Golden Gate Cloning-Compatible DNA Replicon/2A-Mediated Polycistronic Vectors for Plants

Abstract

The expression of multiple proteins and high-throughput vector assembly system are highly relevant in the field of plant genetic engineering and synthetic biology. Deployment of the self-cleaving 2A peptide that mediates polycistronic gene expression has been an effective strategy for multigene expression, as it minimizes issues in coordinated transgene regulation and trait staking in plants. However, efficient vector assembly systems optimized for 2A peptide-mediated polycistronic expression are currently unavailable. Furthermore, it is unclear whether protein expression levels are influenced by the transgene position in the polycistronic expression cassette. In this article, we present Golden Gate cloning-compatible modular systems allowing rapid and flexible construction of polycistronic expression vectors applicable for plants. The genetic modules comprised 2A peptides (T2A and P2A)-linked tricistron expression cassette and its acceptor backbones, named pGO-DV1 and pGO-DV2. While both acceptor backbones were binary T-DNA vectors, pGO-DV2 was specially designed to function as a DNA replicon enhancing gene expression levels. Using the Golden Gate cloning, a set of six tricistronic vectors was constructed, whereby three transgenes encoding fluorescent proteins (mCherry, eYFP, and eGFP) were combinatorially placed along the expression cassette in each of the binary vectors. Transient expression of the construct in tobacco leaves revealed that the expression levels of three fluorescent proteins were comparable each other regardless of the gene positions in the tricistronic expression cassette. pGO-DV2-based constructs were able to increase protein expression level by up to 71%, as compared to pGO-DV1-based constructs.

Q-SHINE: a versatile sensor for glutamine measurement via ligand-induced dimerization

“…Agrobacterium-mediated transient expression analysis was performed as described by Lee et al[31] Agrobacterium tumefaciens LBA4404 cells carrying different sensor constructs were adjusted to OD600 0.4 in resuspension solution (10 mM MgCl 2 , 10 mM MES-KOH (pH 5.6), 100 μM acetosyringone) and infiltrated into leaves of 4 to 5-week-old Nicotiana tabacum cv. Samsun.…”

Compendium on Food Crop Plants as a Platform for Pharmaceutical Protein Production

“…Advances in the assembly of large DNA fragments, where the exact number and arrangement of transgenes are determined, as well as increase in the efficiency of DNA cloning methods, have contributed significantly to the acceleration of the process of constructing a multigene expression cassette construct [ 37 ]. Currently, scientists have at their disposal a large array of kits/systems for implementing the intended strategy of constructing an expression cassette for plant transformation [ 38 , 39 ].…”

“…Currently, scientists have at their disposal a large array of kits/systems for implementing the intended strategy of constructing an expression cassette for plant transformation [ 38 , 39 ]. The most commonly used include Golden Gate and derived methods such as MoClo (for chloroplast engineering), Golden Braid, and Green Gate, which are based on the use of type II restriction enzymes [ 37 ]. However, the latter feature is not entirely desirable in the case of large multigene constructs, due to numerous recognition sites.…”

Challenges and Approaches to Crop Improvement Through C3-to-C4 Engineering

“…Our ability to deal with these technical challenges has been significantly enhanced by a burst of novel gene cloning and genome editing technologies in recent years. The Golden Gate cloning method is of particular interest because it allows the stacking of multiple genes in a straightforward manner ( Lee et al, 2020 ). Using this method, Ermakova et al recently demonstrated that a C4 photosynthetic pathway can be installed in rice by simultaneously expressing five maize photosynthetic enzymes using a single construct ( Ermakova et al, 2021 ).…”

“…Progress in C3-to-C4 engineering has been slow, however, due to a lack of understanding of the mechanisms underlying C4 photosynthesis. This status has changed recently with a number of important findings in C4 research ( Furbank, 2017 ; Sedelnikova et al, 2018 ) and technical breakthroughs ( Zhang et al, 2019 ; Lee et al, 2020 ; Zhu et al, 2020 ). Several new model plant species have also been introduced, such as Setaria viridis (a C4 monocot; Brutnell et al, 2010 ), Cleome gynandra (a C4 dicot; Newell et al, 2010 ), and the grass Alloteropsis semialata ( Lundgren et al, 2016 ).…”