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Synthetic biology is an interdisciplinary research field that standardizes and repurposes biological components to better understand life and solve complex problems. As synthetic biology has developed, the goal has become to generate fully controllable biological systems through whole genome engineering (WGE), the cumulation of standardized genome engineering, and DNA delivery methods. In eukaryotes, genetic tools for WGE are limited to the nucleus and present a need to expand to include mitochondria, which maintains its own unique genome. The work presented here begins developing the resources needed to enable whole mitochondrial genome engineering. First, to standardize mitochondrial genome engineering protocols, I cloned the mitochondrial genomes of two diatomaceous algae, Phaeodactylum tricornutum and Thalassiosira pseudonana, as plasmids in bacteria and yeast. Next, a PCR-based engineering method was optimized to generate derivative algal mitochondrial genomes rapidly and inexpensively. After, I sought to adapt an entirely in vivo DNA delivery method, bacterial conjugation, for mitochondrial DNA-delivery. In any scenario modifying bacterial conjugation’s specificity will likely decrease its efficiency of DNA transfer beyond the level of detection. Therefore, I first improved DNA transfer to eukaryotes by generating and screening a deletion plasmid library for the conjugative plasmid, pTA-Mob 2.0. From this data, pSC5 was created that improved DNA delivery to yeast. pSC5 was used to create the pSC5-toxic plasmids that effectively killed yeast and established a novel first-in-the-world conjugation-based antifungal. Together these resources for mitochondrial genome engineering and improved DNA delivery to eukaryotes should improve the feasibility of future endeavors in whole mitochondrial genome engineering. |
