School of Biomedical Engineering, Colorado State University, Fort Collins, Colorado, USA.
Department of Biology, Colorado State University, Fort Collins, Colorado, USA.
Nat Methods. 2016 Jan;13(1):94-100. doi: 10.1038/nmeth.3659. Epub 2015 Nov 16.
Plant synthetic biology promises immense technological benefits, including the potential development of a sustainable bio-based economy through the predictive design of synthetic gene circuits. Such circuits are built from quantitatively characterized genetic parts; however, this characterization is a significant obstacle in work with plants because of the time required for stable transformation. We describe a method for rapid quantitative characterization of genetic plant parts using transient expression in protoplasts and dual luciferase outputs. We observed experimental variability in transient-expression assays and developed a mathematical model to describe, as well as statistical normalization methods to account for, this variability, which allowed us to extract quantitative parameters. We characterized >120 synthetic parts in Arabidopsis and validated our method by comparing transient expression with expression in stably transformed plants. We also tested >100 synthetic parts in sorghum (Sorghum bicolor) protoplasts, and the results showed that our method works in diverse plant groups. Our approach enables the construction of tunable gene circuits in complex eukaryotic organisms.
植物合成生物学有望带来巨大的技术效益，包括通过预测性设计合成基因回路，开发可持续的基于生物的经济。这些回路是由定量表征的遗传元件构建而成的；然而，由于稳定转化所需的时间，这一特征在植物研究中是一个重大障碍。我们描述了一种使用原生质体瞬时表达和双荧光素酶输出对遗传植物元件进行快速定量表征的方法。我们观察到瞬时表达测定中的实验变异性，并开发了一个数学模型来描述这种变异性，并提出了统计归一化方法来解释和补偿这种变异性，从而使我们能够提取定量参数。我们对拟南芥中的 120 多个合成元件进行了表征，并通过比较瞬时表达与稳定转化植物中的表达验证了我们的方法。我们还在高粱（Sorghum bicolor）原生质体中测试了 100 多个合成元件，结果表明我们的方法适用于不同的植物群体。我们的方法能够在复杂的真核生物中构建可调谐的基因回路。
