Functional Interrogation Of Ribosomal Biology Using Continuous Evolution

利用连续进化研究核糖体生物学的功能 项目摘要/摘要： 在自然界中，对细胞生命至关重要的基本生物现象天生就受到阻碍 探测和审问，因为这些动态系统不能轻易地与直接的人工产物分离 整个活细胞的破坏。核糖体就是这样一个例子，核糖体是一种巨大的多组分蛋白质 作为细胞信息和信号事件的纽带，整合营养供应的工厂 增长动力和资源配置。尽管经过了几十年的研究，这种生物分子组装 由于与脱钩相关的困难，仍然表面上理解和探索不足 翻译装置来自细胞的生存能力。事实上，目前还没有可推广的实验工具包 对核糖体的结构-活性和功能关系的无偏见高通量询问， 核糖体-小分子相互作用的预测，或破坏抗性机制的识别。 这里提出的工作试图克服与体内核糖体操纵相关的挑战， 为了阐明rRNA和有效翻译起始/延伸率之间的关系， 与生长适合性有关，并为询问核糖体-小的提供一个创新的框架 分子相互作用。拟议的工作重点是开发完全正交的核糖体系统。 通过工程转录-翻译实时监测活细胞中的核糖体活性 在所有阶段基于可独立调节的遗传成分的网络。所设计的正交型传感器 核糖体将经历定向进化，产生动力增强或减弱的新变体 属性。为了实现这一点，正交核糖体电路将与一项新兴技术相结合 基于一种称为噬菌体辅助持续进化(PACE)的持续培养方法，促进 在最少的研究人员干预下，在短短几天内进行了数百轮定向进化。最后，为了 展示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-核糖体的相互作用，并产生可操作的耐药性图谱 用于延缓或逃避微生物耐药性。该平台将扩展到高通量筛选 能够通过潜在地调节核糖体翻译的新型化学支架的活动 未被发现的行动模式。广泛地说，我们为生物医学和生物材料利用细菌的能力 未来的应用将取决于对机械控制和优化的详细了解 这些研究使核糖体输出参数成为可能。这里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，并将为 开发新机制的途径将阐明和加强新的方法 生物医学研究和靶向抗菌疗法。

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.