Cultivation and Genetic Manipulation of Saccharolobus islandicus

Saccharolobus islandicus REY15A was grown aerobically at 75°C in STV medium containing mineral salt, 0.2% (w/v) sucrose (S), 0.2% (w/v) tryptone (T), and a mixed vitamin solution (V). Saccharolobus islandicus REY15A(E233S)(ΔpyrEFΔlacS), hereafter E233S, was grown in STVU (STV supplemented with 0.01% (w/v) uracil) medium. The medium was adjusted to pH 3.3 with sulfuric acid, as described previously (29 (link)). SCV medium containing 0.2% (w/v) casamino acid (C) was used for screening and cultivating uracil prototrophic transformants. ATV medium containing 0.2% (w/v) D-arabinose (A) was used for protein expression. Culture plates were prepared using gelrite (0.8% [w/v]) by mixing 2 × STV and an equal volume of 1.6% gelrite. The strains constructed and used in this study are listed in the Supplementary information (Supplementary Table S1).