Sarah Mattson, Graduate Student, Rodrigues Lab, GW Department of Chemistry
Single PCR Mammalian Expression Generated Array Directed Evolution of Biomolecules
Directed evolution is a widely used technique for engineering biomolecules to locate and treat disease, remove plastic or carbon dioxide from the environment, develop synthetic biological systems, and synthesize complex molecules without organic solvents. Previous directed evolution methods are time-consuming, limited in biomolecule number and size, and often require hazardous viruses. In this work, I developed a novel directed evolution protocol with a single error prone polymerase chain reaction (PCR) to amplify an entire plasmid containing a fluorescent protein and heme oxygenase-1 to produce biliverdin. The biomolecules are expressed in mammalian and bacterial cells. The novel method removes most molecular biology steps for faster evolution with fewer errors. The evolution of fluorescent proteins that attach biliverdin is necessary in mammalian cells to compete with high affinity, endogenous proteins. High-throughput screening is performed by fluorescence activated cell sorting (FACS) to screen 50 million cells per hour. Using the new method, we created billions of small Ultra-Red Fluorescent Protein (smURFP) variants and selected new variants that are brighter without biliverdin addition. We developed an entirely new class of fluorescent protein from a frog pigmentation protein, which was initially non-fluorescent and now is 40-fold brighter than smURFP in mammalian cells. The patent-pending directed evolution method allows for the evolution of multiple biomolecules inside cells quickly and efficiently, which is fully automatable in biotechnology companies.
Bio:
Sara Mattson received her M.S. in Chemistry from American University in Washington DC. She performed graduate research with Dr. Monika Konaklieva to synthesize electrophilic small molecules to activate lipoprotein lipase to treat heart disease. Ms. Mattson received her B.S. in Chemistry from the State University of New York, Plattsburgh. Sara Mattson joined Professor Erik Rodriguez’s research group in Spring 2021 and developed new directed evolution methods to create new fluorescent proteins for biological applications.