Multigene Engineering by GoldenBraid Cloning: From Plants to Filamentous Fungi and Beyond

This is the peer reviewed version of the following article: Vazquez-Vilar, M., Gandía, M., García-Carpintero, V., Marqués, E., Sarrion-Perdigones, A., Yenush, L., Polaina, J., Manzanares, P., Marcos, J. F., & Orzaez, D. (2020). Multigene engineering by goldenbraid cloning: from plants to filamentous fungi and beyond. Current Protocols in Molecular Biology, 130, e116, doi: 10.1002/cpmb.116, which has been published in final form at https://doi.org/10.1002/cpmb.116. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.
[EN] Many synthetic biologists have adopted methods based on Type IIS restriction enzymes and Golden Gate technology in their cloning procedures, as these enable the combinatorial assembly of modular elements in a very efficient way following standard rules. GoldenBraid (GB) is a Golden Gate¿based modular cloning system that, in addition, facilitates the engineering of large multigene constructs and the exchange of DNA parts as result of its iterative cloning scheme. GB was initially developed specifically for plant synthetic biology, and it has been subsequently extended and adapted to other organisms such as Saccharomyces cerevisiae, filamentous fungi, and human cells by incorporating a number of host¿specific features into its basic scheme. Here we describe the general GB cloning procedure and provide detailed protocols for its adaptation to filamentous fungi¿a GB variant known as FungalBraid. The assembly of a cassette for gene disruption by homologous recombination, a fungal¿specific extension of the GB utility, is also shown. Development of FungalBraid was relatively straightforward, as both plants and fungi can be engineered using the same binary plasmids via Agrobacterium¿mediated transformation. We also describe the use of a set of web¿based tools available at the GB website that assist users in all cloning procedures. The availability of plant and fungal versions of GB will facilitate genetic engineering in these industrially relevant organisms.