Letícia F. Ferigolo , Mateus H. Vicente , Fabio T.S. Nogueira
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引用次数: 0
Abstract
Gateway system is one of the most known cloning systems, which makes it compatible with several expression vectors, including those used for Yeast Two-Hybrid (Y2H) and Bimolecular Fluorescence Complementation (BiFC) assays. However, this system is laborious and expensive due to its two-step cloning. In this research, we developed a new cloning strategy named Brick into the Gateway (BiG). This approach uses GoldenBraid/Gate assemblies to create a DNA fragment of interest flanked by attL sites, which can be directly recombined into Gateway destination vectors. BiG method showed a high recombination efficiency and ensured the correct reading frame, which was successfully tested in Y2H and BiFC assays. BiG has proven to be a rapid, low-cost, reusable, and directional cloning method which allows the merged use of systems.
Gateway系统是最著名的克隆系统之一，它使其与多种表达载体兼容，包括用于酵母双杂交(Y2H)和双分子荧光互补(BiFC)测定的表达载体。然而，由于它的两步克隆，该系统既费力又昂贵。在本研究中，我们开发了一种名为Brick into the Gateway (BiG)的克隆策略。该方法使用GoldenBraid/Gate程序集来创建attL位点两侧的感兴趣的DNA片段，该片段可以直接重组为Gateway目的向量。BiG方法具有较高的重组效率，并确保了正确的阅读框，在Y2H和BiFC试验中成功验证。BiG已被证明是一种快速、低成本、可重用和定向克隆的方法，它允许系统的合并使用。
期刊介绍：
Plasmid publishes original research on genetic elements in all kingdoms of life with emphasis on maintenance, transmission and evolution of extrachromosomal elements. Objects of interest include plasmids, bacteriophages, mobile genetic elements, organelle DNA, and genomic and pathogenicity islands.
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