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: 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
• 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

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.

使用连续进化对核糖体生物学的功能询问 项目摘要/摘要： 在本质上，基本的生物学现象是细胞生命的核心 探测和审讯，因为这些动态系统不能轻易与直接的人为 整个活细胞中的破坏。一种情况是核糖体，一种巨大的多组分蛋白 充当蜂窝信息和信号事件的联系的工厂，整合营养的可用性 随着增长动态和资源分配。尽管进行了数十年的研究，但这种生物分子组装 由于与将其解耦相关的困难，保持表面上的理解和尚未得到充实的理解 来自细胞活力的翻译设备。实际上，目前尚无可用的实验工具套件 对核糖体的结构活性和功能关系的无偏高通量询问， 预测核糖体 - 小分子相互作用或鉴定破坏性抗性机制。 本文提出的工作试图克服与体内核糖体操纵相关的挑战， 阐明rRNA与有效的翻译启动/伸长率之间的关系 与生长健康相关，并为核糖小all的询问提供创新的框架 分子相互作用。拟议的工作着重于完全正交核糖体系统的开发 通过工程转录翻译对活细胞中核糖体活动的实时监测 在各个阶段基于独立可调遗传成分的网络。设计的正交传感器 核糖体将受到定向进化，产生具有增强或减小动力学的新型变体 特性。为此，正交核糖体电路将与新兴技术连接 基于一种连续培养方法，称为噬菌体辅助连续进化（速度），促进 在短短几天内，研究人员的干预措施就在短短几天之内进行了数百发指导进化。最后，到 这项技术演示了新开发的核糖体传感器和进化平台的实用性 将被杠杆化以告知抗生素 - 核糖体相互作用，并产生可行的耐药性概况 用于延迟或逃避微生物抗性。该平台将扩展到高通量筛选 针对能够通过潜在调节核糖体翻译的新型化学脚手架的运动 未发现的行动方式。从广义上讲，我们利用细菌的生物医学和生物材料的能力 将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 扩展我们对核糖体功能和动态关键因素的理解的潜力，并将铺路 开发新型机制的方式，这些机制将阐明并增强新方法 生物医学研究和靶向抗菌治疗剂。

项目成果

Orthogonal translation enables heterologous ribosome engineering in E. coli.

• DOI: 10.1038/s41467-020-20759-z
• 发表时间: 2021-01-26
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Kolber NS;Fattal R;Bratulic S;Carver GD;Badran AH
• 通讯作者: Badran AH

Directed evolution of rRNA improves translation kinetics and recombinant protein yield.

• DOI: 10.1038/s41467-021-25852-5
• 发表时间: 2021-09-24
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Liu F;Bratulić S;Costello A;Miettinen TP;Badran AH
• 通讯作者: Badran AH

Modern methods for laboratory diversification of biomolecules.

• DOI: 10.1016/j.cbpa.2017.10.010
• 发表时间: 2017-12
• 期刊: Current opinion in chemical biology
• 影响因子: 7.8
• 作者: Bratulic S;Badran AH
• 通讯作者: Badran AH

Synthetic Biological Circuits within an Orthogonal Central Dogma.

• DOI: 10.1016/j.tibtech.2020.05.013
• 发表时间: 2021-01
• 期刊: Trends in biotechnology
• 影响因子: 17.3
• 作者: Costello A;Badran AH
• 通讯作者: Badran AH

A Deep Learning Approach to Antibiotic Discovery.

• DOI: 10.1016/j.cell.2020.01.021
• 发表时间: 2020-02-20
• 期刊: Cell
• 影响因子: 64.5
• 作者: Stokes JM;Yang K;Swanson K;Jin W;Cubillos-Ruiz A;Donghia NM;MacNair CR;French S;Carfrae LA;Bloom-Ackermann Z;Tran VM;Chiappino-Pepe A;Badran AH;Andrews IW;Chory EJ;Church GM;Brown ED;Jaakkola TS;Barzilay R;Collins JJ
• 通讯作者: Collins JJ