RUI: Complex control in C. elegans using de novo designed protein switches

合成生物学的一个基本目标是通过靶向降解重要的调节蛋白来控制细胞过程。该项目的目的是开发和测试一个特定的和可调的系统，蛋白质降解的模式真核生物。该项目被视为朝着将该工具应用于更复杂的生物系统并最终应用于生物医学研究和生物技术迈出的重要一步。该项目将在学年和夏季期间吸引不同群体的本科生研究人员进行跨学科研究，提高他们创造性解决问题的能力、独立思考能力以及与科学受众和公众沟通的能力。这项研究的部分内容将被整合到沃巴什学院的分子遗传学和生物化学课程中，吸引大量学生参与新的科学发现。目前控制秀丽隐杆线虫必需蛋白和多效性蛋白的方法存在局限性，包括不完全外显率、假阴性和无法同时用同一系统差异控制多个基因。项目团队将率先采用锁存正交笼/关键蛋白（LOCKR）方法对秀丽隐杆线虫进行蛋白控制。LOCKR包含一个全新设计的蛋白质“Switch”，能够捕获一个线性生物活性基序和一个诱导肽“Key”。肽键打开开关中被囚禁的基序，激发其生物活性功能。LOCKR系统的全新起源允许将多个开关和键设计为正交对，以同时控制多个蛋白质。线虫优化的degronLOCKR开关（NemDegronLOCKR）将为秀丽隐杆线虫研究人员提供一种强大的蛋白质控制方法，具有组织特异性和在发育的不同时间点独立控制多个基因的能力。本项目采用计算设计、体外生物物理分析和体内实验，使用CRISPR-Cas9将NemDegronLOCKR融合到感兴趣的基因上。这项研究的结果将由项目小组通过演讲和出版物广泛传播。秀丽隐杆线虫菌株和DNA构建体将分别存放在隐杆线虫遗传中心和Addgene。LOCKR系统是高度可调的，因为几乎任何线性图案都可以被囚禁。LOCKR系统的灵活性及其控制信号通路和细胞靶向的能力，将支持合成生物学家寻求产生新的类生命系统。LOCKR系统在线虫中的应用为其在其他更复杂的生物体中的应用奠定了坚实的基础。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。

A fundamental goal in synthetic biology is to control cellular process via the targeted degradation of important regulatory proteins. The aim of this project is to develop and test a specific and tunable system for protein degradation in a model eukaryotic organism. This project is viewed as an important step towards the application of this tool in more complex biological systems and ultimately towards its application in biomedical research and biotechnology. This project will engage a diverse group of undergraduate student researchers in interdisciplinary research during the academic year and summer, improving their creative problem-solving skills, independent thinking, and ability to communicate with both scientific audiences and the general public. Portions of this research will be integrated into the Molecular Genetics and Biochemistry courses at Wabash College, engaging a large number of students in novel scientific discovery. Current methods of controlling essential and pleiotropic proteins in C. elegans suffer from limitations, including incomplete penetrance, false negatives, and the inability to differentially control multiple genes with the same system simultaneously. The project team will pioneer the Latching Orthogonal Cage/Key pRoteins (LOCKR) method of protein control in C. elegans. LOCKR encompasses a de novo designed protein “Switch” capable of caging a linear bioactive motif and an inducible peptide “Key.” The peptide Key unlocks the caged motif in the Switch, eliciting its bioactive function. The LOCKR system’s de novo origins allow the design of multiple Switches and Keys as orthogonal pairs to control multiple proteins simultaneously. A nematode-optimized degronLOCKR Switch (NemDegronLOCKR) will afford C. elegans researchers a robust protein control method with tissue specificity and the ability to independently control multiple genes at various time points in development. This project applies computational design, in vitro biophysical analysis, and in vivo experiments using CRISPR-Cas9 to fuse NemDegronLOCKR to genes of interest. The results of this study will be broadly disseminated by the project team through presentations and publications. C. elegans strains and DNA constructs will be deposited at the Caenorhabditis Genetics Center and Addgene, respectively. The LOCKR system is highly tunable, as virtually any linear motif can be caged. The flexibility of the LOCKR system, and its ability to control signaling pathways and cellular targeting, will support synthetic biologists seeking to produce novel lifelike systems. Demonstration of the utility of the LOCKR system in nematodes lays a firm foundation for its use in other, even more complex organisms.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.