Integrative Mathematical and Experimental Approaches to Understanding Robust Activation of Gene Expression by Light Color

这项研究定义了一种新发现的细菌光色感应系统，该系统有可能调节合成创造的细菌生命形式的反应。这种定制的生物有可能为人类生产一系列有益的产品，但它们的活动必须受到严格控制。目前，合成生物学家很少有高鲁棒性的控制系统，因此发现和开发额外的调控途径是至关重要的。该项目建立在最近发现的一种高度丰富的光合海洋细菌的基础上，利用数学模型来预测将环境光颜色从绿色变为蓝色导致细菌基因表达增加35-40倍的机制。该项目还涉及使用分子、遗传和生化工具来测试模型并定义光调节过程。该系统在微生物生物技术、绿色化学和新兴的快速发展的光遗传学领域具有巨大的潜力。除了该项目的基本科学价值外，还有三名研究生接受了建模和分子遗传学研究方面的培训。高中教师也接受了科学教学法方面的培训。来自科学领域代表性不足群体的学生受益于印第安纳大学（Indiana University）的暑期浸入式研究项目。通过数学建模来预测新发现的信号转导通路的运作机制。用分子生物学和生物化学方法对模型的预测进行检验。这个简单但强大的信号系统是独一无二的，它由蓝光和绿光直接调节，被聚珠球菌广泛使用。该调控元件由FciA、FciB和FciC三个调控蛋白组成，这三个基因在蓝光下表达高度上调，在绿光下表达下调。FciA要么与FciC合作，要么独立于FciC，对这些组件进行建模为这两种模型中的一种提供了加权证据。利用RNAseq和Western blot分析，研究了从绿光下生长切换到蓝光下生长后RNA和蛋白质反应的时间过程。体外和体内方法均用于确定FciA、FciB和FciC结合的特定DNA序列。方法包括Electromobility Shift assay， dna足迹分析和exoChIPseq。最后，信号系统被转移到另一种蓝藻物种和一种非光合细菌，开始发展这种调节系统，用于微生物生物技术中常用的物种。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

This research defines a newly discovered light color sensing system in bacteria that has the potential to regulate responses in synthetically created bacterial life forms. Such customized organisms have the potential to produce a wide range of beneficial products for humankind, but their activities must be strictly controlled. At present, there are few highly robust control systems available to synthetic biologists, therefore the discovery and development of additional regulatory pathways is of paramount importance. Building on recent discoveries in a highly abundant, photosynthetic marine bacterium, this project uses mathematical modeling to predict the mechanism through which shifting ambient light color from green to blue results in a 35-40-fold increase in the bacterium's gene expression. The project also involves the use of molecular, genetic, and biochemical tools to test the models and define the photo-regulation process. This system has tremendous potential for significantly contributing to the areas of microbial biotechnology, green chemistry and the new and rapidly growing field of optogenetics. In addition to the basic scientific value of the project there is training received, by three graduate students, in modeling and molecular genetics research. There is also training received, in science pedagogy, by high school teachers. Students from underrepresented groups in science benefit from a summer research immersion program at Indiana University.Mathematical modeling is used to predict the mechanism through which a newly discovered signal transduction pathway operates. Predictions from the model are tested using molecular biological and biochemical approaches. Diametrically regulated by blue and green light and widely used by Synechococcus, this simple but robust signaling system is unique. The regulation element consists of three regulatory proteins named FciA, FciB, and FciC, and three genes whose expression is highly upregulated in blue light and downregulated in green light. FciA either operates with, or independently of, FciC, and modeling these components provides weighted evidence for one of these two models. Using RNAseq and Western blot analyses a time course of RNA and protein responses is examined after a switch from growth in green light to growth in blue light. Both in vitro and in vivo approaches are used to determine the specific DNA sequences that are bound by FciA, FciB, and FciC. Methods include Electromobility Shift Assays, DNaseI footprinting, and exoChIPseq. Finally, the signaling system is transferred to another cyanobacterial species and to a non-photosynthetic bacterium to begin to develop this regulatory system for use in species that are commonly used in microbial biotechnology.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.