAC1 insert is cloned directly into the iREX genome via homologous recombination, the recombinants become resistant to neomycin and sensitive to spectinomycin due to the replacement of the cI-spc cassette with the BAC1 insert. Because two I-PpoI recognition sequences are introduced at the both ends of the cloning site, the cloned BAC1 insert could be excised by digesting the genomic DNA of the iREX recombinant with I-PpoI and analyzed by contour-clamped homogeneous electric field (CHEF) gel electrophoresis (Figure 3b). One of 20 candidate clones (resistant to neomycin and sensitive to spectinomycin) contained the BAC1 insert. The cloned insert was confirmed by Southern blot analysis using the original BAC1 clone as a probe (Figure 3c). The digestion patterns of iREX/BAC1 were identical to those of the original BAC clone, except for the fragments derived from the ends of the insert. Thus, the iREX was able to clone a giant DNA sequence of over 100 kb, similar to the conventional BGM vector system.Figure 3


An inducible recA expression Bacillus subtilis genome vector for stable manipulation of large DNA fragments.

Ogawa T, Iwata T, Kaneko S, Itaya M, Hirota J - BMC Genomics (2015)

Cloning of BAC1 into the iREX. (a) One-step cloning of BAC1 into the iREX. NmS, neomycin sensitive; NmR, neomycin resistant; SpcS, spectinomycin sensitive; SpcR, spectinomycin resistant; I, I-PpoI recognition sequence. (b) iREX/BAC1 was digested with I-PpoI followed by CHEF gel electrophoresis. The BAC1 insert is indicated as an open arrowhead, and the BGM vector is indicated as a closed arrowhead. A lambda DNA concatemer was used as a size marker in lane M. (c) Original BAC1 and genomic DNA of the iREX recombinant were digested with EcoRI or HindIII and hybridized with the original BAC1 clone as a probe. Band patterns identical to the original BAC1 clones were confirmed in the iREX recombinant, except for the bands derived from the BAC end sequences. The closed arrowhead indicates a BAC1-specific signal, and the open arrowheads indicate BGM-specific signals. In lane M, lambda/HindIII fragments were used as a size marker.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4374399&req=5

Fig3: Cloning of BAC1 into the iREX. (a) One-step cloning of BAC1 into the iREX. NmS, neomycin sensitive; NmR, neomycin resistant; SpcS, spectinomycin sensitive; SpcR, spectinomycin resistant; I, I-PpoI recognition sequence. (b) iREX/BAC1 was digested with I-PpoI followed by CHEF gel electrophoresis. The BAC1 insert is indicated as an open arrowhead, and the BGM vector is indicated as a closed arrowhead. A lambda DNA concatemer was used as a size marker in lane M. (c) Original BAC1 and genomic DNA of the iREX recombinant were digested with EcoRI or HindIII and hybridized with the original BAC1 clone as a probe. Band patterns identical to the original BAC1 clones were confirmed in the iREX recombinant, except for the bands derived from the BAC end sequences. The closed arrowhead indicates a BAC1-specific signal, and the open arrowheads indicate BGM-specific signals. In lane M, lambda/HindIII fragments were used as a size marker.
Mentions: One of the most attractive properties of the BGM vector system is its capacity to clone very large DNA fragments. To examine this important feature in the iREX, we conducted one-step cloning of BAC DNA into the iREX. The BAC clone, designated BAC1, carried a 114 kb mouse genomic DNA fragment containing two class I odorant receptor genes [10]. We transformed the iREX with BAC1 to construct iREX/BAC1 (Figure 3a). Briefly, the iREX is resistant to spectinomycin and sensitive to neomycin because the CI repressor represses the Pr-neo cassette. Once the BAC1 insert is cloned directly into the iREX genome via homologous recombination, the recombinants become resistant to neomycin and sensitive to spectinomycin due to the replacement of the cI-spc cassette with the BAC1 insert. Because two I-PpoI recognition sequences are introduced at the both ends of the cloning site, the cloned BAC1 insert could be excised by digesting the genomic DNA of the iREX recombinant with I-PpoI and analyzed by contour-clamped homogeneous electric field (CHEF) gel electrophoresis (Figure 3b). One of 20 candidate clones (resistant to neomycin and sensitive to spectinomycin) contained the BAC1 insert. The cloned insert was confirmed by Southern blot analysis using the original BAC1 clone as a probe (Figure 3c). The digestion patterns of iREX/BAC1 were identical to those of the original BAC clone, except for the fragments derived from the ends of the insert. Thus, the iREX was able to clone a giant DNA sequence of over 100 kb, similar to the conventional BGM vector system.Figure 3

Bottom Line: We developed a novel BGM vector with inducible recA expression system, iREX, which enables us to manipulate large DNA fragments more stably than the conventional BGM vector by suppressing undesirable recombination.In addition, we demonstrate that the iREX can be applied to handling the DNA, which has several homologous sequences, such as multiple-reporter expression cassettes.Thus, the iREX expands the utility of the BGM vector as a platform for engineering large DNA fragments.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Graduate School of Bioscience and Bioengineering, Tokyo Institute of Technology, Yokohama, 226-8501, Japan. togawa@bio.titech.ac.jp.

ABSTRACT

Background: The Bacillus subtilis genome (BGM) vector is a novel cloning system based on the natural competence that enables B. subtilis to import extracellular DNA fragments into the cell and incorporate the recombinogenic DNA into the genome vector by homologous recombination. The BGM vector system has several attractive properties, such as a megabase cloning capacity, stable propagation of cloned DNA inserts, and various modification strategies using RecA-mediated homologous recombination. However, the endogenous RecA activity may cause undesirable recombination, as has been observed in yeast artificial chromosome systems. In this study, we developed a novel BGM vector system of an inducible recA expression BGM vector (iREX), in which the expression of recA can be controlled by xylose in the medium.

Results: We constructed the iREX system by introducing the xylose-inducible recA expression cassette followed by the targeted deletion of the endogenous recA. Western blot analysis showed that the expression of recA was strictly controlled by xylose in the medium. In the absence of xylose, recA was not expressed in the iREX, and the RecA-mediated recombination reactions were greatly suppressed. By contrast, the addition of xylose successfully induced RecA expression, which enabled the iREX to exploit the same capacities of transformation and gene modifications observed with the conventional BGM vector. In addition, an evaluation of the stability of the cloned DNA insert demonstrated that the DNA fragments containing homologous sequences were more stably maintained in the iREX by suppressing undesirable homologous recombination.

Conclusions: We developed a novel BGM vector with inducible recA expression system, iREX, which enables us to manipulate large DNA fragments more stably than the conventional BGM vector by suppressing undesirable recombination. In addition, we demonstrate that the iREX can be applied to handling the DNA, which has several homologous sequences, such as multiple-reporter expression cassettes. Thus, the iREX expands the utility of the BGM vector as a platform for engineering large DNA fragments.

Show MeSH
Related in: MedlinePlus