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

项目总结 该项目旨在提高操纵哺乳动物微生物区系的精密工具的总体可及性。 在本地肠道环境中，即在原位。尽管哺乳动物肠道群落具有丰富的微生物多样性 随着时间的推移，自然地获得并保持通过实验和治疗改变他们的 与抗生素或粪便移植的成分已明确记录在案。然而，这些都是可以广泛获得的 治疗被发现可以广泛地改变微生物群的组成，这限制了它们对 确定微生物区系的特定种群如何影响宿主的健康。可供选择的方法 允许精确消除或基因编辑内源性肠道原位共生现象最近已被 证明使用水平可传播移动遗传元件(MGES)，包括温带噬菌体， 但还没有得到广泛的确立。噬菌体被认为是对原位微生物组最精确的MGES之一 由于对其寄主范围的大多数研究都未能检测到跨属传染性，因此对其进行了操纵。 在这项申请中提出的研究利用了温带噬菌体的自然丰度和精确度 建立可推广的方法，以重新设计它们，以便在微生物组编辑应用程序中更可靠地使用。 也就是说，我提出的定向进化和理性工程方法将简化 (1)致命性致死性突变体，其必须裂解并能够超级感染其溶原性亲缘，以及(2) 非致命性温带噬菌体衍生物，可消除内源性原噬菌体或稳定地溶原化 频率升高。这项研究的初始指导K99阶段包括原则证明工程 用Lambda进行实验，Lambda是大肠杆菌最具特征的温带噬菌体。除了……之外 为我提供实际的lambda经验，这项至关重要的k99培训将为我的独立研究做好准备。 在R00阶段和我未来的职业生涯中的活动，使我能够熟练地掌握关键 技术-例如在酵母中大规模组装DNA以构建噬菌体基因组文库和整体 噬菌体基因组测序-可应用于非模式微生物区系的研究。培训将是 在两位导师的监督下，他们拥有必要的资源和经验，确保我在这个项目上取得成功， Jef Boeke博士和Marcus Noyes博士以及四名咨询委员会成员 专业知识。来自我的导师和顾问的以职业为导向的指导，以及职业发展活动 在K99阶段，包括赠款编写和项目管理的正式课程，将进一步 促进我向R00阶段的过渡，以及作为一名独立学术研究员的长期工作效率。 最终，这一拟议项目的完成将为在 微生物组领域，同时为微生物基因组工程提供基础工具，奠定了强大的 为我未来实验室的研究项目奠定了基础。

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.