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.Įver since the first construction of a recombinant plasmid, , genetic engineering and molecular cloning mostly rely on the use of type II restriction endonucleases and DNA ligases. 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 was funded by the Collaborative Research Centre SFB873 of the DFG ( and the ERC () grant 282139 “StemCellAdapt”. Received: AugAccepted: NovemPublished: December 20, 2013Ĭopyright: © 2013 Lampropoulos et al.
PLoS ONE 8(12):Įditor: Paul Jaak Janssen, Belgian Nuclear Research Centre SCK/CEN, Belgium The system is cheap and reliable and when combined with a library of modules considerably speeds up cloning and transgene stacking for plant transformation.Ĭitation: Lampropoulos A, Sutikovic Z, Wenzl C, Maegele I, Lohmann JU, Forner J (2013) GreenGate - A Novel, Versatile, and Efficient Cloning System for Plant Transgenesis. GreenGate cloning is simple and efficient since it uses only one type IIS restriction endonuclease, depends on only six types of insert modules (plant promoter, N-terminal tag, coding sequence, C-terminal tag, plant terminator and plant resistance cassette), but at the same time allows assembling several expression cassettes in one binary destination vector from a collection of pre-cloned building blocks. Here we introduce our GreenGate system for rapidly assembling plant transformation constructs, which is based on the Golden Gate method. Several alternative cloning systems have been developed in recent years to overcome these issues, including the type IIS enzyme based Golden Gate technique. Especially in case of long inserts and applications requiring high-throughput, this approach is limited by the number of available unique restriction sites and the need for designing individual cloning strategies for each project. Traditionally it is based on a multitude of type II restriction endonucleases and T4 DNA ligase. Building expression constructs for transgenesis is one of the fundamental day-to-day tasks in modern biology.
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