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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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Construction of therecA-inducible BGM vector system. (a) The BEST310 and iREX constructs that possess two antibiotic resistance gene cassettes for BAC cloning: Pr-neo, a lambda Pr promoter fused to the neomycin resistance gene (neo), and cI-spc, which contains cI encoding the CI repressor protein, which binds to the Pr promoter, fused to the spectinomycin resistance gene (spc). The closed and open arrows indicate the BAC cloning site, and the red and blue lines indicate the pBR322 sequence. (b) The inducible recA expression cassette, pX-recA, was inserted at the amyE locus of the BEST310 genome via homologous recombination. amyE is not essential for the viability of B. subtilis [15]. cat, chloramphenicol acetyltransferase; H, HindIII; X, XhoI. (c) After introducing the pX-recA, the endogenous recA was replaced with the tetracycline resistance gene (tet) via homologous recombination. X, XhoI. (d) Southern blot analysis using an amyE probe indicated the correct insertion of pX-recA. The genomic DNA of the represented clones was digested with HindIII. The open arrowhead indicates the intact amyE in BEST310. The closed arrowheads indicate 5’-amyE and 3’-amyE divided by the insertion of pX-recA. (e) Southern blot analysis using a recA probe indicated the correct insertion of pCTP. The genomic DNA of the represented clones was digested with XhoI. The open arrowheads indicate the endogenous recA. The closed arrowheads indicate the inducible recA derived from pX-recA.
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Fig1: Construction of therecA-inducible BGM vector system. (a) The BEST310 and iREX constructs that possess two antibiotic resistance gene cassettes for BAC cloning: Pr-neo, a lambda Pr promoter fused to the neomycin resistance gene (neo), and cI-spc, which contains cI encoding the CI repressor protein, which binds to the Pr promoter, fused to the spectinomycin resistance gene (spc). The closed and open arrows indicate the BAC cloning site, and the red and blue lines indicate the pBR322 sequence. (b) The inducible recA expression cassette, pX-recA, was inserted at the amyE locus of the BEST310 genome via homologous recombination. amyE is not essential for the viability of B. subtilis [15]. cat, chloramphenicol acetyltransferase; H, HindIII; X, XhoI. (c) After introducing the pX-recA, the endogenous recA was replaced with the tetracycline resistance gene (tet) via homologous recombination. X, XhoI. (d) Southern blot analysis using an amyE probe indicated the correct insertion of pX-recA. The genomic DNA of the represented clones was digested with HindIII. The open arrowhead indicates the intact amyE in BEST310. The closed arrowheads indicate 5’-amyE and 3’-amyE divided by the insertion of pX-recA. (e) Southern blot analysis using a recA probe indicated the correct insertion of pCTP. The genomic DNA of the represented clones was digested with XhoI. The open arrowheads indicate the endogenous recA. The closed arrowheads indicate the inducible recA derived from pX-recA.

Mentions: The inducible recA expression BGM vector (iREX) was constructed based on a BGM vector, BEST310, that was designed for BAC cloning [7], by introducing the inducible recA expression cassette followed by the targeted deletion of the endogenous recA (Figure 1a). For the inducible expression of recA, we used the gene expression cassette pX [15], in which the inducible promoter is regulated by xylose, and this cassette was designed to integrate into the amyE locus of B. subtilis. The inducible recA expression cassette pX-recA was constructed by cloning the B. subtilis recA into the BamHI site of pX and was integrated into the amyE locus of BEST310 to generate BEST310/pX-recA (Figure 1b). To delete the endogenous recA from BEST310/pX-recA, the targeted replacement of the endogenous recA with a tetracycline resistance gene (tet) was performed using pCTP, in which tet was inserted between the flanking sequences of the endogenous recA designated cinA and pbpX (Figure 1c). The resulting recombinant construct was designated as the inducible recA expression BGM vector, iREX. The insertion of pX-recA and the replacement of the endogenous recA with tet were confirmed by Southern blot analysis using an amyE probe and a recA probe (Figure 1d and e).Figure 1


