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The Impact of Laboratory Conditions on the Estimation of Nucleotide Mutation Rates.

Description
Mutation is the source of heritable variation of genotype and phenotype, on which selection may act. Mutation rates describe a fundamental parameter of living things, which influence the rate at which evolution may occur, from viral pathogens to human crops and even to aging cells and the emergence of cancer.…

Mutation is the source of heritable variation of genotype and phenotype, on which selection may act. Mutation rates describe a fundamental parameter of living things, which influence the rate at which evolution may occur, from viral pathogens to human crops and even to aging cells and the emergence of cancer. An understanding of the variables which impact mutation rates and their estimation is necessary to place mutation rate estimates in their proper contexts. To better understand mutation rate estimates, this research investigates the impact of temperature upon transcription rate error estimates; the impact of growing cells in liquid culture vs. on agar plates; the impact of many in vitro variables upon the estimation of deoxyribonucleic acid (DNA) mutation rates from a single sample; and the mutational hazard induced by expressing clustered regularly interspaced short palindromic repeat (CRISPR) proteins in yeast. This research finds that many of the variables tested did not significantly alter the estimation of mutation rates, strengthening the claims of previous mutation rate estimates across the tree of life by diverse experimental approaches. However, it is clear that sonication is a mutagen of DNA, part of an effort which has reduced the sequencing error rate of circle-seq by over 1,000-fold. This research also demonstrates that growth in liquid culture modestly skews the mutation spectrum of MMR- Escherichia coli, though it does not significantly impact the overall mutation rate. Finally, this research demonstrates a modest mutational hazard of expressing Cas9 and similar CRISPR proteins in yeast cells at an un-targeted genomic locus, though it is possible the indel rate has been increased by an order of magnitude.
ContributorsBaehr, Stephan (Author) / Lynch, Michael (Thesis advisor) / Geiler-Samerotte, Kerry (Committee member) / Mangone, Marco (Committee member) / Wilson, Melissa (Committee member) / Arizona State University (Publisher)
Created2023
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Computational Genomics of DNA Viruses: Novel Insights into Bacteriophage and Human Cytomegalovirus Evolution

Description
Viruses are the most abundant biological entities on Earth, infecting all types of cellular organisms. Yet less than 1% of the virosphere on our planet has been characterized to date. Viruses are both an important driver of bacterial evolution and have significant implications for human health, therefore understanding the relative…

Viruses are the most abundant biological entities on Earth, infecting all types of cellular organisms. Yet less than 1% of the virosphere on our planet has been characterized to date. Viruses are both an important driver of bacterial evolution and have significant implications for human health, therefore understanding the relative contributions of various evolutionary forces in shaping their genomic landscapes is of critical importance both mechanistically as well as clinically. In my thesis I use computational genomic approaches to gain novel insights into bacteriophage and human cytomegalovirus evolution. In my first two chapters and associated appendices I characterized the complete genomes of the Cluster P bacteriophage Phegasus and Cluster DR bacteriophage BiggityBass, whose isolation hosts were Mycobacterium smegmatis mc²155 and Gordonia terrae CAG3, respectively. I also determined the bacteriophages' phylogenetic placement and computationally inferred their putative host ranges. For my fourth chapter I assessed the performance of several of these computational host range prediction tools using a dataset of bacteriophages whose host ranges have been experimentally validated. Finally, in my fifth chapter I reviewed the key parameters for developing an evolutionary baseline model of another virus, human cytomegalovirus.
ContributorsHowell, Abigail Ann (Author) / Pfeifer, Susanne P (Thesis advisor) / Jensen, Jeffrey (Committee member) / Snyder-Mackler, Noah (Committee member) / Geiler-Samerotte, Kerry (Committee member) / Arizona State University (Publisher)
Created2023
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Characterizing and Releasing Biological Constraints for Lignocellulosic Bioconversion

Description
Lignocellulose, the major structural component of plant biomass, represents arenewable substrate of enormous biotechnological value. Microbial production of chemicals from lignocellulosic biomass is an attractive alternative to chemical synthesis. However, to create industrially competitive strains to efficiently convert lignocellulose to high-value chemicals, current challenges must be addressed. Redox constraints, allosteric regulation, and transport-related limitations…

Lignocellulose, the major structural component of plant biomass, represents arenewable substrate of enormous biotechnological value. Microbial production of chemicals from lignocellulosic biomass is an attractive alternative to chemical synthesis. However, to create industrially competitive strains to efficiently convert lignocellulose to high-value chemicals, current challenges must be addressed. Redox constraints, allosteric regulation, and transport-related limitations are important bottlenecks limiting the commercial production of renewable chemicals from lignocellulose. Advances in metabolic engineering techniques have enabled researchers to engineer microbial strains that overcome some of these challenges but new approaches that facilitate the commercial viability of lignocellulose valorization are needed. Biological systems are complex with a plethora of regulatory systems that must be carefully modulated to efficiently produce and excrete the desired metabolites. In this work, I explore metabolic engineering strategies to address some of the biological constraints limiting bioproduction such as redox, allosteric, and transport constraints to facilitate cost-effective lignocellulose bioconversion.
ContributorsOnyeabor, Moses Ekenedilichukwu (Author) / Wang, Xuan (Thesis advisor) / Varman, Arul M (Committee member) / Nannenga, Brent (Committee member) / Nielsen, David R (Committee member) / Geiler-Samerotte, Kerry (Committee member) / Arizona State University (Publisher)
Created2023