Antibodies are key proteins of the immune system that help fight disease. In people, immune cells called B cells create antibodies and then evolves them. B cells take months to do this, which makes it difficult to study antibody creation and evolution. This CAREER project will design a method to create and evolve antibodies “from scratch” in yeast, which will open new avenues for exploring antibody creation, evolution, and function. The results of the project will provide insights into the formulation of more effective disease-targeting therapeutics. The project will support project-based course development and the creation of educational Apps to train students in synthetic biology and biotechnology. Inside a person, antibodies are created and evolved at massive scale (millions of new antibodies per day). It is the combined functionality of this ‘evolved antibody repertoire’ that is important towards fighting off disease-causing agents. Unfortunately, it is difficult to study antibody generation and evolution in a way that mimics how they occur naturally. Yeasts are excellent alternate hosts for antibody production because they can display antibody proteins on their surface. This project will fill this gap by engineering yeast to efficiently recapitulate key aspects of recombination and antibody affinity maturation. The project will employ cellular engineering, protein targeting, synthetic biology, and antibody yeast display. Multiplex genetic engineering and enzyme localization studies will be employed to identify the key B cell and native yeast cell enzymes that enhance yeast-mediated recombination, which is a DNA cleavage and repair process. Bespoke selection strategies and genetic arrays will allow for generation of, and controlled selection for, functional antibodies made from scratch in yeast cells. Further incorporation of highly efficient and targeted in vivo mutation of antibody genes will allow for analyses of the evolution of antibody function.