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

Takafumi Ogawa,Mitsuhiro Itaya,Shinya Kaneko,Tetsuo Iwata,Junji Hirota

doi:10.1186/s12864-015-1425-4

Takafumi Ogawa, Mitsuhiro Itaya + Show 3 more

Open Access

https://doi.org/10.1186/s12864-015-1425-4

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Journal: BMC genomics	Publication Date: Mar 18, 2015
Citations: 12	License type: CC BY 4.0

Affiliation: Keio University, Tokyo Institute of Technology

Abstract

BackgroundThe 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.ResultsWe 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.ConclusionsWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1425-4) contains supplementary material, which is available to authorized users.

Highlights

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
Construction of the inducible recA expression BGM vector The inducible recA expression BGM vector was constructed based on a BGM vector, BEST310, that was designed for Bacterial artificial chromosome (BAC) cloning [7], by introducing the inducible recA expression cassette followed by the targeted deletion of the endogenous recA (Figure 1a)
For the inducible expression of recA, we used the gene expression cassette pX [15], in which the inducible promoter is regulated by xylose, and this cassette was designed to integrate into the amyE locus of B. subtilis

Summary

Introduction

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 Bacillus subtilis genome (BGM) vector system has been developed as a novel cloning system for handling large DNA fragments [5,6,7]. B. subtilis can import extracellular DNA molecules into the cytoplasm in a singlestranded form through its transformation machinery, and the recombinogenic DNA is integrated into the genome via RecA-mediated homologous recombination [8]. These sequential events are called “natural competence”. Various types of genomic DNA inserts, including cyanobacteria, Arabidopsis and mouse, have been cloned into the BGM vector [5,6,7,9]

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