Functional Interrogation Of Ribosomal Biology Using Continuous Evolution

利用连续进化对核糖体生物学进行功能探究

• 批准号: 9593383
• 负责人: Ahmed Hussein Badran
• 金额: $ 44.5万
• 依托单位: BROAD INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9593383
• 关键词: Functional; Interrogation; Ribosomal; Biology; Using

FUNCTIONAL INTERROGATION OF RIBOSOMAL BIOLOGY USING CONTINUOUS EVOLUTION PROJECT SUMMARY/ABSTRACT: In nature, fundamental biological phenomena that are central to cellular life are inherently hindered from probing and interrogation, as these dynamic systems cannot be easily decoupled from immediate artifactual disruptions throughout the living cell. One such case is the ribosome, a colossal multi-component protein factory that functions as the nexus for cellular information and signaling events, integrating nutrient availability with growth dynamics and resource allocation. Despite decades of research, this biomolecular assembly remains superficially understood and underexplored, owing to the difficulty associated with decoupling the translational apparatus from cellular viability. In fact, there is currently no generalizable experimental tool-kit for unbiased high-throughput interrogation of the structure-activity and functional relationships of the ribosome, the prediction of ribosome-small molecule interactions, or the identification of disruptive resistance mechanisms. The work proposed herein seeks to overcome the challenges associated with ribosomal manipulation in vivo, to illuminate the relationship between the rRNA and the effective translation initiation/elongation rates as they relate to growth fitness, and to provide an innovative framework for the interrogation of ribosome-small molecule interactions. The proposed work focuses on the development of a fully orthogonal ribosomal system for the real-time monitoring of ribosome activity in living cells through engineered transcription-translation networks based on independently tunable genetic components at all stages. The designed orthogonal sensor ribosomes will be subjected to directed evolution yielding novel variants with enhanced or diminished kinetic properties. To achieve this, the orthogonal ribosome circuit will be interfaced with an emergent technique based on a continuous culturing methodology called Phage-Assisted Continuous Evolution (PACE), facilitating hundreds of rounds of directed evolution in just a few days with minimal researcher intervention. Finally, to demonstrate the utility of the newly developed ribosomal sensors and the evolutionary platform, this technology will be leveraged to inform antibiotic-ribosome interactions, and to generate actionable drug resistance profiles for delaying or evading microbial resistance. This platform will be extended to high-throughput screening campaigns for novel chemical scaffolds capable of modulating ribosomal translation through potentially undiscovered modes of action. Broadly, our ability to harness bacteria for biomedical and biomaterial applications in the future will hinge on the detailed understanding of the mechanistic control and optimization of ribosomal output parameters enabled by these studies. The technological advances proposed herein have the potential to extend our understanding of key factors governing ribosomal function and dynamics, and will pave the way towards the development of novel mechanisms that will illuminate and enhance new approaches in biomedical research and targeted antimicrobial therapeutics.

基于连续进化的核糖体生物学功能查询 项目总结/摘要： 在自然界中，作为细胞生命中心的基本生物现象固有地受到阻碍， 探测和询问，因为这些动态系统不能轻易地与直接的人为因素脱钩。 破坏整个活细胞。其中一个例子是核糖体，一种巨大的多组分蛋白质 作为细胞信息和信号事件的联系的工厂，整合营养物质的可用性 增长动力和资源分配。尽管经过了几十年的研究， 由于与经济和社会脱钩的困难， 细胞活力的翻译装置。事实上，目前还没有可推广的实验工具包， 无偏见的高通量审讯的结构活性和功能的关系的核糖体， 核糖体-小分子相互作用的预测，或破坏性抗性机制的鉴定。 本文提出的工作试图克服与体内核糖体操作相关的挑战， 为了阐明rRNA和有效翻译起始/延伸速率之间的关系， 与生长适应性有关，并为小核糖体的询问提供了一个创新的框架。 分子相互作用拟议的工作重点是发展一个完全正交的核糖体系统 用于通过工程化转录-翻译实时监测活细胞中的核糖体活性 网络的基础上独立可调的遗传组件在所有阶段。所设计的正交传感器 核糖体将进行定向进化，产生具有增强或减弱的动力学的新变体。 特性.为了实现这一点，正交核糖体电路将与一种新兴技术相结合 基于称为噬菌体辅助连续进化（PACE）的连续培养方法， 数百轮的定向进化在短短几天内，研究人员的干预最少。最后为 展示了新开发的核糖体传感器和进化平台的实用性，这项技术 将被用来告知抗生素-核糖体相互作用，并产生可操作的耐药谱， 来延缓或避免微生物的抗药性。该平台将扩展到高通量筛选 能够通过潜在的调节核糖体翻译的新型化学支架的活动 未被发现的行为模式。从广义上讲，我们利用细菌用于生物医学和生物材料的能力 未来的应用将取决于对机械控制和优化的详细理解， 核糖体输出参数使这些研究。本文提出的技术进步具有 有可能扩大我们对控制核糖体功能和动力学的关键因素的理解，并将铺平道路。 发展新机制的方式，将阐明和加强新的方法， 生物医学研究和有针对性的抗菌治疗。