The assembly of synthetic operon in YEp352 was performed according to the BioBrick principles23 (link) described by Zelcbuch et al.24 (link) To further simplify the cloning process, a seamless, recombination-based cloning strategy was applied for the synthetic operon construction in YEplac181 using the ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China). The knockout of ROX1 and knockdown of ERG9 were achieved via CRISPR/Cas9 technologies.25 (link) For the CRISPR genome editing, yeast cells were pre-transformed with p414-TEF1p-Cas9-CYC1t plasmid (Addgene). All the PCR products were analyzed on 1% agarose gels and positive colonies were grown overnight for plasmid extraction using the Tianprep Rapid Mini Plasmid Kit (Tiangen, Beijing China) according to the manufacture's instructions. All clones were verified through the expression cassette sequencing (Sangon, China). The constructed plasmids containing the whole expression operon were chemically transformed into the yeast by S. cerevisiae EasyComp™ Transformation Kit (Invitrogen, CA, United States) and aliquots were plated on the corresponding auxotrophic minimal media. Table S4† lists the primers used to construct these plasmids and strains.
S cerevisiae easycomp transformation kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The S. cerevisiae EasyComp™ Transformation Kit is a laboratory tool designed for the transformation of Saccharomyces cerevisiae, a species of yeast. The kit provides the necessary components and protocols to efficiently introduce foreign DNA into S. cerevisiae cells.
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2 protocols using s cerevisiae easycomp transformation kit
1
Synthetic Operon Assembly and CRISPR Genome Editing
2
Heterologous Expression of CbFAD3 in Yeast
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The coding region of CbFAD3 was cloned into pYES2.0 (Invitrogen, USA) using specific primers (P13 and P14; Supplementary Table S1 ), to construct the expression plasmid pYES2-CbFAD3. pYES2-CbFAD3 and pYES2.0 were transformed into Saccharomyces cerevisiae strain INVSc1 (Invitrogen, USA) using S. cerevisiae EasyComp transformation kit (Invitrogen, USA). The yeast transformants were selected and cultured according to the method of Román et al. (2012) (link). When the OD600 of the culture reached 0.2–0.3, gene expression was induced by adding 2% (w/v) galactose. Yeast cells were harvested by centrifugation at 1500 g for 5 min at 4 °C and washed with distilled water. The extraction and SDS-PAGE of total yeast proteins were performed as described by Horvath and Riezman (1994) (link). The production of C18:3 was induced by adding 2% (w/v) galactose, 50 μM C18:2 (Sigma-Aldrich, USA) and 0.1% (w/v) NP-40, and was measured after growth at 20 °C for 3 d.
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