Abstract
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.
Highlights
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
On the basis of prior knowledge of dominant mutators alleles that interfere with DNA replication fidelity, we sought to design a series of small-molecule inducible mutagenesis plasmids (MPs) that offer broad mutational spectra and high levels of mutagenesis in bacterial cells
We recently reported the development and application of an MP for in vivo mutagenesis during phage-assisted continuous evolution (PACE)[10]
Summary
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 o24 h, outperforming chemical mutagens, ultraviolet light and the mutator strain XL1-Red under similar conditions. This system outperforms commonly used in vivo mutator and chemical mutagenesis methods in mutational efficiency and spectrum, and can even achieve mutational potency and spectrum comparable to that of state-of-the-art in vitro mutagenesis methods We apply this system to two directed evolution case studies in bacteria and bacteriophage, validating their ability to access biomolecule variants with novel properties with greater effectiveness than previously described methods. This system substantially advances in vivo mutagenesis capabilities and increases the effectiveness of laboratory evolution efforts
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