Description

Traditional binary shuttle vectors used for creating plant T-DNA insertion mutants can be difficult to work with for several reasons. First, their large size (> 10 kB) and low-copy origin of replication result in low plasmid yields under typical growth conditions. This makes time consuming, and costly, midi or maxi scale plasmid isolation a requirement. Second, accurate sequence information is often not available, rendering vector modification difficult. Finally, cloning techniques that are reliant on restriction enzyme sites prohibit deliberate design of vectors resulting in haphazard configuration of components. Newer cloning techniques, such as Gibson (Gibson et al., 2009) and In-Fusion^TM assembly, offer unlimited flexibility but rely on sufficient vector fragment quantity; this can be achieved either by restriction digest or by PCR. However, the large size and low DNA yield of binary shuttle vectors make their use in assembly reactions challenging; this is especially true for large batch DNA library cloning.

Movement of features not essential to cloning helped to alleviate the large size of the Ti plasmid; further modification to ‘helper’ plasmids resulted in the pGREEN system (Hellens et al., 1999). The pGREEN binary vector system offers small plasmid size (5.5 kb) and high copy number of the shuttle vector in Escherichia coli (E. coli). Although the pGREEN plasmid collection has aged well, many new molecular tools have been introduced. To provide the research community with an updated toolbox, we created a suite of streamlined pGREEN-based vectors that contain several features frequently used in molecular plant science. We designated the plasmids as pG20, short for pGREEN2020, to maintain a reference to the original hallmark work (Hellens et al., 1999). The small size and high DNA yield provide ease of use, while modular construction allows for easy replacement of the promoter and terminator. The inclusion of a multiple cloning site (MCS) with 6‐bp palindromic unique restriction enzymes sites (Waadt et al., 2008) allows full flexibility with restriction-based cloning, either for ligations or to prepare generic low cost Gibson fragments. To lower design costs further, a single primer pair (or one primer within the pair) can be used to yield several different protein tag fusions. Our vectors contain a variety of C-terminal protein-fusion tags; each one comes with two different antibiotic resistance markers allowing for use in many different plant mutant backgrounds. The addition of N-terminal tags could easily be achieved using the ‘tag less’ construct in a Gibson or In-Fusion^TM assembly. Plasmids can be obtained from Addgene, the Arabidopsis Biological Resource Center (ABRC), or by contacting our group directly.

A total of 16 constructs were created, using the constitutive AtUBQ10 promoter in combination with the AtHSP18.2 terminator, resulting in strong, tissue-independent expression. The following C-terminal fusions are available: RFP, YFP, GUS, luciferase2, His-tag, FLAG-tag and Myc-tag. In addition, there is a tag-free construct with only the MCS between promoter and terminator (Figure 1A, image generated using Snapgene^TM software from GSL Biotech). Each plasmid is available with either hygromycin or glufosinate resistance genes, driven by the CaMV35S promoter. Following confirmation of successful cloning by Sanger sequencing, functionality of the newly designed constructs was demonstrated through transient expression in infiltrated Nicotiana benthamiana leaves (Figure 1B-F). Additionally, we have used these constructs to create numerous stable transgenic Arabidopsis thaliana lines in a variety of on-going projects.