A User's Guide to Golden Gate Cloning Methods and Standards.
A User's Guide to Golden Gate Cloning Methods and Standards.
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DOI：
10.1021/acssynbio.2c00355
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发表时间：
2022-11-18
影响因子：
4.7
通讯作者：
Giachino, Andrea
中科院分区：
文献类型：
--
作者：
Bird, Jasmine E.;Marles-Wright, Jon;Giachino, Andrea
对适合广泛应用的专门分子克隆技术的持续需求推动了许多不同克隆策略的发展。一种获得显著关注的方法是金门组装，它通过利用IIS型限制性内切酶在切割的DNA片段上产生用户指定的粘端来实现DNA部分的分层组装。该技术已被模块化和标准化，包括不同的方法亚族，其中最广泛采用的是MoClo和Golden Braid标准。此外，为特定应用程序或生物体量身定制的专用工具箱也可用。尽管如此，组装方法的数量和范围可能构成新用户采用的障碍，甚至有经验的科学家也可能发现很难辨别哪些工具最适合他们的目标。在这篇综述中，我们提供了一个初学者友好的金门组装指南，比较了不同的可用标准，并详细介绍了常用工具箱的具体功能和特点。我们还提供了最新的金门技术，讨论了最新的进展和挑战，以告知现有用户并促进标准实践。
The continual demand for specialized molecular cloning techniques that suit a broad range of applications has driven the development of many different cloning strategies. One method that has gained significant traction is Golden Gate assembly, which achieves hierarchical assembly of DNA parts by utilizing Type IIS restriction enzymes to produce user-specified sticky ends on cut DNA fragments. This technique has been modularized and standardized, and includes different subfamilies of methods, the most widely adopted of which are the MoClo and Golden Braid standards. Moreover, specialized toolboxes tailored to specific applications or organisms are also available. Still, the quantity and range of assembly methods can constitute a barrier to adoption for new users, and even experienced scientists might find it difficult to discern which tools are best suited toward their goals. In this review, we provide a beginner-friendly guide to Golden Gate assembly, compare the different available standards, and detail the specific features and quirks of commonly used toolboxes. We also provide an update on the state-of-the-art in Golden Gate technology, discussing recent advances and challenges to inform existing users and promote standard practices.
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