Frozen ez yeast transformation 2 kittm

The gpdA promoter and hygromycin-B resistance gene were amplified from plasmid pAN7-1 using NewPgpdA-F and TtrpC-R [32 (link)]. Homologous flanks included in the HDR cassettes were amplified from DNA flanking the sgRNA site in the targeted gene (Tox3) (primers are listed in Additional file 2: Table S2). The 5^′ flank, upstream of the PAM sequence, only included seven bases of sgRNA rather than twenty bases and the HDR cassette encountered an NGG sequence 20–25 bp away from the targeted cut sites avoiding the generation of a potential PAM site in the cassette (Fig. 2b). The HDR cassettes comprising the homologous flanks and selectable markers were then assembled in yeast (Saccharomyces cerevisiae) as previously reported [33 (link)]. The yeast transformation was conducted using the Frozen-EZ Yeast Transformation II Kit^Tm (ZymoResearch) as per manufacturer’s instructions. Yeast colonies harbouring the transformed plasmid were confirmed on selective drop out media (minus uracil). Yeast plasmid extraction was performed using the Zymoprep™ II mini prep protocol (ZymoResearch). Plasmids were then transformed into E. coli and subsequently screened by colony PCR. Takara ExTaq was used for all PCR reactions in this study.

Sterol-producing yeasts were kindly provided from the Riezman lab (Supplementary Table 5)^{35 (link)}. Competent cells of these strains were prepared using the Frozen EZ Yeast Transformation II Kit^TM (Zymo Research, Irvine, CA, USA). Starter cultures were grown in the SD-Leu medium at 30 °C overnight and then used to inoculate 25 mL SD-leu medium in triplicates in a shaking flask with an initial OD₆₀₀ of 0.2–0.4. Samples were harvested at 18 h and pelleted 3000 g for 5 min. Yeast cells were resuspended in 200 μL of TES buffer (50 mM Tris-HCl pH = 7, 600 mM sorbitol, 10 g/L bovine serum albumin, 1.5 mM β-mercaptoethanol) and homogenized with an equal volume of 0.5 mm glass beads in a BBX24 Bullet Blender® homogenizer (Next Advance, Troy, NY, USA) at setting 8 at 4 °C for 4 min. A total of 300 μL of TES buffer was added to the lysed cells, and 400–500 μL of the yeast lysate was transferred into a capped glass tube, followed by adding 1 mL chloroform immediately. The sample was vortexed for 1 min, and the organic phase was transferred into a new glass test tube and dried under a stream of air. The sample was resuspended in 100 μL methanol, centrifuged at 17,000 g for 10 min, and the supernatant was transferred into a LC/MS vial and stored at −20 °C until use.