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RoL: EAGER: DESYN-C3 Enzyme cascades in synthetic membraneless organelles

RoL：EAGER：DESYN-C3 合成无膜细胞器中的酶级联

基本信息

批准号：
1844149
负责人：
Allie Obermeyer
金额：
$ 29.62万
依托单位：
Columbia University
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2021-08-31
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1844149&HistoricalAwards=false
关键词：
RoL EAGER DESYN C3 Enzyme

项目摘要

Like factories, cells organize processes by placing them in distinct rooms (organelles). The project will create a model system to understand how cells utilize compartments without distinct walls. Design rules for the formation and function of these compartments will be established. The rules will be tested by constructing an membraneless organelle that houses enzymes that direct a series of reactions. The project will also focus efforts to foster a diverse STEM workforce through student recruiting, training, and teaching at all levels. Graduate and undergraduate students will be recruited. Outreach activities on enzymes will be developed for middle school students and summer research experiences will be provided to local high school students.Membraneless organelles (M-organelles) provide spatial and temporal organization through selective phase separation. They are important in cell signaling, transcriptional control, and stress response. However, a robust model system that replicates their function does not yet exist. This project will develop a model system based on electrostatically driven liquid-liquid phase separation of enzymes. A model enzyme cascade of glucose oxidase and horseradish peroxidase will be incorporated into these synthetic M-organelles. Protein engineering of the enzymes will be used to determine the design rules for multiple protein partitioning. A library of fluorescent fusion proteins will be biosynthesized and their incorporation in the synthetic M-organelles will be quantified by fluorescence microscopy. The development of these design principles will enable control over the relative ratio of the sequenced enzymes in the synthetic M-organelles. Importantly, the impact of the synthetic M-organelle microenvironment on the efficiency of the enzyme cascade will be evaluated. The spatial distribution of the enzymes, substrates, and product will be monitored and used to determine detailed reaction kinetics. These combined efforts will help to establish rules for the formation and function of M-organelles.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.

像工厂一样，细胞通过将过程放置在不同的房间(细胞器)来组织过程。该项目将创建一个模型系统，以了解细胞如何利用没有明显墙壁的隔间。将制定这些舱室的组成和功能的设计规则。这些规则将通过构建一个无膜细胞器来测试，该细胞器含有指导一系列反应的酶。该项目还将重点努力通过招生、培训和各级教学来培养多样化的STEM劳动力队伍。将招收研究生和本科生。将为中学生开展关于酶的推广活动，并向当地高中生提供暑期研究经验。无膜细胞器(M-细胞器)通过选择性相分离提供空间和时间组织。它们在细胞信号、转录调控和应激反应中起着重要作用。然而，复制它们的功能的健壮的模型系统还不存在。本项目将开发一种基于静电驱动的酶的液液相分离的模型系统。葡萄糖氧化酶和辣根过氧化物酶的模型级联酶将被加入到这些合成的M细胞器中。酶的蛋白质工程将被用来确定多重蛋白质分配的设计规则。一个荧光融合蛋白文库将被生物合成，它们在合成的M细胞器中的掺入将通过荧光显微镜进行量化。这些设计原则的发展将能够控制合成M细胞器中已测序酶的相对比例。重要的是，将评估合成M细胞器微环境对酶级联效率的影响。将监测酶、底物和产物的空间分布，并用于确定详细的反应动力学。这些共同努力将有助于建立M组织的形成和功能规则。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。