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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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Stable gene manipulation in the iREX by preventing the deletion. (a) Schematic diagram of the insertion of IRES-tauEGFP-cI-spc and IRES-tauLacZ into MOR42-3 and MOR42-2, respectively, and the assumed deletion at the IRES-tau sequences. (b) The signal derived from the deletion was observed in BEST310/BAC1-GL (open arrowhead). The same result was obtained in iREX/BAC1-GL in the presence of xylose because of the induced RecA. In contrast, the signal derived from the deletion was not observed for iREX/BAC1-GL in the absence of xylose due to the strong repression of recA. The closed arrowheads indicate the signals derived from the intact inserts of BEST310/BAC1-GL and iREX/BAC1-GL. BEST310/BAC1-GL-deletion, which was screened from BEST310/BAC1-GL culture by neomycin resistance and spectinomycin sensitivity, was used as a control of deletion. The BGM vector is indicated as a closed arrow. (c) The proportions of the spectinomycin-resistant clones containing two IRES-tau sequences were 69% and 73% in the BEST310/BAC1-GL and iREX/BAC1-GL with xylose, respectively. In contrast, the proportion of the spectinomycin-resistant clones was 93% in the iREX/BAC1-GL without xylose. Error bars, s.d. n = 3.
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Fig5: Stable gene manipulation in the iREX by preventing the deletion. (a) Schematic diagram of the insertion of IRES-tauEGFP-cI-spc and IRES-tauLacZ into MOR42-3 and MOR42-2, respectively, and the assumed deletion at the IRES-tau sequences. (b) The signal derived from the deletion was observed in BEST310/BAC1-GL (open arrowhead). The same result was obtained in iREX/BAC1-GL in the presence of xylose because of the induced RecA. In contrast, the signal derived from the deletion was not observed for iREX/BAC1-GL in the absence of xylose due to the strong repression of recA. The closed arrowheads indicate the signals derived from the intact inserts of BEST310/BAC1-GL and iREX/BAC1-GL. BEST310/BAC1-GL-deletion, which was screened from BEST310/BAC1-GL culture by neomycin resistance and spectinomycin sensitivity, was used as a control of deletion. The BGM vector is indicated as a closed arrow. (c) The proportions of the spectinomycin-resistant clones containing two IRES-tau sequences were 69% and 73% in the BEST310/BAC1-GL and iREX/BAC1-GL with xylose, respectively. In contrast, the proportion of the spectinomycin-resistant clones was 93% in the iREX/BAC1-GL without xylose. Error bars, s.d. n = 3.

Mentions: We inserted IRES-tauEGFP-cI-spc and IRES-tauLacZ 3 bp downstream of the stop codon of the class I odorant receptor genes of MOR42-3 and MOR42-2 in iREX/BAC1 and BEST310/BAC1 to generate iREX/BAC1-GL and BEST310/BAC1-GL, respectively (Figure 5a). Fusion of the microtubule-associated protein tau with the reporter protein enables the visualization of the axonal projections of neurons expressing the reporter gene. Both iREX/BAC1-GL and BEST310/BAC1-GL contain two homologous sequences of IRES-tau. First, we spread these recombinants on LB plates containing spectinomycin, and then we inoculated the formed colonies into liquid spectinomycin-free LB medium. The genomic DNA of the overnight cultures was digested with I-PpoI and analyzed using CHEF gel electrophoresis to examine the digestion pattern derived from the deletion. In BEST310/BAC1-GL, there was an additional signal, indicating that the deletion derived from the RecA activity had occurred (Figure 5b). The same result was obtained in the iREX/BAC1-GL with xylose due to the induced RecA. By contrast, no deletion signal was shown by the iREX/BAC1-GL in the absence of xylose. To examine the deletion event further, we quantified the event by estimating the proportions of the spectinomycin-resistant clones containing two homologous sequences of IRES-tau. The proportions of the intact clones were 69% and 73% in the BEST310/BAC1-GL and iREX/BAC1-GL with xylose, respectively (Figure 5c). On the other hand, the proportion of the intact clones was 93% in the iREX/BAC1-GL without xylose. These results indicate that the iREX can stably maintain the cloned DNA insert by preventing deletion at two homologous sequences.Figure 5


