Directed evolution of antimutator E. coli




Leon, Dacia

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Biological systems are essential tools for addressing societal challenges. There have been several successes in this field, however, a strong hindrance lies in the ephemeral nature of these systems – cells are tiny factories that evolve. Evolution poses a problem because when a desired function is encoded into the DNA of the host organism, the host uses its own resources to perform the function and there is likely no associated fitness benefit. Therefore, there is strong selection for inactivation of the function due to the metabolic load imposed on cellular resources. One way to address this problem is to engineer evolutionary stability by lowering a host organism’s basal mutation rate and concomitantly reducing the probability that an encoded function will become mutated.
In Chapter 1 of this dissertation, I discuss the nature of the metabolic cost associated with engineering biology and mechanisms by which host adaptation occurs. I also explore cellular pathways involved in genetic stability and examine previously characterized antimutators. Chapter 2 describes the first iteration of a directed evolution method used to engineer antimutators in Escherichia coli, Periodic Reselection for Evolutionarily Reliable Variants (PResERV). In this first PResERV experiment, I observe that the antimutator phenotype is due to mutations in genes involved DNA replication and RNA metabolism (polA, polB, and rne). In Chapter 3, I perform the same PResERV experiment on a greater scale and characterize a series of antimutator strains. The causative alleles in many of these strains are in genes involved in the tricarboxylic acid cycle and electron transport chain (sucD and sdhA). These alleles are shown to reduce oxidative stress. Chapter 4 demonstrates results from another PResERV experiment using a clean-genome E. coli strain, MDS42, as the host organism. In sum, this work shows the many mechanisms that lead to an antimutator phenotype, and these findings are used to build stable strains for reliable engineering of biology. Finally, there are two appendices (Appendix A and B) which discuss my work in examining the evolutionary path to citrate utilization in Lenski’s long-term evolution experiment (LTEE) and a do-it-yourself method for using gellan gum as an alternative to microbial agar media.



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