S99: Tools for higher throughput continuous culture in yeast

Continuous culture is a powerful approach for microbial fermentation, cultivation, and selection.  Chemostats, one such platform, maintain cells in tightly controlled, nutrient-limited, steady state environments, making them ideal for experimental evolution, physiology characterization, and modeling applications.  Despite these advantages, chemostats have not found a large user base, due in part to their expense and technical difficulty.  These problems have also limited high throughput experimentation in chemostats.  As a solution, we have developed a low cost, small footprint chemostat array built entirely from off-the-shelf parts.  The “ministats” operate at a practical volume for convenient sampling, and at a high enough population size for effective experimental evolution.  The ministat compares favorably to a modified commercial fermentor in measurements of device robustness, cell density, genome-wide gene expression, and evolution outcomes.  Using the ministats, we recently completed 64 independent 300 generation evolution experiments with yeast propagated under three selective regimes, a much higher throughput than was previously practical.  In parallel, we used a second set of ministats to measure population fitness every 50 generations via co-culture with a fluorescently marked ancestral competitor. Our results show that different selective pressures demonstrate different capacities for improvement, with sulfate-limited cultures moving to high fitness much more quickly than phosphate or glucose limitation.  We are now coupling the ministats with deep sequencing to better understand the genetic makeup of these populations.  Ministats give us an unprecedented ability to monitor and compare adaptation of microbes in real time, and we anticipate that they will be useful for many applications.