Domestication of Nonconventional Gram-negative Bacteria as Synthetic Biology Chassis

Keating, Kevin

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Domestication of Nonconventional Gram-negative Bacteria as Synthetic Biology Chassis

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Bacteria have evolved to thrive in an astonishing range of conditions, to produce chemicals and materials useful to humans, and to sense and respond to changes in their environment in ways that could be leveraged by synthetic biologists. Yet, several challenges exist which slow the onboarding of new strains of bacteria into the laboratory. Advanced cloning systems, which have been developed for model bacteria such as Escherichia coli, do not contain destination vectors which can replicate across a broad range of bacteria. Genetic parts which control transcription and translation are either generated de novo for new organisms or are transferred from E. coli ad hoc. In addition, basic biological knowledge is lacking. In this work, I describe a modification to the advanced cloning system CIDAR MoClo which both enables an efficient, automation-ready cloning method for use with bacteria across Pseudomonadota and unlocks a large set of genetic parts compatible with the same cloning standard. I describe a systematic approach to screening these parts for suitability in new hosts in order to rapidly generate a parts set achieving constitutive expression levels across several orders of magnitude. I further the basic biological understanding of three bacterial cellulose producing strains by generating complete genomes with manually-curated bacterial cellulose synthase operons. With these efforts, I have advanced the ability of the field to rapidly onboard new synthetic biology chassis and have laid basic biology groundwork for future synthetic biologists to work more effectively in Komagataeibacter bacteria.

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