Abstract
Synthetic Biology requires efficient and versatile DNA assembly systems to facilitate the building of new genetic modules/pathways from basic DNA parts in a standardized way. Here we present GoldenBraid (GB), a standardized assembly system based on type IIS restriction enzymes that allows the indefinite growth of reusable gene modules made of standardized DNA pieces. The GB system consists of a set of four destination plasmids (pDGBs) designed to incorporate multipartite assemblies made of standard DNA parts and to combine them binarily to build increasingly complex multigene constructs. The relative position of type IIS restriction sites inside pDGB vectors introduces a double loop (“braid”) topology in the cloning strategy that allows the indefinite growth of composite parts through the succession of iterative assembling steps, while the overall simplicity of the system is maintained. We propose the use of GoldenBraid as an assembly standard for Plant Synthetic Biology. For this purpose we have GB-adapted a set of binary plasmids for A. tumefaciens-mediated plant transformation. Fast GB-engineering of several multigene T-DNAs, including two alternative modules made of five reusable devices each, and comprising a total of 19 basic parts are also described.
Highlights
Synthetic Biology adapts the general engineering principle of assembling standard components, dating back to the Industrial Revolution, to biological components
GoldenBraid is an adaptation of Golden Gate to Synthetic Biology
We initially considered three categories of basic parts, namely promoters (PR), coding sequences (CDS) and terminators (TM)
Summary
Synthetic Biology adapts the general engineering principle of assembling standard components, dating back to the Industrial Revolution, to biological components This discipline aims at the design of artificial living forms displaying new traits not existing in nature [1,2]. Increasingly efficient homologous recombination methods have enormously facilitated the assembly of large DNA sequences up to the genome range [6], with the synthesis of a complete bacterial genome serving as best example [7,8] Despite these technical advances, many critical engineering issues as the exhaustive characterization of new genetic modules, their re-adaptation for additional purposes or their combination with other devices to produce combined traits still require from increasingly efficient and versatile DNA assembly methods operating at intermediate range. Standardization favors reusability, as any standard pieces can be exchanged for assembling different constructs following common rules of assembly
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