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)

Construction of therecA-inducible BGM vector system. (a) The BEST310 and iREX constructs that possess two antibiotic resistance gene cassettes for BAC cloning: Pr-neo, a lambda Pr promoter fused to the neomycin resistance gene (neo), and cI-spc, which contains cI encoding the CI repressor protein, which binds to the Pr promoter, fused to the spectinomycin resistance gene (spc). The closed and open arrows indicate the BAC cloning site, and the red and blue lines indicate the pBR322 sequence. (b) The inducible recA expression cassette, pX-recA, was inserted at the amyE locus of the BEST310 genome via homologous recombination. amyE is not essential for the viability of B. subtilis [15]. cat, chloramphenicol acetyltransferase; H, HindIII; X, XhoI. (c) After introducing the pX-recA, the endogenous recA was replaced with the tetracycline resistance gene (tet) via homologous recombination. X, XhoI. (d) Southern blot analysis using an amyE probe indicated the correct insertion of pX-recA. The genomic DNA of the represented clones was digested with HindIII. The open arrowhead indicates the intact amyE in BEST310. The closed arrowheads indicate 5’-amyE and 3’-amyE divided by the insertion of pX-recA. (e) Southern blot analysis using a recA probe indicated the correct insertion of pCTP. The genomic DNA of the represented clones was digested with XhoI. The open arrowheads indicate the endogenous recA. The closed arrowheads indicate the inducible recA derived from pX-recA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Construction of therecA-inducible BGM vector system. (a) The BEST310 and iREX constructs that possess two antibiotic resistance gene cassettes for BAC cloning: Pr-neo, a lambda Pr promoter fused to the neomycin resistance gene (neo), and cI-spc, which contains cI encoding the CI repressor protein, which binds to the Pr promoter, fused to the spectinomycin resistance gene (spc). The closed and open arrows indicate the BAC cloning site, and the red and blue lines indicate the pBR322 sequence. (b) The inducible recA expression cassette, pX-recA, was inserted at the amyE locus of the BEST310 genome via homologous recombination. amyE is not essential for the viability of B. subtilis [15]. cat, chloramphenicol acetyltransferase; H, HindIII; X, XhoI. (c) After introducing the pX-recA, the endogenous recA was replaced with the tetracycline resistance gene (tet) via homologous recombination. X, XhoI. (d) Southern blot analysis using an amyE probe indicated the correct insertion of pX-recA. The genomic DNA of the represented clones was digested with HindIII. The open arrowhead indicates the intact amyE in BEST310. The closed arrowheads indicate 5’-amyE and 3’-amyE divided by the insertion of pX-recA. (e) Southern blot analysis using a recA probe indicated the correct insertion of pCTP. The genomic DNA of the represented clones was digested with XhoI. The open arrowheads indicate the endogenous recA. The closed arrowheads indicate the inducible recA derived from pX-recA.
Mentions: The inducible recA expression BGM vector (iREX) was constructed based on a BGM vector, BEST310, that was designed for BAC cloning [7], by introducing the inducible recA expression cassette followed by the targeted deletion of the endogenous recA (Figure 1a). For the inducible expression of recA, we used the gene expression cassette pX [15], in which the inducible promoter is regulated by xylose, and this cassette was designed to integrate into the amyE locus of B. subtilis. The inducible recA expression cassette pX-recA was constructed by cloning the B. subtilis recA into the BamHI site of pX and was integrated into the amyE locus of BEST310 to generate BEST310/pX-recA (Figure 1b). To delete the endogenous recA from BEST310/pX-recA, the targeted replacement of the endogenous recA with a tetracycline resistance gene (tet) was performed using pCTP, in which tet was inserted between the flanking sequences of the endogenous recA designated cinA and pbpX (Figure 1c). The resulting recombinant construct was designated as the inducible recA expression BGM vector, iREX. The insertion of pX-recA and the replacement of the endogenous recA with tet were confirmed by Southern blot analysis using an amyE probe and a recA probe (Figure 1d and e).Figure 1

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