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)

Stable gene manipulation in the iREX by preventing the deletion. (a) Schematic diagram of the insertion of IRES-tauEGFP-cI-spc and IRES-tauLacZ into MOR42-3 and MOR42-2, respectively, and the assumed deletion at the IRES-tau sequences. (b) The signal derived from the deletion was observed in BEST310/BAC1-GL (open arrowhead). The same result was obtained in iREX/BAC1-GL in the presence of xylose because of the induced RecA. In contrast, the signal derived from the deletion was not observed for iREX/BAC1-GL in the absence of xylose due to the strong repression of recA. The closed arrowheads indicate the signals derived from the intact inserts of BEST310/BAC1-GL and iREX/BAC1-GL. BEST310/BAC1-GL-deletion, which was screened from BEST310/BAC1-GL culture by neomycin resistance and spectinomycin sensitivity, was used as a control of deletion. The BGM vector is indicated as a closed arrow. (c) The proportions of the spectinomycin-resistant clones containing two IRES-tau sequences were 69% and 73% in the BEST310/BAC1-GL and iREX/BAC1-GL with xylose, respectively. In contrast, the proportion of the spectinomycin-resistant clones was 93% in the iREX/BAC1-GL without xylose. Error bars, s.d. n = 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig5: Stable gene manipulation in the iREX by preventing the deletion. (a) Schematic diagram of the insertion of IRES-tauEGFP-cI-spc and IRES-tauLacZ into MOR42-3 and MOR42-2, respectively, and the assumed deletion at the IRES-tau sequences. (b) The signal derived from the deletion was observed in BEST310/BAC1-GL (open arrowhead). The same result was obtained in iREX/BAC1-GL in the presence of xylose because of the induced RecA. In contrast, the signal derived from the deletion was not observed for iREX/BAC1-GL in the absence of xylose due to the strong repression of recA. The closed arrowheads indicate the signals derived from the intact inserts of BEST310/BAC1-GL and iREX/BAC1-GL. BEST310/BAC1-GL-deletion, which was screened from BEST310/BAC1-GL culture by neomycin resistance and spectinomycin sensitivity, was used as a control of deletion. The BGM vector is indicated as a closed arrow. (c) The proportions of the spectinomycin-resistant clones containing two IRES-tau sequences were 69% and 73% in the BEST310/BAC1-GL and iREX/BAC1-GL with xylose, respectively. In contrast, the proportion of the spectinomycin-resistant clones was 93% in the iREX/BAC1-GL without xylose. Error bars, s.d. n = 3.
Mentions: We inserted IRES-tauEGFP-cI-spc and IRES-tauLacZ 3 bp downstream of the stop codon of the class I odorant receptor genes of MOR42-3 and MOR42-2 in iREX/BAC1 and BEST310/BAC1 to generate iREX/BAC1-GL and BEST310/BAC1-GL, respectively (Figure 5a). Fusion of the microtubule-associated protein tau with the reporter protein enables the visualization of the axonal projections of neurons expressing the reporter gene. Both iREX/BAC1-GL and BEST310/BAC1-GL contain two homologous sequences of IRES-tau. First, we spread these recombinants on LB plates containing spectinomycin, and then we inoculated the formed colonies into liquid spectinomycin-free LB medium. The genomic DNA of the overnight cultures was digested with I-PpoI and analyzed using CHEF gel electrophoresis to examine the digestion pattern derived from the deletion. In BEST310/BAC1-GL, there was an additional signal, indicating that the deletion derived from the RecA activity had occurred (Figure 5b). The same result was obtained in the iREX/BAC1-GL with xylose due to the induced RecA. By contrast, no deletion signal was shown by the iREX/BAC1-GL in the absence of xylose. To examine the deletion event further, we quantified the event by estimating the proportions of the spectinomycin-resistant clones containing two homologous sequences of IRES-tau. The proportions of the intact clones were 69% and 73% in the BEST310/BAC1-GL and iREX/BAC1-GL with xylose, respectively (Figure 5c). On the other hand, the proportion of the intact clones was 93% in the iREX/BAC1-GL without xylose. These results indicate that the iREX can stably maintain the cloned DNA insert by preventing deletion at two homologous sequences.Figure 5

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