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知系统，该系统有可能调节合成细菌生命形式的反应。这种定制的生物体有可能为人类生产各种有益的产品，但必须严格控制其活动。目前，合成生物学家几乎没有高度稳健的控制系统，因此发现和开发其他调控途径至关重要。基于最近在一种高度丰富的光合作用海洋细菌中的发现，该项目使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，遗传和生物化学工具来测试模型并定义光调节过程。该系统具有巨大的潜力，为微生物生物技术，绿色化学和新的和快速发展的光遗传学领域作出重大贡献。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州大学的暑期研究沉浸计划。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测试从模型的预测。由蓝光和绿色光进行直径调节，并被聚球藻广泛使用，这种简单但强大的信号系统是独特的。调控元件由三种名为FciA、FciB和FciC的调控蛋白以及三种表达在蓝光中高度上调而在绿色光中下调的基因组成。FciA与FciC一起或独立于FciC操作，并且对这些组件进行建模为这两个模型中的一个提供加权证据。使用RNAseq和蛋白质印迹分析，在从绿色光中生长转换为蓝光中生长之后，检查RNA和蛋白质应答的时间过程。使用体外和体内方法来确定由FciA、FciB和FciC结合的特异性DNA序列。方法包括电迁移率变化测定、DNaseI足迹法和exoChIPseq。最后，信号系统被转移到另一个蓝藻物种和一个非光合细菌开始开发这种监管系统，用于在微生物生物技术中常用的物种。这个奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

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.