Vazquez-Vilar Marta, Quijano-Rubio Alfredo, Fernandez-Del-Carmen Asun, Sarrion-Perdigones Alejandro, Ochoa-Fernandez Rocio, Ziarsolo Peio, Blanca José, Granell Antonio, Orzaez Diego
Instituto de Biología Molecular y Celular de Plantas (IBMCP), Consejo Superior de Investigaciones Científicas (CSIC), Universidad Politécnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain.
Centro de Conservación y Mejora de la Agrodiversidad Valenciana (COMAV), Universidad Politécnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain.
Nucleic Acids Res. 2017 Feb 28;45(4):2196-2209. doi: 10.1093/nar/gkw1326.
Modular DNA assembly simplifies multigene engineering in Plant Synthetic Biology. Furthermore, the recent adoption of a common syntax to facilitate the exchange of plant DNA parts (phytobricks) is a promising strategy to speed up genetic engineering. Following this lead, here, we present a platform for plant biodesign that incorporates functional descriptions of phytobricks obtained under pre-defined experimental conditions, and systematically registers the resulting information as metadata for documentation. To facilitate the handling of functional descriptions, we developed a new version (v3.0) of the GoldenBraid (GB) webtool that integrates the experimental data and displays it in the form of datasheets. We report the use of the Luciferase/Renilla (Luc/Ren) transient agroinfiltration assay in Nicotiana benthamiana as a standard to estimate relative transcriptional activities conferred by regulatory phytobricks, and show the consistency and reproducibility of this method in the characterization of a synthetic phytobrick based on the CaMV35S promoter. Furthermore, we illustrate the potential for combinatorial optimization and incremental innovation of the GB3.0 platform in two separate examples, (i) the development of a collection of orthogonal transcriptional regulators based on phiC31 integrase and (ii) the design of a small genetic circuit that connects a glucocorticoid switch to a MYB/bHLH transcriptional activation module.
模块化DNA组装简化了植物合成生物学中的多基因工程。此外，最近采用通用语法来促进植物DNA部件（植物砖）的交换是加速基因工程的一种有前景的策略。在此基础上，我们提出了一个植物生物设计平台，该平台纳入了在预定义实验条件下获得的植物砖的功能描述，并将所得信息系统地注册为文档的元数据。为便于处理功能描述，我们开发了GoldenBraid（GB）网络工具的新版本（v3.0），该版本整合了实验数据并以数据表的形式显示。我们报告了在本氏烟草中使用荧光素酶/海肾荧光素酶（Luc/Ren）瞬时农杆菌浸润测定法作为评估调控植物砖赋予的相对转录活性的标准，并展示了该方法在基于花椰菜花叶病毒35S启动子的合成植物砖表征中的一致性和可重复性。此外，我们在两个独立的例子中说明了GB3.0平台的组合优化和渐进创新潜力，（i）基于phiC31整合酶开发一组正交转录调节因子，（ii）设计一个将糖皮质激素开关与MYB/bHLH转录激活模块连接的小型遗传电路。
