Peripheral infrastructure vectors and an extended set of plant parts for the Modular Cloning system.

Johannes Gantner,Johannes Stuttmann,Ramona Gruetzner,Katharina Bürstenbinder,Carola Kretschmer,Yasin Dagdas,Sylvestre Marillonnet,Theresa Ilse,Christian Löfke,Jana Ordon,Alvaro Galli

doi:10.1371/journal.pone.0197185

Abstract

Standardized DNA assembly strategies facilitate the generation of multigene constructs from collections of building blocks in plant synthetic biology. A common syntax for hierarchical DNA assembly following the Golden Gate principle employing Type IIs restriction endonucleases was recently developed, and underlies the Modular Cloning and GoldenBraid systems. In these systems, transcriptional units and/or multigene constructs are assembled from libraries of standardized building blocks, also referred to as phytobricks, in several hierarchical levels and by iterative Golden Gate reactions. Here, a toolkit containing further modules for the novel DNA assembly standards was developed. Intended for use with Modular Cloning, most modules are also compatible with GoldenBraid. Firstly, a collection of approximately 80 additional phytobricks is provided, comprising e.g. modules for inducible expression systems, promoters or epitope tags. Furthermore, DNA modules were developed for connecting Modular Cloning and Gateway cloning, either for toggling between systems or for standardized Gateway destination vector assembly. Finally, first instances of a “peripheral infrastructure” around Modular Cloning are presented: While available toolkits are designed for the assembly of plant transformation constructs, vectors were created to also use coding sequence-containing phytobricks directly in yeast two hybrid interaction or bacterial infection assays. The presented material will further enhance versatility of hierarchical DNA assembly strategies.

Highlights

Molecular cloning belongs to the unbeloved, yet inevitable everyday tasks of many wet lab molecular biologists
The vectors pJOG130 and pJOG131 are based on a common backbone (Kanamycin resistance, M13fwd/rev priming sites), and contain a ccdB negative selection cassette flanked by Golden Gate cloning sites (BsaI) and attL1/2 sites
We show how the Modular Cloning assembly standard may, by integrating just a few modules, be used for inexpensive generation of Gateway entry clones, toggling between cloning systems, or standardized assembly of Gateway destination vectors

Summary

Introduction

Molecular cloning belongs to the unbeloved, yet inevitable everyday tasks of many wet lab molecular biologists. Inserts may be mobilized from entry clones into a wide array of destination vectors by basically failsafe, highly efficient and unified recombination reactions. Gateway cloning is relatively costly, as it relies on use of the proprietary BP/LR enzyme blends, and represents a rather binary approach to molecular cloning where a single insert is mobilized into a new sequence context. To some extent, this was overcome by the invention of multisite Gateway systems [4]. Most popular strategies for combinatorial DNA assembly rely on enzymatic reaction assembly (Gibson assembly, In-FusionTM Cloning [6,7,8]) or Golden Gate cloning

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Results

Conclusion