Doctor of Philosophy
Karas, Bogumil J.
Edgell, David R.
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.
Summary for Lay Audience
Cells can be viewed as microscopic machines, such as a computer. Like a software program, their DNA encodes instructions, which the operating system interprets as commands based on various inputs, similar to a cell sensing its environment. A cell’s complete set of instructions is referred to as a genome, and scientists can modify genomes to alter how and what the cell does, including producing medicines or storing carbon, for instance. However, with current technologies in most cells, typically, only a few edits to a genome can be made at a time, making the process tedious and labor-intensive.
Recently, technologies that enable researchers to modify entire genomes simultaneously have become available. These technologies are referred to in their entirety as whole genome engineering (WGE). WGE is accomplished by writing a new desired genome and building it from scratch using chemical methods called DNA synthesis. The synthetic genome’s instructions are verified and then delivered into a cell lacking DNA. These technologies have been developed for relatively simple bacteria; however, more complex cells lack WGE methods and can each contain 1–3 different genomes that could be modified. The main limiting factors are an absence of standardized methods for modifying entire genomes of more complex cell types and difficulties delivering them where they need to go.
This thesis develops foundational technologies required to enable future WGE of the genome located within a cellular compartment called the mitochondria, widely known for its role in energy production. Specifically, I developed standardized methods for modifying mitochondrial genomes in approximately ten days at a fraction of the cost of DNA synthesis. After, I sought to improve DNA delivery to complex cells using a method called bacterial conjugation. I improved this DNA delivery method to fungi 23-fold and showed it could be used as an antifungal. Future research should attempt to adapt this DNA delivery method’s specificity in complex cells, from indiscriminate DNA delivery anywhere inside the cell to specifically their mitochondrial compartment. Now, WGE of mitochondria requires developing innovative ways to identify what cells have successfully received a genome and deciding what to make them do.
Cochrane, Ryan R., "Genetic Tools Towards A Synthetic Biology Approach For Whole Mitochondrial Genome Engineering" (2023). Electronic Thesis and Dissertation Repository. 9294.
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