2.6 Determination of plasmid copy number and gene expression
level
Total DNA and RNA of the engineered yeast HygL8-G418G1 under different
extraction conditions were collected and used for determining the
plasmid copy number and the gene expression level, respectively. DNA was
extracted by hydroxybenzene-chloroform-isoamyl alcohol (25:24:1) method.
Firstly, cells were harvested by centrifugation (12,000 g, 5 min) and
suspended with 500 µl PBS buffer. Added with a few glass beads and 500
µl phenol/chloroform/isoamyl alcohol (25:24:1), the cells were broken
through the tissuelyser (JingXin, Shanghai, China), then centrifuged at
12,000 g, 10 min, 4 °C. The aqueous phase (top) was transferred to a new
tube, added with 400 µl chloroform, and mixed gently. Then, it was
centrifuged at 12,000 g, 10 min, 4 °C, and the supernatant was
transferred to another new tube. It was added with 800 µl ethanol and
mixed gently, kept at -20 °C for 2 h, then centrifuged at 12,000 g, 10
min, 4 °C. The resultant precipitation pellet was washed by 75% ethanol
twice, and air-dried at 37 °C to evaporate the ethanol. Finally, the
genome DNA was dissolved in 100 µl sterilized water for qPCR analysis.
Total RNA was obtained by using yeast total RNA isolation kit (Sangon
Biotech, Shanghai, China). The quantitative real-time polymerase chain
reaction (qPCR) was carried out as previously reported (Lan et al.,
2019) to determine the plasmid copy number and gene expression level.
Primer pairs qCYP5150L8-F/qCYP5150L8-R, qCYP5139G1-F/qCYP5139G1-R,
qiGLCPR-F/qiGLCPR-R and qERG2-F/qERG2-R, qTAF10-F/q-TAF10-R were
designed for target genes and reference genes in qPCR reactions,
respectively (Table S1). SYBR Green Real-Time PCR Master Mix (NEB,
Beijing, China) on a qPCR apparatus (qTOWER3G touch, Analytikjena,
Germany) was used for the analysis.