Limits...
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.

Show MeSH

Related in: MedlinePlus

Evaluation of the stability of the cloned DNA using inversion. (a) Schematic diagram of the evaluation of the cloned DNA stability using inversion. TetS, tetracycline sensitive; TetR, tetracycline resistant. (b) Numbers of tetracycline-resistant colonies in the BEST310 system and the iREX system. Error bars, s.d. n = 3. (c) Many tetracycline-resistant recombinants were observed in the BEST310 system. In the iREX system, the same results were obtained in the presence of xylose because of the induced RecA. In contrast, few colonies were observed using the iREX in the absence of xylose. This result indicates that the cloned DNA insert of the iREX is stably maintained due to the strong repression of recA. (d and e) Southern blot analysis using a tet probe revealed changes in the sizes of the signals, indicating the inversion of the BAC1 insert. The genomic DNA of the represented clones was digested with BamHI. In lane M, lambda/HindIII fragments were used as a size marker.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Evaluation of the stability of the cloned DNA using inversion. (a) Schematic diagram of the evaluation of the cloned DNA stability using inversion. TetS, tetracycline sensitive; TetR, tetracycline resistant. (b) Numbers of tetracycline-resistant colonies in the BEST310 system and the iREX system. Error bars, s.d. n = 3. (c) Many tetracycline-resistant recombinants were observed in the BEST310 system. In the iREX system, the same results were obtained in the presence of xylose because of the induced RecA. In contrast, few colonies were observed using the iREX in the absence of xylose. This result indicates that the cloned DNA insert of the iREX is stably maintained due to the strong repression of recA. (d and e) Southern blot analysis using a tet probe revealed changes in the sizes of the signals, indicating the inversion of the BAC1 insert. The genomic DNA of the represented clones was digested with BamHI. In lane M, lambda/HindIII fragments were used as a size marker.

Mentions: To examine this, we first evaluated the stability of the cloned insert in terms of homologous recombination-mediated inversion using the tet-inversion cassette system [10]. Because the tet-inversion cassette system is dependent on the construction of the tet, we deleted tet in iREX/BAC1 by replacing tet with the cI-bsr cassette, which consisted of the CI repressor gene and the blasticidin S resistance gene. Then, two incomplete fragments of the tetracycline resistance gene, te (5’ end) and et (3’ end), which shared an overlapping region of approximately 1.1 kb, were inserted at the ends of the BAC1 sequences of iREX/BAC1 and of BEST310/BAC1. The recombination between the overlapping homologous sequences between the incomplete tet fragments resulted in the inversion of the cloned inserts as well as the acquisition of tetracycline resistance because of the formation of the complete tet (Figure 4a). Overnight cultures of iREX/BAC1 or BEST310/BAC1 with the tet-inversion cassette were spread on LB plates containing tetracycline, and the numbers of tetracycline-resistant colonies were counted. Notably, many tetracycline-resistant colonies were formed in the BEST310 system. The same tendency was observed in the iREX system in the presence of xylose. In contrast, there were few colonies in the iREX system in the absence of xylose, indicating that the cloned DNA insert was maintained much more stably in the iREX system than in the conventional BGM vector system (Figure 4b and c). The inversion of the inserts was confirmed by Southern blot analysis using a tet probe (Figure 4d and e).Figure 4


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)

Evaluation of the stability of the cloned DNA using inversion. (a) Schematic diagram of the evaluation of the cloned DNA stability using inversion. TetS, tetracycline sensitive; TetR, tetracycline resistant. (b) Numbers of tetracycline-resistant colonies in the BEST310 system and the iREX system. Error bars, s.d. n = 3. (c) Many tetracycline-resistant recombinants were observed in the BEST310 system. In the iREX system, the same results were obtained in the presence of xylose because of the induced RecA. In contrast, few colonies were observed using the iREX in the absence of xylose. This result indicates that the cloned DNA insert of the iREX is stably maintained due to the strong repression of recA. (d and e) Southern blot analysis using a tet probe revealed changes in the sizes of the signals, indicating the inversion of the BAC1 insert. The genomic DNA of the represented clones was digested with BamHI. In lane M, lambda/HindIII fragments were used as a size marker.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Evaluation of the stability of the cloned DNA using inversion. (a) Schematic diagram of the evaluation of the cloned DNA stability using inversion. TetS, tetracycline sensitive; TetR, tetracycline resistant. (b) Numbers of tetracycline-resistant colonies in the BEST310 system and the iREX system. Error bars, s.d. n = 3. (c) Many tetracycline-resistant recombinants were observed in the BEST310 system. In the iREX system, the same results were obtained in the presence of xylose because of the induced RecA. In contrast, few colonies were observed using the iREX in the absence of xylose. This result indicates that the cloned DNA insert of the iREX is stably maintained due to the strong repression of recA. (d and e) Southern blot analysis using a tet probe revealed changes in the sizes of the signals, indicating the inversion of the BAC1 insert. The genomic DNA of the represented clones was digested with BamHI. In lane M, lambda/HindIII fragments were used as a size marker.
Mentions: To examine this, we first evaluated the stability of the cloned insert in terms of homologous recombination-mediated inversion using the tet-inversion cassette system [10]. Because the tet-inversion cassette system is dependent on the construction of the tet, we deleted tet in iREX/BAC1 by replacing tet with the cI-bsr cassette, which consisted of the CI repressor gene and the blasticidin S resistance gene. Then, two incomplete fragments of the tetracycline resistance gene, te (5’ end) and et (3’ end), which shared an overlapping region of approximately 1.1 kb, were inserted at the ends of the BAC1 sequences of iREX/BAC1 and of BEST310/BAC1. The recombination between the overlapping homologous sequences between the incomplete tet fragments resulted in the inversion of the cloned inserts as well as the acquisition of tetracycline resistance because of the formation of the complete tet (Figure 4a). Overnight cultures of iREX/BAC1 or BEST310/BAC1 with the tet-inversion cassette were spread on LB plates containing tetracycline, and the numbers of tetracycline-resistant colonies were counted. Notably, many tetracycline-resistant colonies were formed in the BEST310 system. The same tendency was observed in the iREX system in the presence of xylose. In contrast, there were few colonies in the iREX system in the absence of xylose, indicating that the cloned DNA insert was maintained much more stably in the iREX system than in the conventional BGM vector system (Figure 4b and c). The inversion of the inserts was confirmed by Southern blot analysis using a tet probe (Figure 4d and e).Figure 4

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