GATEWAY™ cloning in particular was readily adopted in the plant field – as large binary vectors for Agrobacterium tumefaciens mediated plant transformations were difficult to handle via classical cloning.ĭespite the benefits, current recombination-based cloning approaches are limited in their flexibility. Once the master clone sequence has been confirmed, the target can be transferred to any compatible expression vector via site-specific recombination. gene of interest) either by homologous recombination, classical cloning or TOPO cloning (Invitrogen) based on topoisomerases. Typically, master or entry vectors are created by insertion of a target PCR sequence (i.e. Today several commercial and non-commercial systems are available, including Invitrogen’s GATEWAY™ system, Clontech’s Creator system, as well as the Univector cloning system developed in the lab of Stephen Elledge. Second generation cloning technologies based on homologous site-specific recombination enable very effective high-throughput construction of recombinant DNA and are not dependent on particular restriction sites. For each construct a custom cloning strategy has to be adapted, which is limited by the available restriction sites. By today’s standards this first generation of cloning is relatively time consuming, inflexible, and assembly of large or multiple fragments can be ineffective. Ĭlassical DNA cloning was initiated by the discovery of bacterial type II endonucleases, which in combination with ligases allowed researchers to cleave and rejoin given DNA fragments at restriction sites. Transgenic plants carrying synthetic DNA sequences are indispensable tools for fundamental research and offer great promise for crop improvements that cannot be achieved by classical breeding. Manipulation and creation of custom DNA sequences is also central in the emerging field of synthetic biology, which aims to engineer new biological components, networks, pathways or even complete organisms for a variety of biotechnological applications. Recombinant DNA is required to analyze gene and protein function by complementation, localization, overexpression, gene silencing (knockdown) and knockout as well as for the production of proteins or other biomolecules in transgenic organisms. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The authors have declared that no competing interests exist.ĭNA cloning technologies are instrumental to the functional dissection of biological systems. were additionally funded by the Emmy Noether grant of the German Research Foundation (Deutsche Forschungsgemeinschaft DFG ). This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.įunding: The work of all authors was funded as part of the Sonderforschungsbereich 924 (SFB924 ). Received: OctoAccepted: JanuPublished: February 13, 2014Ĭopyright: © 2014 Binder et al. (2014) A Modular Plasmid Assembly Kit for Multigene Expression, Gene Silencing and Silencing Rescue in Plants. As proof of principle, we silenced a destabilized GFP gene ( dGFP) and restored GFP fluorescence by expression of a recoded version of dGFP, which was not targeted by the silencing construct.Ĭitation: Binder A, Lambert J, Morbitzer R, Popp C, Ott T, Lahaye T, et al. By combination of the silencing construct together with a codon adapted rescue construct into one vector, our system facilitates genetic complementation and thus confirmation of the causative gene responsible for a given RNAi phenotype.
We assembled an RNA interference (RNAi) module for the construction of intron-spliced hairpin RNA constructs and demonstrated silencing of GFP in N. As an example, we created T-DNA constructs encoding multiple fluorescent proteins targeted to distinct cellular compartments (nucleus, cytosol, plastids) and demonstrated simultaneous expression of all genes in Nicotiana benthamiana, Lotus japonicus and Arabidopsis thaliana.
Starting from a common set of modules, such as promoters, protein tags and transcribed regions of interest, synthetic genes are assembled, which can be further combined to multigene constructs.
We developed a GG based toolkit for the flexible construction of binary plasmids for transgene expression in plants. The Golden Gate (GG) modular assembly approach offers a standardized, inexpensive and reliable way to ligate multiple DNA fragments in a pre-defined order in a single-tube reaction.
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