Lauritsen Ida, Kim Se Hyeuk, Porse Andreas, Nørholm Morten H H
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Lyngby, Denmark.
Methods Mol Biol. 2018;1772:469-476. doi: 10.1007/978-1-4939-7795-6_28.
Plasmids are highly useful tools for studying living cells and for heterologous expression of genes and pathways in cell factories. Standardized tools and operating procedures for handling such DNA vectors are core principles in synthetic biology. Here, we describe protocols for molecular cloning and exchange of genetic parts in the Standard European Vectors Architecture (SEVA) vector system. Additionally, to facilitate rapid testing and iterative bioengineering using different vector designs, we provide a one-step protocol for a universal CRISPR-Cas9-based plasmid curing system (pFREE) and demonstrate the application of this system to cure SEVA constructs (all vectors are available at SEVA/Addgene).
质粒是研究活细胞以及在细胞工厂中进行基因和途径异源表达的非常有用的工具。处理此类DNA载体的标准化工具和操作程序是合成生物学的核心原则。在这里，我们描述了标准欧洲载体架构（SEVA）载体系统中分子克隆和遗传元件交换的方案。此外，为了便于使用不同的载体设计进行快速测试和迭代生物工程，我们提供了一种基于CRISPR-Cas9的通用质粒消除系统（pFREE）的一步方案，并展示了该系统在消除SEVA构建体中的应用（所有载体可在SEVA/Addgene获得）。
