Tikh Ilya B, Samuelson James C
Protein Expression and Modification Division, New England BioLabs, Inc., Ipswich, MA, 01938-2723, USA.
Biol Methods Protoc. 2016 Dec 27;1(1):bpw004. doi: 10.1093/biomethods/bpw004. eCollection 2016 Mar.
The ability to alter the genomic material of a prokaryotic cell is necessary for experiments designed to define the biology of the organism. In addition, the production of biomolecules may be significantly improved by application of engineered prokaryotic host cells. Furthermore, in the age of synthetic biology, speed and efficiency are key factors when choosing a method for genome alteration. To address these needs, we have developed a method for modification of the genome named FAST-GE for ast ssembly-mediated carless argeted enome diting. Traditional cloning steps such as plasmid transformation, propagation and isolation were eliminated. Instead, we developed a DNA assembly-based approach for generating scarless strain modifications, which may include point mutations, deletions and gene replacements, within 48 h after the receipt of polymerase chain reaction primers. The protocol uses established, but optimized, genome modification components such as I-SceI endonuclease to improve recombination efficiency and SacB as a counter-selection mechanism. All DNA-encoded components are assembled into a single allele-exchange vector named pDEL. We were able to rapidly modify the genomes of both B and K-12 strains with high efficiency. In principle, the method may be applied to other prokaryotic organisms capable of circular dsDNA uptake and homologous recombination.
改变原核细胞基因组物质的能力对于旨在定义生物体生物学特性的实验来说是必要的。此外，通过应用工程化原核宿主细胞，生物分子的产量可能会显著提高。再者，在合成生物学时代，速度和效率是选择基因组改变方法时的关键因素。为满足这些需求，我们开发了一种名为FAST-GE（即单链组装介导的无痕靶向基因组编辑）的基因组修饰方法。传统的克隆步骤，如质粒转化、扩增和分离都被省去了。相反，我们开发了一种基于DNA组装的方法来产生无痕菌株修饰，在收到聚合酶链反应引物后的48小时内，这些修饰可能包括点突变、缺失和基因替换。该方案使用已确立但经过优化的基因组修饰元件，如I-SceI内切核酸酶来提高重组效率，并使用SacB作为反选择机制。所有DNA编码元件都组装到一个名为pDEL的单等位基因交换载体中。我们能够高效快速地修饰B菌株和K-12菌株的基因组。原则上，该方法可应用于其他能够摄取环状双链DNA并进行同源重组的原核生物。
