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

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.

Microtubule-associated protein IQ67 DOMAIN5 regulates morphogenesis of leaf pavement cells inArabidopsis thaliana

“…Confocal imaging was performed with a Zeiss LSM 780 inverted microscope using a ×40 water immersion objective, unless stated otherwise. Generation of yellow fluorescent protein (YFP)–IQD5, Y N –TRM1, red fluorescent protein (RFP)–TUA5, and mCherry–CaM2 is described in Bürstenbinder et al (2017 b ) and Gantner et al (2018). GFP was excited using a 488 nm laser and emission was detected between 493 nm and 555 nm.…”

Targeting specificity of nuclear-encoded organelle proteins with a self-assembling split-fluorescent protein toolkit

“…The modular cloning principle and DNA fragments of the Plant Parts I and II toolkits were used for vector construction (Weber et al, 2011;Engler et al, 2014;Gantner et al, 2018). The modules utilized for cloning of Golden Gate-based vectors are illustrated in Fig.…”

Highly efficient multiplex editing: One-shot generation of 8xNicotiana benthamianaand 12x Arabidopsis mutants

“…Representative pictures of markers with high predictive power are shown. To query for presence/absence of Bs3 , approximately 4 weeks-old plants were infiltrated with a Pseudomonas fluorescens strain for translocation of AvrBs3 (Gantner et al, 2018). Phenotypes were recorded 3 dpi, images show a Bs3 -transgenic plant in comparison to wild-type control.…”

“…The GoldenGate technique following the Modular Cloning syntax for hierarchical DNA assembly was used for most clonings (Engler et al , 2008, Weber et al , 2011). Previously reported plasmids belonging to the Modular Cloning Toolkit and the MoClo Plant Parts I and II collections were used (Engler et al , 2014, Gantner et al , 2018). For domestication of new DNA modules, respective fragments were amplified using Polymerase X (Roboklon), and ligated into Level 0 vectors.…”