Continuous Directed Evolution of Enzymes with Novel Substrate Specificity
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CitationCarlson, Jacob Charles. 2013. Continuous Directed Evolution of Enzymes with Novel Substrate Specificity. Doctoral dissertation, Harvard University.
AbstractMethodological advances in directed evolution have already made it possible to discover useful biomolecules within months to years. A further acceleration of this process might make it possible to address outstanding challenges, or needs for which the current timescale is a fundamental barrier. To realize these goals would require transformative methodological advances in directed evolution. In Chapter One, current methods in directed evolution are briefly reviewed. In Chapter Two, a general platform for continuous directed evolution is presented. The method is used to evolve T7 RNA polymerase enzymes with novel promoter activity on the days timescale. In Chapter Three, a method is developed for tuning selection stringency during continuous evolution, thus obviating the requirement for a minimal starting library activity. In Chapter Four, a method is developed for simultaneous positive and negative selection, thus allowing explicit selection for substrate specific enzymes. In Chapter Five, the advances in stringency modulation and negative selection are combined to evolve highly substrate specific enzymes starting from an inactive starting library. In a continuous evolutionary arc of less than three days, we discover T7 RNA polymerase enzymes with a degree of specificity for the T3 promoter exceeding that of the wild type enzyme for its native substrate.
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