Ferigolo Letícia F, Vicente Mateus H, Nogueira Fabio T S
Laboratory of Molecular Genetics of Plant Development, Escola Superior de Agricultura 'Luiz de Queiroz' (ESALQ), University of São Paulo, 13418-900 Piracicaba, São Paulo, Brazil.
Laboratory of Molecular Genetics of Plant Development, Escola Superior de Agricultura 'Luiz de Queiroz' (ESALQ), University of São Paulo, 13418-900 Piracicaba, São Paulo, Brazil.
Plasmid. 2022 May;121:102630. doi: 10.1016/j.plasmid.2022.102630. Epub 2022 Apr 8.
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系统是最知名的克隆系统之一，它能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）分析的载体。然而，由于其两步克隆过程，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）”的新克隆策略。这种方法使用GoldenBraid/Gate组装来创建一个两侧带有attL位点的目标DNA片段，该片段可直接重组到Gateway目的载体中。BiG方法显示出高重组效率并确保了正确的阅读框，这在Y2H和BiFC分析中得到了成功验证。BiG已被证明是一种快速、低成本、可重复使用且定向的克隆方法，它允许系统的合并使用。
