A Golden Gate Modular Cloning Toolbox for Plants

Abstract

Plant Synthetic Biology requires robust and efficient methods for assembling multigene constructs. Golden Gate cloning provides a precision module-based cloning technique for facile assembly of multiple genes in one construct. We present here a versatile resource for plant biologists comprising a set of cloning vectors and 96 standardized parts to enable Golden Gate construction of multigene constructs for plant transformation. Parts include promoters, untranslated sequences, reporters, antigenic tags, localization signals, selectable markers, and terminators. The comparative performance of parts in the model plant Nicotiana benthamiana is discussed.

Local Auxin Biosynthesis Mediated by a YUCCA Flavin Monooxygenase Regulates Haustorium Development in the Parasitic Plant Phtheirospermum japonicum

“…For the DEX-inducible CRE-lox system construct, Golden Gate cloning technology was used (Engler et al, 2014). The Arabidopsis PGP4 and RPS5a promoter region and the Pj-YUC3 sequence from the start codon to the 39 untranslated region were divided into two parts and amplified by PCR from Arabidopsis genomic DNA and P. japonicum genomic DNA, respectively.…”

Bile Acid Sodium Symporter BASS6 Can Transport Glycolate and Is Involved in Photorespiratory Metabolism in Arabidopsis thaliana

“…Constructs were then assembled using the Golden Gate cloning protocol then subcloned into a binary vector (EC50505) (Werner et al, 2012;Engler et al, 2014;Marillonnet and Werner, 2015;Patron et al, 2015). For stable transformation, the binary vectors were transformed into Agrobacterium tumefaciens C58C1 by electroporation and then transformed into the Col-0 wild-type strain, plgg1-1, or bass6-1 T-DNA insertion lines using the floral dip method (Clough and Bent, 1998).…”

Synthetic Botany

“…For instance, Nicotiana benthamiana leaves can be infiltrated with Agrobacterium at multiple regions of a single leaf and screened for gene expression a few days after infection. This allows relatively fast characterization of libraries of genetic components in a common genetic background (Sparkes et al 2006;Engler et al 2014;Brückner et al 2015). Transient leaf agroinfiltration in Nicotiana has been adopted as an efficient method for the optimization of metabolic pathways, such as artemisinin (van Herpen et al 2010) and triterpene biosynthesis pathways (reviewed in Thimmappa et al 2014), and the bioproduction of vaccines (D'Aoust et al 2008, Mardanova et al 2015.…”

“…The application of Type IIS restriction enzymes for assembling standard parts, known widely as "Golden Gate Cloning," has become widely used as an alternative approach because parts can be exchanged and assembled cheaply, easily, and in an automatable way without proprietary tools and reagents (Engler et al 2009;Sarrion-Perdigones et al 2011;Werner et al 2012). Many commonly used sequences have been adapted for Type IIS assembly by various plant research laboratories (Sarrion-Perdigones et al 2011;Weber et al 2011;Emami et al 2013;Lampropoulos et al 2013;Binder et al 2014;Engler et al 2014;Vafaee et al 2014), and a common syntax to enable the exchange of interoperable DNA parts for plants has recently been agreed by a large section of the plant research community (see Fig. 1; Patron et al 2015).…”