CAREER: Tunable Dynamics from Interlinked Feedback Loops in Synthetic and Natural Gene Circuits

智力优势：由于营养供应的波动、化学应激源和环境变化，生物体必须应对广泛的时变压力。基因相同的细胞群体可以使用随机、随机的基因表达来使它们的反应多样化，并对这种不确定性进行对冲。然而，当环境变化缓慢，可以感知和应对时，就几乎没有必要采取多样化的应对措施。这就提出了一个基本问题，即细胞如何协调这两个潜在的矛盾需求：需要使用表型多样性来预测压力，并采用定义明确的确定性反应。要做到这一点，需要可调的基因调控网络，根据环境条件在随机和确定性反应之间切换。最近的理论研究表明，相互关联的正反馈和负反馈(“混合反馈”)网络可以提供这种类型的可调响应。使用单细胞延时显微镜和微流控实验，并辅之以数学模型，该项目将量化合成和自然混合反馈电路的可调性，并将测试细胞如何处理时变环境。这项工作意义重大，因为它将揭示遗传机制，允许原本相同的细胞在某些情况下选择随机命运，在其他情况下展示确定性反应，使有机体能够应对环境不确定性。广泛影响：研究计划与社区、高中和本科生层面的系统和合成生物学教育的扩展计划相结合：首先，通过与佛蒙特州伯灵顿的Echo科学中心的合作，PI将制作一个特别的夏季展览和演讲，旨在向社区受众交流尖端系统和合成生物学主题。其次，PI将在她的研究小组中接待本科生巴雷特学者，并将为州长工程学院的高中生提供学生报告的指导。第三，PI将在目前专注于传统机械和电气工程应用的两门核心工程课程中引入系统和合成生物学示例。

ABSTRACTIntellectual Merit: Organisms must cope with a wide range of time-varying stresses, owing to fluctuations in nutrient availability, chemical stressors, and environmental changes. Genetically identical populations of cells can use random, stochastic expression of genes to diversify their response and hedge against this uncertainty. However, when environmental changes are slow and can be sensed and responded to, there is little need for a diversified response. This raises a fundamental question as to how cells reconcile these two potentially contradictory demands: the need to use phenotypic diversity to anticipate stress and also employ well-defined deterministic responses. Achieving this requires gene regulatory networks that are tunable, switching between stochastic and deterministic responses depending on environmental conditions. Recent theoretical studies have suggested that interlinked positive and negative feedback ("mixed feedback") networks, can provide this type of tunable response. Using single-cell time-lapse microscopy and microfluidic experiments supported by mathematical modeling, this project will quantify the tunability of synthetic and natural mixed feedback circuits and will test how cells handle time-varying environments. This work is significant because it will reveal genetic mechanisms that allow otherwise identical cells to choose random fates in some circumstances and exhibit deterministic responses in others, allowing organisms to cope with environmental uncertainty.Broader Impacts: The research plan is integrated with an outreach program focused on systems and synthetic biology education at the community, high school, and undergraduate levels: First, through a partnership with ECHO Science Center in Burlington, Vermont, the PI will produce a special summer exhibit and talk aimed at communicating cutting-edge systems and synthetic biology topics to community audiences. Second, the PI will host undergraduate Barrett Scholars in her research group and will provide mentorship for student presentations to high school students in the Governor's Institute in Engineering. Third, the PI will introduce systems and synthetic biology examples into two core engineering courses that currently focus on traditional mechanical and electrical engineering applications.