Date of Award
Doctor of Philosophy (PhD)
Microbes naturally coexist in complex, multi-strain communities that are valuable assets for their host. Commensal and probiotic microbes prevent pathogen colonization, reduce the frequency and severity of various ailments, provide essential nutrients, and offer various additional benefits. Understanding the dynamics of and tailoring microbial communities to provide additional beneficial functions is a primary focus of researchers in medicine and agriculture. To date, consortia have primarily been manipulated by supplementing the communities with microbes that were engineered in vitro or by introducing stimuli that alter the metabolism or composition of the community. This method has proven successful, with numerous microbes engineered for diverse diagnostic and therapeutic applications. However, these microbes often lack acute environmental awareness and input stimuli often lack strain-specificity. These deficiencies can lead to unwanted activity and adaptations in the engineered and bystander microbes with adverse consequences on the host and the environment. As such, consortia should be manipulated using stimuli-specific sensors and strain-specific kill switches to ensure spatiotemporally responses and on-demand removal of the engineered microbes. However, manipulating microbial consortia to respond to specific in situ stimuli with strain-specificity remains challenging. To address these challenges, this dissertation describes the development of 1) a semi-rational, high-throughput protein engineering approach and the first ligand-specific sensors for phenylalanine, tyrosine, phenylethylamine, and tyramine, 2) multi-input CRISPR-based microbial kill switches that regulate the survival of probiotic Escherichia coli in the mammalian gut and environment, and 3) a computational program to design efficient, strain-specific CRISPR guide RNAs with applications including the isolation, killing, and engineering of microbes in situ. Together, this work will facilitate the safe and effective management of microbial consortia for diverse applications.
Tae Seok Moon
Gautam Dantas, Fangqiong Ling, Thaddeus Stappenbeck, Joshua Yuan,
Available for download on Tuesday, June 20, 2023