Biomedical Engineering Reference
In-Depth Information
Chapter 10
Design and Construction of Multigenic Constructs for Plant
Biotechnology Using the GoldenBraid Cloning Strategy
Alejandro Sarrion-Perdigones , Jorge Palaci , Antonio Granell ,
and Diego Orzaez
Abstract
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 effi cient: 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 words
Synthetic biology, DNA assembly, Type IIS enzymes, Plant biotechnology, Multigene
constructs, Multigene engineering
1
Introduction
Synthetic Biology aims to engineer living systems with functions
not found in nature, thus incorporating new features to existing
living organisms [
1
,
2
]. Complex protein production, metabolic
engineering, or genetic networks often need the integration of sev-
eral transgenes into the biological model, what requires more than
single transcriptional units (TUs) building but the construction of
complex multigenic structures. Custom DNA synthesis [
3
] as well
as other strategies involving homologous and site-specifi c recom-
bination [
4
-
6
] have been reported as successful methodologies for
multigene engineering. However, neither they produce reusable
units nor facilitate the set up of combinatorial strategies. An alter-
native approach is the modular construction of genetic devices using
standardized DNA parts. Modular design facilitates combinatorial
