An efficient cloning method to expand vector and restriction site compatibility of Golden Gate Assembly.
An efficient cloning method to expand vector and restriction site compatibility of Golden Gate Assembly.
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DOI：
10.1016/j.crmeth.2023.100564
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发表时间：
2023-08-28
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Golden Gate Assembly is an efficient and rapid cloning method but requires dedicated vectors. Here, we modified Golden Gate to expand its compatibility to a broader range of destination vectors while maintaining its strengths. Our Expanded Golden Gate (ExGG) assembly adds to the insert(s) type IIS restriction sites that generate protruding ends compatible with traditional type IIP sites on the recipient vector. The ligated product cannot be cleaved again, owing to a single-base change near the junction. This allows the reaction to proceed in a single tube without an intermediate purification step. ExGG can be used to introduce multiple fragments into a vector simultaneously, including shorter fragments (<100 bp) and fragments with shared sequences, which can be difficult to assemble with other fast cloning strategies. Thus, ExGG extends the convenience of Golden Gate to a much larger space of pre-existing vectors designed for conventional cloning. We expanded the compatibility of Golden Gate Assembly to a broader range of vectors Expanded Golden Gate retains the efficiency and speed of Golden Gate Assembly Expanded Golden Gate allows for digestion and ligation in a single reaction Expanded Golden Gate can assemble multiple insert fragments Molecular cloning is a technique fundamental to many fields of biological research. Conventional cloning proceeds by separate digestions of vector and insert, followed by gel purification, ligation, and transformation. Golden Gate Assembly is a fast and efficient cloning method, wherein digestion and ligation of all fragments occur in a single reaction. However, Golden Gate Assembly requires dedicated destination vectors and cannot be used on the majority of existing plasmids. We have developed an Expanded Golden Gate (ExGG) strategy that retains the strengths of Golden Gate Assembly and extends them to a much larger range of destination vectors. Sorida and Bonasio develop an Expanded Golden Gate (ExGG) strategy that allows rapid insertion of one or more inserts in a broader range of destination plasmids with high efficiency and accuracy. ExGG is performed in a single tube, making routine molecular cloning fast and easy.
