Ogawa Takafumi, Iwata Tetsuo, Kaneko Shinya, Itaya Mitsuhiro, Hirota Junji
Department of Bioengineering, Graduate School of Bioscience and Bioengineering, Tokyo Institute of Technology, Yokohama, 226-8501, Japan.
Department of Molecular Bioscience, Graduate School of Bioscience and Bioengineering, Tokyo Institute of Technology, Yokohama, 226-8501, Japan.
BMC Genomics. 2015 Mar 18;16(1):209. doi: 10.1186/s12864-015-1425-4.
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.
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.
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.
枯草芽孢杆菌基因组（BGM）载体是一种基于自然感受态的新型克隆系统，它能使枯草芽孢杆菌将细胞外DNA片段导入细胞，并通过同源重组将重组DNA整合到基因组载体中。BGM载体系统具有几个吸引人的特性，比如兆碱基的克隆能力、克隆DNA插入片段的稳定增殖以及使用RecA介导的同源重组的各种修饰策略。然而，正如在酵母人工染色体系统中所观察到的那样，内源性RecA活性可能会导致不良重组。在本研究中，我们开发了一种新型的可诱导recA表达的BGM载体（iREX）系统，其中recA的表达可由培养基中的木糖控制。
我们通过引入木糖诱导的recA表达盒，随后对内源性recA进行靶向缺失，构建了iREX系统。蛋白质免疫印迹分析表明，recA的表达受到培养基中木糖的严格控制。在没有木糖的情况下，recA在iREX中不表达，RecA介导的重组反应受到极大抑制。相比之下，添加木糖成功诱导了RecA表达，这使得iREX能够发挥与传统BGM载体相同的转化和基因修饰能力。此外，对克隆DNA插入片段稳定性的评估表明，通过抑制不良同源重组，含有同源序列的DNA片段在iREX中得到更稳定的维持。
我们开发了一种具有可诱导recA表达系统的新型BGM载体iREX，它使我们能够通过抑制不良重组比传统BGM载体更稳定地操作大的DNA片段。此外，我们证明iREX可应用于处理具有多个同源序列的DNA，比如多报告基因表达盒。因此，iREX扩展了BGM载体作为工程化大DNA片段平台的用途。
