Department of Cell and Metabolic Biology, Leibniz Institute of Plant Biochemistry, Halle, Germany.
Nambawan Biotech GmbH, Halle, Germany.
Methods Mol Biol. 2020;2205:125-141. doi: 10.1007/978-1-0716-0908-8_8.
Modular cloning systems that rely on type IIS enzymes for DNA assembly have many advantages for complex pathway engineering. These systems are simple to use, efficient, and allow users to assemble multigene constructs by performing a series of one-pot assembly steps, starting from libraries of cloned and sequenced parts. The efficiency of these systems also facilitates the generation of libraries of construct variants. We describe here a protocol for assembly of multigene constructs using the Modular Cloning system MoClo. Making constructs using the MoClo system requires users to first define the structure of the final construct to identify all basic parts and vectors required for the construction strategy. The assembly strategy is then defined following a set of standard rules. Multigene constructs are then assembled using a series of one-pot assembly steps with the set of identified parts and vectors.
模块化克隆系统依赖于 II 型酶进行 DNA 组装，对于复杂途径的工程具有许多优势。这些系统使用简单、高效，并且允许用户通过执行一系列一锅组装步骤，从克隆和测序的文库部分开始，组装多基因构建体。这些系统的效率也有助于构建变体文库的生成。我们在这里描述了使用模块化克隆系统 MoClo 组装多基因构建体的方案。使用 MoClo 系统构建构建体，用户首先需要定义最终构建体的结构，以确定构建策略所需的所有基本部分和载体。然后，根据一组标准规则定义组装策略。然后，使用一组鉴定的部分和载体，通过一系列一锅组装步骤来组装多基因构建体。
