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

基本信息

批准号：
10459135
负责人：
Ahmed Hussein Badran
金额：
$ 44.38万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2022-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10459135
关键词：
Address Affect Animal Model Antibiotic Resistance Antibiotics Bacteria Bacteriophages Biochemical Biocompatible Materials Biogenesis Biological Biological Phenomena Biology Biomedical Research Cells Chemicals Clinical Trials Code Combined Modality Therapy Defect Development Directed Molecular Evolution Drug resistance Engineering Escherichia coli Event Evolution Future Gap Junctions Generations Genes Genetic Genetic Transcription Glean Growth Intervention Kinetics Knowledge Life Link Mediating Medical Methodology Minor Modeling Modification Molecular Morphologic artifacts Multi-Drug Resistance Mutation Natural Products Nature Network-based Nutrient Output Peptidyltransferase Play Production Property Protein Synthesis Inhibition Proteins Real-Time Systems Regulation Research Research Personnel Resistance Resistance profile Resource Allocation Ribosomal Interaction Ribosomal RNA Ribosomes Role Signal Transduction Stimulus Structure Structure-Activity Relationship System Techniques Technology Therapeutic Time Translating Translation Initiation Translations Variant Work antimicrobial base design dynamic system fitness high throughput screening improved in vivo in vivo monitoring innovation microbial novel novel strategies novel therapeutics pathogenic bacteria rapid technique real time monitoring resistance mechanism response scaffold sensor small molecule

项目摘要

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.

利用持续进化对核糖体生物学进行功能研究项目摘要/摘要：在自然界中，作为细胞生命核心的基本生物现象本质上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工制品脱钩整个活细胞的破坏。其中一个例子是核糖体，一种巨大的多组分蛋白质工厂充当细胞信息和信号事件的纽带，整合营养物质的可用性增长动力和资源配置。尽管经过了数十年的研究，这种生物分子组装由于与解耦相关的困难，仍然被肤浅地理解和探索不足来自细胞活力的翻译装置。事实上，目前还没有通用的实验工具包对核糖体的结构-活性和功能关系进行无偏高通量询问，预测核糖体-小分子相互作用，或识别破坏性抗性机制。本文提出的工作旨在克服与体内核糖体操作相关的挑战，阐明 rRNA 与有效翻译起始/延伸率之间的关系与生长适应性相关，并为小核糖体的研究提供创新框架分子相互作用。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基于各个阶段独立可调遗传成分的网络。正交传感器设计核糖体将进行定向进化，产生具有增强或减弱动力学的新变体特性。为了实现这一目标，正交核糖体电路将与新兴技术相连接基于称为噬菌体辅助连续进化（PACE）的连续培养方法，促进只需最少的研究人员干预，即可在短短几天内完成数百轮定向进化。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，该技术将被用来告知抗生素-核糖体相互作用，并生成可操作的耐药性概况用于延迟或避免微生物耐药性。该平台将扩展到高通量筛选寻找能够通过潜在的方式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。从广义上讲，我们利用细菌进行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这些研究启用了核糖体输出参数。本文提出的技术进步具有有潜力扩展我们对控制核糖体功能和动力学的关键因素的理解，并将铺平道路开发新机制的途径将阐明和加强新方法生物医学研究和靶向抗菌治疗。