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

项目总结

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.