Department of Molecular Biology, University of Potsdam, Potsdam, Germany.
Methods Mol Biol. 2020;2205:49-67. doi: 10.1007/978-1-0716-0908-8_3.
The implementation of complex cloning projects covering the assembly of entire biological pathways or large genetic circuits poses a major challenge in the field of biotechnology and synthetic biology, as such projects can be costly and time-consuming. To overcome these difficulties, we developed the software-assisted AssemblX toolkit, which allows even unexperienced users to design, build, and subsequently test large DNA constructs. Currently, AssemblX allows the assembly of up to 25 functional units (e.g., genes), from 75 or more subunits (e.g., promoters, coding sequences, terminators). At the first assembly level, AssemblX uses overlap-based, scar-free, and sequence-independent cloning methods. This allows the unrestricted design at the gene level without the need for laborious parts domestication. The standardized, polymerase chain reaction-free, and virtually sequence-independent assembly into multigene modules relies on rare cutting homing endonucleases and computationally optimized overlap sequences. Selection and marker switching strategies ensure an effective process, and the assembly product can be transferred to any desired expression host.
在生物技术和合成生物学领域，实施涵盖整个生物途径或大型遗传回路组装的复杂克隆项目是一项重大挑战，因为此类项目既昂贵又耗时。为了克服这些困难，我们开发了软件辅助的 AssemblX 工具包，即使是没有经验的用户也可以使用它来设计、构建和随后测试大型 DNA 构建体。目前，AssemblX 允许组装多达 25 个功能单元（例如基因），由 75 个或更多亚基（例如启动子、编码序列、终止子）组成。在第一个组装级别，AssemblX 使用基于重叠、无痕和序列无关的克隆方法。这允许在无需繁琐的部分驯化的情况下在基因水平上进行无限制的设计。多基因模块的标准化、无聚合酶链反应和几乎序列无关的组装依赖于稀有切割同源定向内切酶和经过计算优化的重叠序列。选择和标记切换策略确保了有效的过程，并且组装产物可以转移到任何所需的表达宿主。
