Leibniz-Institut für Pflanzenbiochemie, Department of Cell and Metabolic Biology, Halle, Germany.
Icon Genetics GmbH, Halle, Germany.
Methods Mol Biol. 2025;2850:21-39. doi: 10.1007/978-1-0716-4220-7_2.
Modular cloning systems that rely on type IIS enzymes for DNA assembly have many advantages for construct engineering for biological research and synthetic biology. 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 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.
依赖于 II 型酶的模块化克隆系统在生物研究和合成生物学的构建工程中具有许多优势。这些系统易于使用、高效，并允许用户通过执行一系列一锅组装步骤，从克隆和测序部分的库开始，组装多基因构建体。这些系统的效率也促进了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 系统构建构建体需要首先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。多基因构建体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进行组装。
