A unified multi-kingdom Golden Gate cloning platform
A unified multi-kingdom Golden Gate cloning platform
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DOI：
10.1038/s41598-019-46171-2
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发表时间：
2019-07-12
影响因子：
4.6
通讯作者：
Parniske, Martin
中科院分区：
文献类型：
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作者：
Chiasson, David;Gimenez-Oya, Victor;Parniske, Martin
由于最近的技术突破，如金门模块化克隆，从定制DNA部分组装复合DNA模块已成为常规。使用Golden Gate，可以有效地组装自定义转录单元和片段单元，以生成更高阶的组装。虽然金门克隆系统已经被开发出来组装DNA质粒，用于模式物种的实验工作，但它们通常不适用于来自其他王国的生物。因此，一个典型的跨王国工作的分子生物学实验室必须使用多克隆策略来组装DNA构建体以进行实验测定。为了简化DNA组装过程，我们开发了一个多王国（MK）金门组装平台，用于真菌、真细菌、原生生物、植物和动物界物种的实验工作。质粒骨架和部分突出端在整个平台上是一致的，节省了实验室的时间和资源。我们通过跨王国进行各种实验来展示该系统的功能，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MK系统简化了模块化DNA构建体的组装，用于跨一系列模型生物体的生物测定。
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.