Institute of Plant Breeding, Genetics and Genomics, University of Georgia, University of Georgia, Athens, Georgia, United States of America.
Center for Applied Genetic Technologies, University of Georgia, Athens, Georgia, United States of America.
PLoS One. 2024 Sep 18;19(9):e0306008. doi: 10.1371/journal.pone.0306008. eCollection 2024.
Genetic modification of plants fundamentally relies upon customized vector designs. The ever-increasing complexity of transgenic constructs has led to increased adoption of modular cloning systems for their ease of use, cost effectiveness, and rapid prototyping. GreenGate is a modular cloning system catered specifically to designing bespoke, single transcriptional unit vectors for plant transformation-which is also its greatest flaw. MultiGreen seeks to address GreenGate's limitations while maintaining the syntax of the original GreenGate kit. The primary limitations MultiGreen addresses are 1) multiplexing in series, 2) multiplexing in parallel, and 3) repeated cycling of transcriptional unit assembly through binary intermediates. MultiGreen efficiently concatenates bespoke transcriptional units using an additional suite of level 1acceptor vectors which serve as an assembly point for individual transcriptional units prior to final, level 2, condensation of multiple transcriptional units. Assembly with MultiGreen level 1 vectors scales at a maximal rate of 2*⌈log6n⌉+3 days per assembly, where n represents the number of transcriptional units. Further, MultiGreen level 1 acceptor vectors are binary vectors and can be used directly for plant transformation to further maximize prototyping speed. MultiGreen is a 1:1 expansion of the original GreenGate architecture's grammar and has been demonstrated to efficiently assemble plasmids with multiple transcriptional units. MultiGreen has been validated by using a truncated violacein operon from Chromobacterium violaceum in bacteria and by deconstructing the RUBY reporter for in planta functional validation. MultiGreen currently supports many of our in-house multi transcriptional unit assemblies and will be a valuable strategy for more complex cloning projects.
植物的基因改造从根本上依赖于定制的载体设计。转基因构建体的日益复杂导致模块化克隆系统因其易用性、成本效益和快速原型制作而得到越来越多的采用。GreenGate 是一种模块化克隆系统，专门用于设计定制的、单一转录单元载体用于植物转化——这也是它最大的缺陷。MultiGreen 旨在解决 GreenGate 的局限性，同时保持原始 GreenGate 工具包的语法。MultiGreen 主要解决的限制有 1）串联多重化，2）并行多重化，以及 3）通过二元中间物重复循环转录单元组装。MultiGreen 使用额外的一级接受载体套件有效地串联定制转录单元，这些载体套件在最终二级多个转录单元的缩合之前，充当单个转录单元的组装点。使用 MultiGreen 一级载体进行组装的速度最快可达 2*⌈log6n⌉+3 天/次组装，其中 n 代表转录单元的数量。此外，MultiGreen 一级接受载体是二元载体，可直接用于植物转化，以进一步最大化原型制作速度。MultiGreen 是原始 GreenGate 架构语法的 1:1 扩展，已被证明可有效地组装具有多个转录单元的质粒。MultiGreen 已通过在细菌中使用来自 Chromobacterium violaceum 的截短的 violacein 操纵子以及通过对 RUBY 报告基因进行体内功能验证得到验证。MultiGreen 目前支持我们的许多内部多转录单元组装，并且将是更复杂克隆项目的有价值策略。
