Person:
Carlson, Jacob Charles

Profile Picture

Email Address

AA Acceptance Date

Birth Date

Research Projects

Organizational Units

Job Title

Last Name

Carlson

First Name

Jacob Charles

Name

Carlson, Jacob Charles

Filters

2013 - 20142

Settings

Author's Publications: search.filters.isAuthorOfPublication.b77c5038-4ef0-46f3-8d57-f412af17acd8

Search Results

Now showing 1 - 2 of 2
  • Thumbnail Image
    Publication
    Continuous Directed Evolution of Enzymes with Novel Substrate Specificity
    (2013-07-05) Carlson, Jacob Charles; Liu, David Ruchien; Kahne, Daniel; Murray, Andrew; Walsh, Christopher
    Methodological 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.
  • Thumbnail Image
    Publication
    Negative selection and stringency modulation in phage-assisted constinuous evolution
    (2014) Carlson, Jacob Charles; Badran, Ahmed; Guggiana-Nilo, Drago A.; Liu, David
    Phage-assisted continuous evolution (PACE) uses a modified filamentous bacteriophage life cycle to dramatically accelerate laboratory evolution experiments. In this work we expand the scope and capabilities of the PACE method with two key advances that enable the evolution of biomolecules with radically altered or highly specific new activities. First, we implemented small molecule-controlled modulation of selection stringency that enables otherwise inaccessible activities to be evolved directly from inactive starting libraries through a period of evolutionary drift. Second, we developed a general negative selection that enables continuous counter-selection against undesired activities. We integrated these developments to continuously evolve mutant T7 RNA polymerase enzymes with ∼10,000-fold altered, rather than merely broadened, substrate specificities during a single three-day PACE experiment. The evolved enzymes exhibit specificity for their target substrate that exceeds that of wild-type RNA polymerases for their cognate substrates, while maintaining wild-type-like levels of activity.