Browsing by Subject "antibiotic resistance"
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Item Competition-Mediated Identification of the First Environmental Protein Responsible for the Degradation of the Lipopeptide Surfactin(2014-05-02) Gorzelnik, Karl VStreptomycetes, as nonmotile microbes, are forced to adapt to environmental conditions they cannot escape. In order to adapt to their environment streptomycetes produce an array of both secondary metabolites to antagonize competitors and degradative enzymes to take advantage of various nutritional sources and to degrade xenobiotics, molecules from foreign organisms. This work shows two instances of streptomycetes adapting to their neighbors. Streptomyces sp. Mg1 was determined to be resistant to surfactin, a molecule produced by Bacillus subtilis which inhibits aerial hyphae development. After identifying possible enzymatic sources of degradation several candidate enzymes were cloned and expressed in Escherichia coli. One candidate, ?secreted hydrolase? was purified under denaturing conditions and refolded. This enzyme was shown to degrade surfactin and another B. subtilis metabolite, plipastatin. Upon alteration of assay conditions the enzyme was also able to degrade daptomycin. The other instance of a streptomycete adapting to its neighbors is of S. coelicolor, which in the presence of some strains of B. subtilis is able to produce undecylprodigiosin earlier than it normally would. The induction of undecylprodigiosin indicates that either B. subtilis is altering a neighbor?s physiology through a secreted compound or that S. coelicolor is able to detect a xenobiotic and responds by producing an antibiotic compound. The inducing compound from B. subtilis was fractionated and conditions for its further purification were determined.Item Overcoming Clonal Interference in Escherichia coli Using Genderless High Frequency Recombination Strains(2014-04-16) Winkler, JamesAdaptive 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.