Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Karas, Bogumil J.

2nd Supervisor

Edgell, David R.

Co-Supervisor

Abstract

Microorganisms can be harnessed for bioproduction and biotechnology to further global efforts in agriculture, health, manufacturing and sustainability. Traditional microbial chassis used for these purposes are the most well-characterized bacteria and yeast, Escherichia coli and Saccharomyces cerevisiae. Synthetic biology can be used to facilitate engineering of microbial chassis with new or improved traits. However, there is a need to expand the number and diversity of available microbial chassis to include microorganisms that have innate genetic, metabolic and physiological characteristics that we could make use of. Two such bacteria include the nitrogen-fixing plant symbiont, Sinorhizobium meliloti, and the polyextremophile, Deinococcus radiodurans. While some progress has been made toward the development of these chassis, further strain and tool developments are required to unlock their full potential. Here, I present the expansion of the genetic toolkits for S. meliloti and D. radiodurans to improve their utility as bacterial chassis for synthetic biology applications.

First, I engineered a genome-reduced strain of S. meliloti as a novel conjugative host and demonstrated the transfer of multi-host shuttle vectors to bacteria, yeast and microalgae. Then, I developed a conjugative protocol to transfer DNA from E. coli to D. radiodurans and showed its utility through the generation of robust systems for conjugation-based genome engineering and whole genome cloning in vivo. Using this method, I cloned the large (178 kb) MP1 megaplasmid from D. radiodurans in E. coli. Finally, I developed a strategy to create seamless gene deletions in D. radiodurans which was demonstrated through the sequential genetic knockout of four restriction-modification systems.

The tools and strains developed in my thesis will add to the growing genetic toolkits of S. meliloti and D. radiodurans. The establishment of S. meliloti as an alternative chassis for interkingdom DNA transfer will allow for the study and engineering of agriculturally-relevant microorganisms and modulation of microbial communities. Likewise, the expansion of genetic tools will establish D. radiodurans as a microbial platform for industrial applications and the study of extremophile biology. These bacterial chassis will complement the use of traditional microbial chassis, broadening the potential solutions synthetic biology could offer.

Summary for Lay Audience

Microorganisms like bacteria, yeast and algae can be used to produce food, medicine, building materials and improve the environment. Well-known examples of this include using yeast to make bread or bacteria to make pharmaceutical drugs like insulin. Sinorhizobium meliloti and Deinococcus radiodurans are two bacteria that naturally have beneficial traits and functions. S. meliloti lives in soil and helps improve plant growth, while D. radiodurans is able to tolerate harsh conditions such as drought or exposure to radiation. However, much like you would use tools to renovate a house, in order to work with these microorganisms, we need to have tools to make changes to their genetic information. Genetic information (i.e., DNA) is composed of biological building blocks in a particular sequence that determines the traits and functions of all living things. Using synthetic biology, we can engineer organisms by adding, removing or changing these building blocks. The purpose of my thesis is to expand the genetic toolbox of S. meliloti and D. radiodurans so that they can be engineered more easily.

To achieve this, I demonstrated efficient methods to introduce DNA into S. meliloti and D. radiodurans, including conjugation. Conjugation is the transfer of DNA directly from one organism to another. Using conjugation, I developed new strategies to alter the genetic information of these organisms through the addition or removal of small or large segments of DNA. Finally, I demonstrated the ability for S. meliloti to conjugate DNA directly to bacteria, yeast and algae to allow for easier engineering of these microorganisms.

The genetic tools described in this thesis, coupled with the unique qualities of S. meliloti and D. radiodurans, make them attractive for research and commercial use. Now, the process to insert DNA encoding a new function, remove unnecessary DNA, or look more closely at DNA to better understand the biology of these bacteria, is simpler and faster. The improved ability to engineer S. meliloti and D. radiodurans will allow us to accelerate their use in creating solutions to global challenges in agriculture or industry, such as increasing crop growth or removing toxins from nuclear waste sites.

Creative Commons License

Creative Commons Attribution-Noncommercial 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial 4.0 License

Available for download on Monday, May 01, 2023

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