Methods in Continuous Culture: Mutation Accumulation, Evolution, and Aging

Abstract

There are many ways to culture a single celled organism, each way uniquely capable of answering a different biological question. Continuous culture, a form of growing organisms in which fresh medium is continually added to dilute out the doubling microbe, has existed for decades. As such, many different methods have been developed that utilize the constant dilution. Chapter 1 includes background and history on the methods used in this thesis and what they have been used to address. Chapter 2 describes a multiplexed assay to determine mutation rate in S. cerevisiae using the chemostat. I first successfully benchmarked the method against Luria-Delbrück fluctuation tests using a collection of published MSH2 variants. Msh2 is a DNA repair enzyme that is associated with Hereditary Nonpolyposis Colorectal Cancer. In Chapter three, I describe the generation of an additional 185 clinically relevant human variants in the yeast ortholog and how I estimated their pathogenicity. I also associated the data with clinical findings. In Chapter 4, I describe the implementation of two previously published devices, the turbidostat and the Miniature-Chemostat Aging Device (MAD). The turbidostat, a device which adds in additional medium as the culture rises above a density set point, successfully determined that aneuploidy is a common suppressor of the slow growth phenotype associated with low ribosomal abundance in S. cerevisiae. The implementation of the MAD – a device which removes daughter cells from an aging population of S. cerevisiae – was used to probe the proteomic changes associated with age. Lastly, chapter 5 focuses on how all these robustly built devices can be used together or separately to answer interesting biological questions going forward.