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

项目摘要 该项目旨在提高操纵哺乳动物微生物群的精确工具的整体可及性 在天然肠道环境中，即，在原地。尽管哺乳动物的肠道群落中有丰富的微生物多样性 随着时间的推移，自然获得和维持，实验和治疗改变其 与抗生素或粪便移植物的组合物已被明确记录。然而，这些广泛存在的 发现治疗广泛地改变了微生物组组成，这限制了它们对 确定特定的微生物群如何影响宿主的健康。的替代方法获得 允许精确消除或原位遗传编辑内源性肠道微生物， 使用水平可传递的移动的遗传元件（MGE），包括温带的， 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之一 由于大多数关于其宿主范围的研究未能检测到跨属感染性，因此， 在这项申请中提出的研究利用了温带气候的自然丰富性和精确性， 建立可推广的方法来重新设计它们，以便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理性工程方法将简化 (1)专性裂解并能重复感染其溶原性亲属的致死性毒性突变体，以及（2） 非致死性温和噬菌体衍生物，其可以消除内源性原噬菌体或在 高频率。本研究的初始指导K99阶段包括原理验证工程 实验与λ，最好的特点温和噬菌体的大肠杆菌。除了 这个重要的K99培训为我提供了实际的lambda经验，使我为独立研究做好了准备 在R 00阶段和我未来的职业生涯中，通过让我掌握关键技能， 技术-例如在酵母中大规模DNA组装以构建噬菌体基因组文库和整个 噬菌体基因组测序-可应用于非模型微生物群的研究。培训将 由两位导师监督，他们拥有必要的资源和经验，以确保我在这个项目上取得成功， 博士Jef Boeke和Marcus Noyes博士，以及四名顾问委员会成员， 专业知识我的导师和顾问提供以职业为导向的指导，并沿着职业发展活动 在K99阶段，包括关于赠款写作和项目管理的正式课程，将进一步 促进我向R 00阶段的过渡，以及我作为一名独立学术研究者的长期生产力。 最终，完成这一拟议的项目将为控制良好的实验开辟新的途径， 微生物组领域，同时为微生物基因组工程提供基础工具， 我未来实验室研究计划的基础

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.