Browsing by Author "Gambill, Lauren Celeste"

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    Development of ribozyme-derived RNA tools for engineering microbes and microbial consortia
    (2022-10-07) Gambill, Lauren Celeste; Chappell, James
    The advent of next generation sequencing has illuminated the members present in microbial communities and we have begun to better characterize these diverse and complex communities by capturing both culturable and unculturable members. With this wealth of information in hand, we can now begin to think about adding control to these environments for numerous applications. Towards this goal, we need to know which members of a given community can be engineered and we need to be able to selectively control subpopulations of microbes within these communities using genetic programs. Here, we have developed two ribozyme-derived tools, which we call RENDR (Ribozyme-ENabled Detector of RNA) and RAM (Ribozyme-Addressable Memory). We built RENDR from the ground up, first showing that the cis-acting ribozyme functionally splices two exons together to enable translation of the transcript. We then split the ribozyme into two fragments and added complimentary RNA guides, which allowed us to probe a library of ribozyme split variants for functionality. The RNA guides were altered to bind to a third RNA, wherein design rules demonstrated the importance of RNA guide length on ribozyme splicing efficiency. Finally, we swapped the GFP output with colorimetric, gas-producing, and transcriptional regulator outputs, and demonstrated RENDR functionality across a diverse set of microbial hosts. With RAM, we first demonstrated functionality of the system and elucidated design rules by using a GFP splicing assay to analyze splicing efficiencies. We then targeted the RNA guide of RAM to 16S rRNA, conjugated a microbial consortium, and used DNA sequencing to determine which microbes were conjugated. These tools will help us to better understand how different conditions – like temperature and the presence of disinfectants – alter conjugation at the consortia level and how we might go about engineering microbes and microbial consortia to be more robust to negative perturbations in the community.