Brick into the Gateway (BiG): A novel approach for faster cloning combining Golden Gate and Gateway methods

Letícia F Ferigolo; Mateus H Vicente; Fabio T S Nogueira

doi:10.1016/j.plasmid.2022.102630

Brick into the Gateway (BiG): A novel approach for faster cloning combining Golden Gate and Gateway methods

Plasmid. 2022 May:121:102630. doi: 10.1016/j.plasmid.2022.102630. Epub 2022 Apr 8.

Authors

Letícia F Ferigolo¹, Mateus H Vicente¹, Fabio T S Nogueira²

Affiliations

¹ 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.
² 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. Electronic address: ftsnogue@usp.br.

PMID: 35398459
DOI: 10.1016/j.plasmid.2022.102630

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.

Keywords: Cloning; DNA construct; Gateway method; Golden Gate; GoldenBraid.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cloning, Molecular
Genetic Vectors* / genetics
Plasmids / genetics