RoL: EAGER: DESYN-C3 Membraneless organelles by design: a biomimetic approach

细胞必须组织大量的反应。它通过隔离细胞器内的某些反应和分子来实现这一目标。有时这些细胞器存在于膜内。有时它们只是以不同的液相存在。控制细胞器的形成和组成是创造合成细胞的核心。合成细胞可用于化学物质、生物燃料和治疗药物的生物制造，以及潜在的应用。这个项目将研究没有膜的合成细胞器是否可以容纳复杂的反应序列。高中生将直接参与这个项目，通过合成生物学的实践培训和参加国际基因工程机器（iGEM）竞赛。他们面临的挑战将是开发能够触发液相细胞器形成的蛋白质标签。他们还会在博客上分享他们的经历。这将有望增加他们在stem相关领域追求事业的热情和兴趣。蛋白质的液-液相分离是活细胞的基本分离过程。它支持形成功能性，非膜结合，液体样室参与细胞功能和发育。例子包括核仁、Cajal小体、核斑点、胚粒、加工小体、应力小颗粒和细胞信号室。这些无膜细胞器可以以高度动态的方式分离和浓缩特定的分子集。它们还能迅速地与微环境交换成分。在液滴内，蛋白质保持或获得折叠结构，并保留特定蛋白质识别等功能。这些结构越来越多地与蛋白质聚集引起的疾病有关。在这个项目中，我们将测试是否可以利用LLPS来增强催化作用。一个汇集酶级联元素的细胞器将被构建和评估。这些无膜细胞器将通过在体内表达模块化蛋白和在显微镜下观察LLPS液滴的形成来评估。此外，我们将扩展这种方法，使酶级联，包括人工金属酶。如果成功，该提议将建立一种创新的，简单的方法来生成合成生物学的细胞器，补充现有的方法，如蛋白质外壳或囊泡封装。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。

The cell has to organize a large number of reactions. It accomplishes this by isolating certain reactions and molecules inside of compartments known as organelles. Sometimes these organelles exist within membranes. Sometimes they exist simply as a distinct liquid phase. Controlling organelle formation and composition is central to creating a synthetic cell. Synthetic cells could be used in the biomanufacturing of chemicals, biofuels, and therapeutics, among potential applications. This project will investigate whether synthetic organelles without membranes can house complex reaction sequences. High school students will be involved in this project directly, through hands-on training in synthetic biology and participation in the international genetically engineered machine (iGEM) competition. Their challenge will be to develop protein tags that can trigger formation of the liquid phase organelles. They will also share their experiences on a blog. This will hopefully increase their enthusiasm and interest in pursuing careers in STEM-related fields.Liquid-liquid phase separation (LLPS) of proteins is a fundamental process in living cells. It underpins the formation of functional, non-membrane bound, liquid-like compartments involved in cell function and development. Examples include the nucleolus, Cajal bodies, nuclear speckles, germ granules, processing bodies, stress granules, and cell signaling compartments. These membraneless organelles can separate and concentrate specific sets of molecules in a highly dynamic manner. They also exchange components with their microenvironments rapidly. Within the liquid droplet, proteins maintain or acquire a folded structure, and conserve functions such as specific protein recognition. Increasingly, these structures are being implicated in diseases caused by protein aggregation. In this project, we will test whether LLPS can be exploited to enhance catalysis. An organelle that brings together elements of an enzyme cascade will be constructed and evaluated. These membraneless organelles will be evaluated by expressing the modular proteins in vivo and visualizing the formation of LLPS droplets by microscopy. Further, we will extend this approach to enable enzymatic cascades that include artificial metalloenzymes. If successful, this proposal will establish an innovative, simple method to generate organelles for synthetic biology, complementing existing methods such as encapsulation by protein shells or by vesicles.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.