Chemostat Culture for Yeast Physiology and Experimental Evolution

Abstract

Continuous culture provides many benefits over the classical batch style of growing yeast cells. Steadystate cultures allow for precise control of growth rate and environment. Cultures can be propagated for weeks or months in these controlled environments, which is important for the study of experimental evolution. Despite these advantages, chemostats have not become a highly used system, in large part because of their historical impracticalities, including low throughput, large footprint, systematic complexity, commercial unavailability, high cost, and insufficient protocol availability. However, we have developed methods for building a relatively simple, low-cost, small footprint array of chemostats that can be run in multiples of 32. This "ministat array" can be applied to problems in yeast physiology and experimental evolution. BATCH CULTUREThe most common method of growing yeast cell cultures is the standard "batch" culture. In this regime, a small number of cells are inoculated into nutrient-rich medium and allowed to divide at maximal growth rate through nutrient exhaustion and into saturation. Sampling of the culture for the experiment of choice is most typically done from the saturated culture or from cells growing in midlog phase before the diauxic shift. Batch culture provides a number of obvious benefits-it is easy, uses glassware commonly available in any laboratory, and is consistent with a vast literature. However, batch cultures can be problematic for certain applications. When comparing cells of different genotypes, for example, differences in the maximal growth rate are a common phenotype. Many other phenotypes co-vary with growth rate, leading to a nonspecific suite of cell biological, gene expression, and other physiological changes that may not be directly related to the mutation of interest (Regenberg et al. 2006;Castrillo et al. 2007;Brauer et al. 2008).Batch culture has also been a standard for long-term evolution experiments. Depending on the question being asked, batch culture with serial dilution can be a simple and scalable solution (e.g., Zeyl et al. 2003;Lang et al. 2011). However, such cultures are only rarely propagated in a relatively constant environment and growth rate regime because doing so can require a heroic sampling regimen where back dilution is performed multiple times per day (e.g., Torres et al. 2010). More typically, cultures are allowed to exhaust nutrients before transfer to new medium, leading to a variation in growth rate and nutrient access over the evolutionary time course. These discontinuities in selective pressure can lead to complex subpopulation structures in which different genotypes specialize for the various stages of the growth cycle (e.g., dividing a few extra times or failing to die after saturation, shortening lag phase,

Yeast grown in continuous culture systems can detect mutagens with improved sensitivity relative to the Ames test

“…In addition, while the culture continued to grow over the course of the month, the data became noisy after a period of about two and a half weeks, perhaps because of evolution occurring within the population [12,23]. For example, if a cell that is wild-type at the canavanine locus acquires a mutation that enables it to compete significantly better for nutrients in the chemostat environment, then the number of observed canavanine-resistant colonies will drop below that expected for the given level of mutagen.…”

“…Continuous culture systems allow for the long-term, controlled growth of microorganisms or cultured cells. Consequently, they have been used for many scientific and industrial uses, including producing biologics like small molecules [1–3] and recombinant proteins [4,5]; assessing the growth rate [6–9] or metabolism [10,11] of microorganisms or cultured cells under defined conditions; and for studying evolution [12–14]. Continuous culture systems typically operate in one of two modes: a chemostat, where a limited amount of nutrients are constantly added to the culture, or a turbidostat, where the culture density is kept constant [15].…”

“…Here, we present yeast grown in continuous culture systems as a novel method of mutagenicity testing. Yeast are an ideal organism for continuous culture systems because they grow robustly at room temperature, are well-established model organisms, perform well in continuous culture systems [12,23], and have been previously functionalized as biosensors [24]. Moreover, as yeast are eukaryotes, their metabolisms and DNA repair pathways are more similar to that of humans, potentially providing an advantage for mutagenicity testing when compared to bacteria.…”

The environmental stress response causes ribosome loss in aneuploid yeast cells

“…The strong correlation between ESR strength and doubling times in complex aneuploid strains suggested that proliferation rate was the primary determinant of ESR strength. To directly test this possibility, we examined whether equalizing proliferation rate among complex aneuploid strains and euploid control strains affected the correlation between ESR strength and degree of aneuploidy by culturing cells in a phosphate-limited chemostat (4,13). When proliferation rate was equalized in this manner, the ESR gene expression signature was no longer evident in aneuploid strains ( Fig.…”

Yeast grown in continuous culture systems can detect mutagens with improved sensitivity relative to the Ames test

“…Continuous culture systems allow for the long-term, controlled growth of microorganisms or cultured cells. Consequently, they have been used for many scientific and industrial uses, including producing biologics like small molecules [1][2][3] and recombinant proteins [4,5]; assessing the growth rate [6][7][8][9] or metabolism [10,11] of microorganisms or cultured cells under defined conditions; and for studying evolution [12][13][14]. Continuous culture systems typically operate in one of two modes: a chemostat, where a limited amount of nutrients are constantly added to the culture, or a turbidostat, where the culture density is kept constant [15].…”