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

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• 作者：Takafumi Ogawa (1)
  Tetsuo Iwata (1)
  Shinya Kaneko (2)
  Mitsuhiro Itaya (3)
  Junji Hirota (1) (4)
  
  1. Department of Bioengineering ; Graduate School of Bioscience and Bioengineering ; Tokyo Institute of Technology ; Yokohama ; 226-8501 ; Japan
  2. Department of Molecular Bioscience ; Graduate School of Bioscience and Bioengineering ; Tokyo Institute of Technology ; Yokohama ; 226-8501 ; Japan
  3. Institute for Advanced Biosciences ; Keio University ; Tsuruoka ; 997-0017 ; Japan
  4. Center for Biological Resources and Informatics ; Tokyo Institute of Technology ; 4259-B63 Nagatsuta-cho ; Midori-ku ; Yokohama ; 226-8501 ; Japan
  
• 关键词：Bacillus subtilis ; BGM vector ; Genome engineering ; RecA ; Homologous recombination
• 刊名：BMC Genomics
• 出版年：2015
• 出版时间：December 2015
• 年：2015
• 卷：16
• 期：1
• 全文大小：1,359 KB
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• 刊物主题：Life Sciences, general; Microarrays; Proteomics; Animal Genetics and Genomics; Microbial Genetics and Genomics; Plant Genetics & Genomics;
• 出版者：BioMed Central
• ISSN：1471-2164

文摘

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.