Design and Construction of Multigenic Constructs for Plant Biotechnology Using the GoldenBraid Cloning Strategy
GoldenBraid (GB) is an iterative and standardized DNA assembling system specially designed for Multigene Engineering in Plant Synthetic Biology. GB is based on restriction–ligation reactions using type IIS restriction enzymes. GB comprises a collection of standard DNA pieces named “GB parts” and a set of destination plasmids (pDGBs) that incorporate the multipartite assembly of standardized DNA parts. GB reactions are extremely efficient: two transcriptional units (TUs) can be assembled from several basic GBparts in one T-DNA less than 24 h. Moreover, larger assemblies comprising 4–5 TUs are routinely built in less than 2 working weeks. Here we provide a detailed view of the GB methodology. As a practical example, a Bimolecular Fluorescence Complementation construct comprising four TUs in a 12 kb DNA fragment is presented.
Key wordsSynthetic biology DNA assembly Type IIS enzymes Plant biotechnology Multigene constructs Multigene engineering
We want to thank Dr. Alejandro Ferrando sharing BiFC vectors and Dr. Cristina Ferrandiz for help with protein-protein interaction examples. This work was supported by the Spanish Ministry of Economy and Competitiveness: Grant BIO2010-15384. A. Sarrion-Perdigones is recipient of a Research Personnel in Training (FPI) fellowship.