Streamlining temperate phage engineering to facilitate precise in situ manipulation of gut microbiota

简化温带噬菌体工程以促进肠道微生物群的精确原位操作

基本信息

批准号：
10700151
负责人：
Gregory William Goldberg
金额：
$ 12.5万
依托单位：
NEW YORK UNIVERSITY SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-07 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10700151
关键词：
Advisory Committees Antibiotics Attention Bacteria Bacterial Genome Bacteriophages Base Sequence Bioinformatics Biology CRISPR/Cas technology Cells Coliphages Committee Members Communities Complex Cosmids Coupled Cues DNA Development Directed Molecular Evolution Elements Engineering Ensure Environment Escherichia coli Excision Feces Foundations Frequencies Future Generations Genes Genetic Genome Genome engineering Genomic Library Germ-Free Grant Health Human Immunity Immunize In Situ Infection Lethal Genes Libraries Lysogeny Lytic Lytic Phase Mentors Methodology Methods Microbe Mobile Genetic Elements Modernization Mutagens Organism Outcome Phase Phylogeny Physiological Population Prevalence Process Productivity Prophage Excisions Prophages Protocols documentation Reagent Research Research Activity Research Personnel Resources Risk Specificity System Techniques Testing Therapeutic Time Training Transgenic Organisms Virulence Virulent Work Writing Yeasts career career development commensal bacteria deep sequencing derepression experience experimental study fecal transplantation fitness genome sequencing gut bacteria gut colonization gut microbiota improved in silico microbial microbial genome microbiome microbiome alteration microbiome composition microbiota mutant prevent programs promoter side effect site-specific integration skills success tool transmission process transplantation therapy

Advisory Committees Antibiotics Attention Bacteria Bacterial Genome Bacteriophages Base Sequence Bioinformatics Biology CRISPR/Cas technology Cells Coliphages Committee Members Communities Complex Cosmids Coupled Cues DNA Development Directed Molecular Evolution Elements Engineering Ensure Environment Escherichia coli Excision Feces Foundations Frequencies Future Generations Genes Genetic Genome Genome engineering Genomic Library Germ-Free Grant Health Human Immunity Immunize In Situ Infection Lethal Genes Libraries Lysogeny Lytic Lytic Phase Mentors Methodology Methods Microbe Mobile Genetic Elements Modernization Mutagens Organism Outcome Phase Phylogeny Physiological Population Prevalence Process Productivity Prophage Excisions Prophages Protocols documentation Reagent Research Research Activity Research Personnel Resources Risk Specificity System Techniques Testing Therapeutic Time Training Transgenic Organisms Virulence Virulent Work Writing Yeasts career career development commensal bacteria deep sequencing derepression experience experimental study fecal transplantation fitness genome sequencing gut bacteria gut colonization gut microbiota improved in silico microbial microbial genome microbiome microbiome alteration microbiome composition microbiota mutant prevent programs promoter side effect site-specific integration skills success tool transmission process transplantation therapy

展开

项目摘要

PROJECT SUMMARY This project aims to improve the overall accessibility of precise tools for manipulating mammalian microbiota within native gut environments, i.e., in situ. Despite the rich microbial diversity that mammalian gut communities natively acquire and maintain over time, the feasibility of experimentally and therapeutically altering their composition with antibiotics or fecal transplants has been clearly documented. However, these widely available treatments are found to broadly alter microbiome composition and this has limited their practical utility for determining how particular populations of microbiota influence their host’s health. Alternative approaches that allow for precise elimination or genetic editing of endogenous gut commensals in situ have recently been demonstrated using horizontally transmissible mobile genetic elements (MGEs), including temperate phages, but are not yet widely established. Phages are thought to be among the most precise MGEs for in situ microbiome manipulation given that the majority of studies on their host range have failed to detect cross-genus infectivity. Studies proposed in this application harness the natural abundance and precision of temperate phages and will establish generalizable methods to re-engineer them for more reliable use in microbiome editing applications. Namely, my proposed directed evolution and rational engineering approaches will streamline the generation of (1) lethal virulent mutants that are obligately lytic and capable of superinfecting their lysogenic kin, as well as (2) non-lethal temperate phage derivatives that can eliminate endogenous prophages or stably lysogenize at elevated frequencies. The initial mentored K99 phase of this research includes proof-of-principle engineering experiments with lambda, the most well-characterized temperate phage of Escherichia coli. In addition to providing me with hands-on lambda experience, this vital K99 training will prepare me for independent research activities during the R00 phase and throughout my future career by allowing me to become skilled in key techniques—such as large-scale DNA assembly in yeast for construction of phage genome libraries and whole phage genome sequencing—that can be applied to the study of non-model microbiota. Training will be supervised by two mentors with the requisite resources and experience to ensure my success on this project, Dr. Jef Boeke and Dr. Marcus Noyes, as well as four advisory committee members with complementary expertise. Career-oriented guidance from my mentors and advisors, along with career development activities during the K99 phase that include formal coursework on grant writing and project management, will further facilitate my transition to the R00 phase and my long-term productivity as an independent academic investigator. Ultimately, completion of this proposed project will open new avenues for well-controlled experimentation in the microbiome field while providing fundamental tools for microbial genome engineering and laying a strong foundation for my future lab’s research program.

项目摘要该项目旨在提高操纵哺乳动物微生物的精确工具的总体可及性在本地肠道环境中，即原位。尽管哺乳动物肠道社区具有丰富的微生物多样性随着时间的流逝，本来就获得和维持的，在实验和治疗上的可行性已清楚地记录了具有抗生素或粪便移植物的组成。但是，这些广泛可用发现治疗可广泛改变微生物组的组成，这限制了其实际实用性确定特定的微生物群如何影响宿主的健康。替代方法最近允许精确消除或遗传性肠道肠道原位的原位使用水平传播的移动遗传元件（MGE）证明，包括温度噬菌体，但尚未广泛建立。噬菌体被认为是原位微生物组最精确的MGE 鉴于大多数关于其宿主范围的研究未能检测到跨发感染的操作。在此应用中提出的研究可以利用温度噬菌体的自然抽象和精度建立可通用的方法来重新设计它们，以在微生物组编辑应用中更可靠地使用。也就是说，我提出的定向进化和合理的工程方法将简化生成（1）具有强制性裂解并且能够超级感染的致命病毒突变体以及（2）非致命的温度噬菌体衍生物，可以消除内源性或稳定的溶菌原频率升高。最初修补了这项研究的K99阶段包括原则工程证明 Lambda的实验，这是大肠杆菌中最良好的温度噬菌体。此外为我提供动手的Lambda经验，这种重要的K99培训将为我准备独立研究在R00阶段和整个未来职业的活动，使我能够熟练掌握关键技术 - 酵母中的大规模DNA组件，用于噬菌体基因组库和整个噬菌体基因组测序 - 可以应用于非模型菌群的研究。培训将是由两位导师的监督，具有所需的资源和经验，以确保我在这个项目上的成功， Jef Boeke博士和Marcus Noyes博士，以及四名咨询委员会成员专业知识。我的导师和顾问的面向职业指导以及职业发展活动在K99阶段，包括赠款写作和项目管理的正式课程，将进一步促进了我向R00阶段的过渡以及作为独立学术研究者的长期生产力。最终，该拟议项目的完成将为实验良好的实验开辟新的途径微生物组领域在提供微生物基因组工程的基本工具并铺设强大的工具我未来实验室研究计划的基础。