University of California Berkeley, Chemical and Biomolecular Engineering, Berkeley, California 94720, United States.
University of California Davis, Department of Plant Biology and Genome Center, Davis, California 95616, United States.
ACS Synth Biol. 2021 Oct 15;10(10):2763-2766. doi: 10.1021/acssynbio.1c00248. Epub 2021 Sep 14.
Plant synthetic biology requires precise characterization of genetic elements to construct complex genetic circuits that can improve plant traits or confer them with new characteristics. Transcriptional reporter assays are essential to quantify the effect of gene expression regulator elements. Additionally, transcriptional reporter systems are a key tool in understanding control of gene expression in biology. In this work, we construct and characterize a dual color luciferase ratiometric reporter system that possesses several advantages over currently used reporters. It is ratiometric, thus reducing variability and increasing consistency between experiments; it is fast, as both reporters can be measured at the same time in a single reaction, and it is less expensive to perform than current dual luciferase reporter assays. We have validated our system quantifying the transcriptional capability of a panel of promoters and terminators commonly used in synthetic biology with a broad range of expression magnitudes, and in a biologically relevant system, nitrate response.
植物合成生物学需要对遗传元件进行精确的特征描述，以构建能够改善植物特性或赋予其新特性的复杂遗传回路。转录报告基因检测是定量分析基因表达调控元件影响的必要手段。此外，转录报告基因系统是理解生物学中基因表达调控的关键工具。在这项工作中，我们构建并表征了一种双色荧光素酶比率报告基因系统，它相对于目前使用的报告基因具有几个优势。它是比率式的，因此减少了实验之间的变异性和增加了一致性；它快速，因为两个报告基因可以同时在单个反应中进行测量；而且与目前的双荧光素酶报告基因检测相比，它的成本更低。我们已经验证了我们的系统，通过对一系列在合成生物学中常用的启动子和终止子的转录能力进行定量分析，这些启动子和终止子具有广泛的表达幅度，并且在一个具有生物学意义的系统中，硝酸盐响应。
