Department of Food Science, University of Tennessee Knoxville, 102 Food Safety and Processing Building 2600 River Dr., Knoxville, Tennessee 37996, United States.
Center for Agricultural Synthetic Biology, University of Tennessee Institute of Agriculture, Knoxville, Tennessee 37996, United States.
ACS Synth Biol. 2022 Aug 19;11(8):2741-2755. doi: 10.1021/acssynbio.2c00147. Epub 2022 Jul 28.
While the installation of complex genetic circuits in microorganisms is relatively routine, the synthetic biology toolbox is severely limited in plants. Of particular concern is the absence of combinatorial analysis of regulatory elements, the long design-build-test cycles associated with transgenic plant analysis, and a lack of naming standardization for cloning parts. Here, we use previously described plant regulatory elements to design, build, and test 91 transgene cassettes for relative expression strength. Constructs were transiently transfected into leaves and expression of a fluorescent reporter was measured from plant canopies, leaves, and protoplasts isolated from transfected plants. As anticipated, a dynamic level of expression was achieved from the library, ranging from near undetectable for the weakest cassette to a ∼200-fold increase for the strongest. Analysis of expression levels in plant canopies, individual leaves, and protoplasts were correlated, indicating that any of the methods could be used to evaluate regulatory elements in plants. Through this effort, a well-curated 37-member part library of plant regulatory elements was characterized, providing the necessary data to standardize construct design for precision metabolic engineering in plants.
虽然在微生物中安装复杂的遗传电路相对常规,但合成生物学工具在植物中受到严重限制。特别令人关注的是缺乏调控元件的组合分析,与转基因植物分析相关的长设计-构建-测试周期,以及克隆部件的命名标准化缺失。在这里,我们使用先前描述的植物调控元件来设计、构建和测试 91 个用于相对表达强度的转基因盒。将构建体瞬时转染到叶片中,并从转染植物的叶肉原生质体中测量荧光报告蛋白的表达。正如预期的那样,从文库中实现了动态的表达水平,从最弱的盒到最强的盒的表达水平增加了约 200 倍。对植物冠层、单个叶片和原生质体中的表达水平进行了相关性分析,表明任何一种方法都可以用于评估植物中的调控元件。通过这项工作,我们对 37 个精心设计的植物调控元件组成的部分文库进行了特征描述,为植物中精确代谢工程的构建设计提供了必要的数据标准化。