Journal
PLASMID
Volume 121, Issue -, Pages -Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.plasmid.2022.102630
Keywords
Cloning; DNA construct; GoldenBraid; Golden Gate; Gateway method
Categories
Funding
- Sao Paulo Research Foundation - FAPESP [2018/17441-3, 2019/20157-8]
- Coordination for the Improvement of Higher Education Personnel - CAPES [88882.461731/2019-01, 88887.309513/2018-00]
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In this study, a new cloning strategy named BiG was developed, which allows the merged use of Gateway system and other systems. The BiG method showed high cloning efficiency and ensured the correct reading frame.
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.
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