Overcoming Clonal Interference in Escherichia coli Using Genderless High Frequency Recombination Strains
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Adaptive laboratory evolution (ALE) is a powerful tool for strain improvement, and has been applied successfully to improve a range of desirable phenotypes in model organisms through continuous cultivation under a selective pressure of interest. Despite its demonstrable utility, one limiting factor for the effectiveness of ALE is competition between beneficial mutants that exist contemporaneously within an evolving population. This phenomenon of clonal interference arises from the fact that the majority of microbes are obligate asexual organisms that cannot exchange DNA between cells. Mutants that arise must therefore compete for resources until the fittest mutant drives the others to extinction. The resulting loss of genetic information from the population slows the overall rate of adaptation, and decreases the amount of information that can be extracted from a given ALE experiment. To overcome these limitations, we have developed a novel in situ mating system based on the F plasmid to allow continuous DNA exchange between E. coli cells in liquid culture, allowing mutants to potentially combine their mutations into a single genetic background. The utility and limitations of an existing recombination method, genome shuffling, are also explored to demonstrate the advantages of this new method. The design and initial testing of the in situ mating system is first validated, and the system is used for a subsequent evolution experiment under osmotic stress to validate the industrial applicability of the mating system. Adaptive mutants generated in the course of these experiments are then used to test whether tolerant mutants can be formed via conjugation. Finally, additional side projects focusing on strain or population characterization tools are discussed, followed by recommendations for future work.
Winkler, James (2014). Overcoming Clonal Interference in Escherichia coli Using Genderless High Frequency Recombination Strains. Doctoral dissertation, Texas A & M University. Available electronically from