2024-03-29T06:36:29Zhttp://digital.csic.es/dspace-oai/requestoai:digital.csic.es:10261/2071252022-12-30T07:52:33Zcom_10261_131com_10261_2com_10261_103com_10261_1col_10261_384col_10261_356
Multigene Engineering by GoldenBraid Cloning: From Plants to Filamentous Fungi and Beyond
Vázquez Vilar, Marta
Gandía, Mónica
García-Carpintero, Víctor
Marqués, Eric
Sarrión Perdigones, Alejandro
Yenush, Lynne
Polaina Molina, Julio
Manzanares, Paloma
Marcos López, José Francisco
Ministerio de Economía y Competitividad (España)
Ministerio de Ciencia, Innovación y Universidades (España)
Generalitat Valenciana
GoldenBraid
Synthetic biology
Synthetic biology
Modular cloning
Agrobacterium‐mediated transformation
FungalBraid
This is the peer reviewed version of the following article: Multigene Engineering by GoldenBraid Cloning: From Plants to Filamentous Fungi and Beyond. Vazquez-Vilar, M.; Gandía, M.; García-Carpintero, V.; Marqués, E.; Sarrion-Perdigones, A.; Yenush, L.; Polaina, J.; Manzanares, P.; Marcos, J.F.; Orzaez, D. Current Protocols in Molecular Biology 130(1): E116 (2020), 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 Use of Self-Archived Versions.
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.
This work was funded by Grant BIO2013‐42193 and Grant BIO2016‐78601‐R, Plan Nacional I+D, Spanish Ministry of Economy and Competitiveness, RTI2018‐101115‐B‐C21 from the “Ministerio de Ciencia, Innovación y Universidades” (Spain) (MICINN/FEDER Funds), and PROMETEO/2018/066 from “Conselleria d'Educació” (Generalitat Valenciana, Comunitat Valenciana, Spain) and SUSPHIRE PCI2018‐092893–ERA CoBioTech (109) (MCIU/FEDER).
Peer reviewed
2020-04-09T10:39:40Z
2020-04-09T10:39:40Z
2020-03-09
artículo
http://purl.org/coar/resource_type/c_6501
Current Protocols in Molecular Biology 130(1): E116 (2020)
10.1002/cpmb.116
http://hdl.handle.net/10261/207125
1934-3647
http://dx.doi.org/10.13039/501100003359
http://dx.doi.org/10.13039/501100003329
en
#PLACEHOLDER_PARENT_METADATA_VALUE#
#PLACEHOLDER_PARENT_METADATA_VALUE#
#PLACEHOLDER_PARENT_METADATA_VALUE#
info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/BIO2013-42193
info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/BIO2016-78601-R
MICIU/ICTI2017-2020/RTI2018-101115-B-C21
Postprint
https://doi.org/10.1002/cpmb.116
Sí
open
John Wiley & Sons