Development of potent in vivo mutagenesis plasmids with broad mutational spectra.
Development of potent in vivo mutagenesis plasmids with broad mutational spectra.
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DOI：
10.1038/ncomms9425
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发表时间：
2015-10-07
影响因子：
16.6
通讯作者：
Liu DR
中科院分区：
文献类型：
--
作者：
Badran AH;Liu DR
增强细胞中随机诱变的方法提供了优于体外诱变的优点，但目前的体内方法存在缺乏控制、基因组不稳定、效率低和突变谱窄的问题。我们采用机制驱动的方法，在大肠杆菌中建立了一个有效的、可诱导的、广谱的、基于载体的突变系统。大肠杆菌，增强突变322，000倍以上的基础水平，超过了广泛使用的体内和体外方法的突变效率和光谱。我们证明，该系统可用于在野生型大肠杆菌中进化抗生素抗性。在相同条件下，诱变剂、紫外线和突变菌株XL 1-Red的诱变效果均优于化学诱变剂。该系统还使得T7 RNA聚合酶变体能够在<10小时内使用T3启动子启动转录。我们的研究结果能够在体内对染色体、游离体和病毒进行广谱诱变，并且适用于细菌和噬菌体介导的实验室进化平台。 随机DNA诱变在自然界和实验室中都提供了遗传多样性。在这里，Badran和Liu提出了一种有效的、可诱导的、广谱的、基于载体的大肠杆菌诱变系统。大肠杆菌的突变效率和光谱的最广泛使用的体内和体外诱变方法。
Methods to enhance random mutagenesis in cells offer advantages over in vitro mutagenesis, but current in vivo methods suffer from a lack of control, genomic instability, low efficiency and narrow mutational spectra. Using a mechanism-driven approach, we created a potent, inducible, broad-spectrum and vector-based mutagenesis system in E. coli that enhances mutation 322,000-fold over basal levels, surpassing the mutational efficiency and spectra of widely used in vivo and in vitro methods. We demonstrate that this system can be used to evolve antibiotic resistance in wild-type E. coli in <24 h, outperforming chemical mutagens, ultraviolet light and the mutator strain XL1-Red under similar conditions. This system also enables the continuous evolution of T7 RNA polymerase variants capable of initiating transcription using the T3 promoter in <10 h. Our findings enable broad-spectrum mutagenesis of chromosomes, episomes and viruses in vivo, and are applicable to both bacterial and bacteriophage-mediated laboratory evolution platforms. Random DNA mutagenesis provides genetic diversity both in nature and the laboratory. Here, Badran and Liu present a potent, inducible, broad-spectrum and vector-based mutagenesis system in E. coli that surpasses the mutational efficiency and spectra of the most widely used in vivo and in vitro mutagenesis methods.