Optimizing S. rosetta Culture Conditions

Strains of S. rosetta were co-cultured with Echinicola pacifica bacteria (Levin and King, 2013 (link)); American Type Culture Collection [ATCC], Manassas, VA; Cat. No. PRA-390) in seawater-based media enriched with glycerol, yeast extract, and peptone to promote the growth of E. pacifica that serve as the choanoflagellate prey (Levin and King, 2013 (link); Booth et al., 2018 (link)). We further supplemented this media with cereal grass (King et al., 2009 (link); Fairclough et al., 2010 (link); Carolina Biological Supply Company, Burlington, NC; Cat. No. 132375), which we call high nutrient media (Supplementary file 1-Table A), as we noticed that this addition promoted S. rosetta growth to a higher cell density (~10⁷ cells/ml [Figure 3—figure supplement 2A] versus ~10⁶ cells/ml (Booth et al., 2018 (link)). To maintain rapidly proliferating cells in an abundance of nutrients, cultures were diluted 1 in 30 daily or 1 in 60 every two days into 6 ml of high nutrient media in 25 cm² vented culture flasks (Corning, Oneonta, NY, USA; Cat. No. 430639) and incubated at 22°C and 60% relative humidity. To prevent an overgrowth of bacteria when S. rosetta experienced stress, such as after transfections or during clonal isolation, we cultured S. rosetta in low nutrient media, which is 0.375x high nutrient media (Supplementary file 1-Table A).