GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology

Abstract

Plant synthetic biology aims to apply engineering principles to plant genetic design. One strategic requirement of plant synthetic biology is the adoption of common standardized technologies that facilitate the construction of increasingly complex multigene structures at the DNA level while enabling the exchange of genetic building blocks among plant bioengineers. Here, we describe GoldenBraid 2.0 (GB2.0), a comprehensive technological framework that aims to foster the exchange of standard DNA parts for plant synthetic biology. GB2.0 relies on the use of type IIS restriction enzymes for DNA assembly and proposes a modular cloning schema with positional notation that resembles the grammar of natural languages. Apart from providing an optimized cloning strategy that generates fully exchangeable genetic elements for multigene engineering, the GB2.0 toolkit offers an evergrowing open collection of DNA parts, including a group of functionally tested, premade genetic modules to build frequently used modules like constitutive and inducible expression cassettes, endogenous gene silencing and protein-protein interaction tools, etc. Use of the GB2.0 framework is facilitated by a number of Web resources that include a publicly available database, tutorials, and a software package that provides in silico simulations and laboratory protocols for GB2.0 part domestication and multigene engineering. In short, GB2.0 provides a framework to exchange both information and physical DNA elements among bioengineers to help implement plant synthetic biology projects.

Bricks and blueprints: methods and standards for DNA assembly

“…To enable using Golden Gate for hierarchical assembly, a variety of physical standards have been developed that adopt a tiered assembly approach: in the first tier genes are assembled from their elementary parts (promoters, open reading frames, terminators, etc. ; Figure 1a) [29][30][31][32] and then in the second tier, these genes are combined to form multi-gene systems (Figure 1b). The MoClo standard, first developed for plant molecular biology, uses a parallel approach for all tiers but requires the use of a large number of entry and destination vectors 29 .…”

“…The MoClo standard, first developed for plant molecular biology, uses a parallel approach for all tiers but requires the use of a large number of entry and destination vectors 29 . The GoldenBraid standard reduces the number of required vectors by applying a pairwise approach for assembly beyond the gene-level, but at the cost of requiring more steps for larger constructs 30,33 .…”

“…Simultaneous digestion and ligation forces incorrectly assembled plasmids to be recut by BsaI while correct ones are protected. (b) Diagram of the MoClo and GoldenBraid 2.0 standards 29,30 . These provide two different approaches for the second tier of Golden Gate assembly, where the genes built in the first tier reaction are combined to form multi-gene constructs.…”

“…The MoClo standard, first developed for plant molecular biology, uses a parallel approach for all tiers but requires the use of a large number of entry and destination vectors 29 . The GoldenBraid standard reduces the number of required vectors by applying a pairwise approach for assembly beyond the gene-level, but at the cost of requiring more steps for larger constructs 30,33 .The power of Golden Gate to perform parallel assembly of multiple parts without PCR has made it very popular and recently a mammalian MoClo (mMoClo) standard was introduced 34 . mMoClo enables the stable integration into defined sites in mammalian chromosomes of very large multigene constructs such as genetic switch programs that encode the regulated expression of counteracting transcription factors that establish hereditable memory in cells 34 .…”

Advances in Crop Synthetic Biology: A Platform that Bridges the Expertise of Crop Molecular Biologists, Engineers, Software Developers and Mathematical Modelers

“…Despite the fact that there are still organizations that oppose GM crops or at least wish that the GM crop products be appropriately and rightlylabeled, yet a new paradigm shift from GM crop technology has emerged, as:plant synthetic biology".Plant synthetic biology has been recently effectively applied in embedding of completely artificial genetic code circuits into naturally existing plants used as framework of "chassis" [1].The solid foundation of synthetic biology is rooted from the system biology i.e. a technology that quantitatively characterizes the molecules and the way that the networks of molecules behave [2].…”