Marillonnet Sylvestre, Werner Stefan
Department of Cell and Metabolic Biology, Leibniz-Institut für Pflanzenbiochemie, Halle, Germany.
Nambawan Biotech GmbH, Halle, Germany.
Methods Mol Biol. 2019;1927:93-109. doi: 10.1007/978-1-4939-9142-6_7.
Efficient DNA assembly methods are essential tools for synthetic biology and metabolic engineering. Among several recently developed methods that allow assembly of multiple DNA fragments in a single step, DNA assembly using type IIS enzymes provides many advantages for complex pathway engineering. In particular, it provides the ability for the user to quickly assemble multigene constructs using a series of simple one-pot assembly steps starting from libraries of cloned and sequenced parts. 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. Basic parts that are not yet available need to be made. Multigene constructs are then assembled using a series of one-pot assembly steps with the set of identified parts and vectors.
高效的DNA组装方法是合成生物学和代谢工程的重要工具。在最近开发的几种能够在一步中组装多个DNA片段的方法中，使用IIS型酶进行DNA组装为复杂的途径工程提供了许多优势。特别是，它为用户提供了一种能力，即从克隆和测序的部件库开始，通过一系列简单的一锅法组装步骤快速组装多基因构建体。我们在此描述一种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件和载体。尚未获得的基本部件需要进行制备。然后使用一系列一锅法组装步骤，将已鉴定的部件和载体组装成多基因构建体。
