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中的光颜色传感系统，该系统有可能调节合成的细菌生命形式的反应。这种定制的有机体有可能生产出广泛的有益于人类的产品，但它们的活动必须受到严格控制。目前，合成生物学家几乎没有高度健壮的控制系统，因此发现和开发额外的调控途径是至关重要的。该项目以最近在一种高度丰富的光合作用海洋细菌中的发现为基础，利用数学建模来预测环境光颜色从绿色转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和定义光调节过程。该系统在微生物生物技术、绿色化学和新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。高中教师还接受了科学教育学方面的培训。印第安纳大学的暑期研究沉浸项目让来自科学领域代表性不足群体的学生受益。数学建模被用来预测一种新发现的信号转导途径的运作机制。使用分子生物学和生物化学方法对该模型的预测进行了检验。这个简单但强大的信号系统受到蓝绿光的径向调节，并被聚球藻广泛使用，是独一无二的。该调控元件由FCIA、FCIB和FCIC三个调控蛋白和三个基因组成，它们在蓝光下表达上调，在绿光下表达下调。FCIA要么与FCIC一起运行，要么独立于FCIC运行，对这些组件进行建模为这两个模型中的一个提供了加权证据。使用RNAseq和Western印迹分析，在从绿光生长切换到蓝光生长后，检测了RNA和蛋白质响应的时间进程。体外和体内方法都被用来确定与FCIA、FCIB和FCIC结合的特定DNA序列。方法包括电子迁移率移位分析、DNaseI足迹和exoChIPseq。最后，信号系统被转移到另一个蓝藻物种和一个非光合作用细菌，以开始开发这种调控系统，用于微生物生物技术中常用的物种。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.