Centre for Organismal Studies, Heidelberg University, Heidelberg, Baden-Württemberg, Germany.
PLoS One. 2013 Dec 20;8(12):e83043. doi: 10.1371/journal.pone.0083043. eCollection 2013.
Building expression constructs for transgenesis is one of the fundamental day-to-day tasks in modern biology. Traditionally it is based on a multitude of type II restriction endonucleases and T4 DNA ligase. Especially in case of long inserts and applications requiring high-throughput, this approach is limited by the number of available unique restriction sites and the need for designing individual cloning strategies for each project. Several alternative cloning systems have been developed in recent years to overcome these issues, including the type IIS enzyme based Golden Gate technique. Here we introduce our GreenGate system for rapidly assembling plant transformation constructs, which is based on the Golden Gate method. GreenGate cloning is simple and efficient since it uses only one type IIS restriction endonuclease, depends on only six types of insert modules (plant promoter, N-terminal tag, coding sequence, C-terminal tag, plant terminator and plant resistance cassette), but at the same time allows assembling several expression cassettes in one binary destination vector from a collection of pre-cloned building blocks. The system is cheap and reliable and when combined with a library of modules considerably speeds up cloning and transgene stacking for plant transformation.
构建转基因的表达构建体是现代生物学中最基本的日常任务之一。传统上，它基于多种类型 II 限制内切酶和 T4 DNA 连接酶。特别是在长插入物和需要高通量的情况下，这种方法受到可用独特限制位点的数量以及为每个项目设计单独克隆策略的需求的限制。近年来已经开发了几种替代的克隆系统来克服这些问题，包括基于 II 型酶的 Golden Gate 技术。在这里，我们介绍了我们的 GreenGate 系统，用于快速组装植物转化构建体，该系统基于 Golden Gate 方法。GreenGate 克隆简单高效，因为它只使用一种类型 II 限制内切酶，只依赖于六种类型的插入模块（植物启动子、N 端标签、编码序列、C 端标签、植物终止子和植物抗性盒），但同时允许从预克隆构建块集合中在一个二元目的载体中组装多个表达盒。该系统价格便宜且可靠，与模块库结合使用时，可大大加快植物转化的克隆和转基因堆叠。
