Faculty of Biology, Genetics, LMU Munich, D-82152, Martinsried, Germany.
Department of Biology, Saint Mary's University, B3H 3C3, Halifax, Canada.
Sci Rep. 2019 Jul 12;9(1):10131. doi: 10.1038/s41598-019-46171-2.
Assembling composite DNA modules from custom DNA parts has become routine due to recent technological breakthroughs such as Golden Gate modular cloning. Using Golden Gate, one can efficiently assemble custom transcription units and piece units together to generate higher-order assemblies. Although Golden Gate cloning systems have been developed to assemble DNA plasmids required for experimental work in model species, they are not typically applicable to organisms from other kingdoms. Consequently, a typical molecular biology laboratory working across kingdoms must use multiple cloning strategies to assemble DNA constructs for experimental assays. To simplify the DNA assembly process, we developed a multi-kingdom (MK) Golden Gate assembly platform for experimental work in species from the kingdoms Fungi, Eubacteria, Protista, Plantae, and Animalia. Plasmid backbone and part overhangs are consistent across the platform, saving both time and resources in the laboratory. We demonstrate the functionality of the system by performing a variety of experiments across kingdoms including genome editing, fluorescence microscopy, and protein interaction assays. The versatile MK system therefore streamlines the assembly of modular DNA constructs for biological assays across a range of model organisms.
由于近年来 Golden Gate 模块化克隆等技术突破，从定制 DNA 元件组装复合 DNA 模块已成为常规操作。使用 Golden Gate，人们可以有效地将定制的转录单元和片段单元组装在一起，以生成更高阶的组装体。尽管 Golden Gate 克隆系统已被开发用于组装实验模型物种所需的 DNA 质粒，但它们通常不适用于其他王国的生物。因此，一个跨越王国的典型分子生物学实验室必须使用多种克隆策略来组装用于实验测定的 DNA 构建体。为了简化 DNA 组装过程，我们开发了一个多王国 (MK) Golden Gate 组装平台，用于真菌、真细菌、原生生物、植物和动物王国的物种的实验工作。该平台的质粒骨架和部分突出端是一致的，在实验室中节省了时间和资源。我们通过在跨越王国的各种实验中展示该系统的功能，包括基因组编辑、荧光显微镜和蛋白质相互作用测定。因此，多功能的 MK 系统简化了跨越一系列模式生物的生物测定用模块化 DNA 构建体的组装。
