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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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Western blot analysis and optimization of RecA induction. (a) Western blot analysis with an anti-RecA antibody indicated the expression of RecA in the presence of xylose. Remarkably, expression of RecA in the absence of xylose was strictly repressed. BEST310/ΔrecA, that was constructed by replacing the endogenous recA of BEST310 with tet of pCTP, was used as a negative control. (b) Schematic diagram of the cloning procedure. erm, erythromycin resistance gene; EmS, erythromycin sensitive; EmR, erythromycin resistant. (c) Numbers of erythromycin-resistant colonies under various induction times after addition of 1.0% xylose. Error bars, s.d. n = 3. (d) Numbers of erythromycin-resistant colonies under various xylose concentrations at 150 min induction. Error bars, s.d. n = 3. (e) Erythromycin-resistant recombinants were obtained only in the presence of xylose. The picture of xylose (+) is representative plate at a final xylose concentration of 1.0% at the induction time of 150 min. (f) All colonies formed on LB plates containing erythromycin were fluorescent due to their GFP gene.
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Fig2: Western blot analysis and optimization of RecA induction. (a) Western blot analysis with an anti-RecA antibody indicated the expression of RecA in the presence of xylose. Remarkably, expression of RecA in the absence of xylose was strictly repressed. BEST310/ΔrecA, that was constructed by replacing the endogenous recA of BEST310 with tet of pCTP, was used as a negative control. (b) Schematic diagram of the cloning procedure. erm, erythromycin resistance gene; EmS, erythromycin sensitive; EmR, erythromycin resistant. (c) Numbers of erythromycin-resistant colonies under various induction times after addition of 1.0% xylose. Error bars, s.d. n = 3. (d) Numbers of erythromycin-resistant colonies under various xylose concentrations at 150 min induction. Error bars, s.d. n = 3. (e) Erythromycin-resistant recombinants were obtained only in the presence of xylose. The picture of xylose (+) is representative plate at a final xylose concentration of 1.0% at the induction time of 150 min. (f) All colonies formed on LB plates containing erythromycin were fluorescent due to their GFP gene.

Mentions: To confirm the xylose-induced RecA expression, we first performed Western blot analysis. In the absence of xylose, there was no immunosignal for RecA detected from the iREX, indicating that the expression of recA was strictly repressed. In contrast, a signal for RecA was detected from the iREX in the presence of xylose as well as from the conventional BGM vector, BEST310. These results indicate that the expression of recA was strictly controlled by xylose (Figure 2a).Figure 2


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)

Western blot analysis and optimization of RecA induction. (a) Western blot analysis with an anti-RecA antibody indicated the expression of RecA in the presence of xylose. Remarkably, expression of RecA in the absence of xylose was strictly repressed. BEST310/ΔrecA, that was constructed by replacing the endogenous recA of BEST310 with tet of pCTP, was used as a negative control. (b) Schematic diagram of the cloning procedure. erm, erythromycin resistance gene; EmS, erythromycin sensitive; EmR, erythromycin resistant. (c) Numbers of erythromycin-resistant colonies under various induction times after addition of 1.0% xylose. Error bars, s.d. n = 3. (d) Numbers of erythromycin-resistant colonies under various xylose concentrations at 150 min induction. Error bars, s.d. n = 3. (e) Erythromycin-resistant recombinants were obtained only in the presence of xylose. The picture of xylose (+) is representative plate at a final xylose concentration of 1.0% at the induction time of 150 min. (f) All colonies formed on LB plates containing erythromycin were fluorescent due to their GFP gene.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Western blot analysis and optimization of RecA induction. (a) Western blot analysis with an anti-RecA antibody indicated the expression of RecA in the presence of xylose. Remarkably, expression of RecA in the absence of xylose was strictly repressed. BEST310/ΔrecA, that was constructed by replacing the endogenous recA of BEST310 with tet of pCTP, was used as a negative control. (b) Schematic diagram of the cloning procedure. erm, erythromycin resistance gene; EmS, erythromycin sensitive; EmR, erythromycin resistant. (c) Numbers of erythromycin-resistant colonies under various induction times after addition of 1.0% xylose. Error bars, s.d. n = 3. (d) Numbers of erythromycin-resistant colonies under various xylose concentrations at 150 min induction. Error bars, s.d. n = 3. (e) Erythromycin-resistant recombinants were obtained only in the presence of xylose. The picture of xylose (+) is representative plate at a final xylose concentration of 1.0% at the induction time of 150 min. (f) All colonies formed on LB plates containing erythromycin were fluorescent due to their GFP gene.
Mentions: To confirm the xylose-induced RecA expression, we first performed Western blot analysis. In the absence of xylose, there was no immunosignal for RecA detected from the iREX, indicating that the expression of recA was strictly repressed. In contrast, a signal for RecA was detected from the iREX in the presence of xylose as well as from the conventional BGM vector, BEST310. These results indicate that the expression of recA was strictly controlled by xylose (Figure 2a).Figure 2

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