Construction method and application of saccharomyces cerevisiae genetically engineered bacterium with high yield of 3-methylthio-propanol
A technology of Saccharomyces cerevisiae and methylthiopropanol, which is applied in the directions of microorganism-based methods, biochemical equipment and methods, enzymes, etc., can solve problems such as toxic by-products and serious environmental pollution, and achieve the effect of increasing the synthesis amount
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[0038] 1. Cloning of ARO8 and ARO10 genes
[0039] 1.1 Extraction of Saccharomyces cerevisiae S288c genomic DNA
[0040] The freshly activated S. cerevisiae S288c strain was inoculated into YPD liquid medium, placed in a shaker at 30°C, 220rpm and shaken overnight.
[0041] (1) Take 1 mL of bacterial liquid into a 1.5 mL centrifuge tube, centrifuge at 12,000 rpm for 1 min at room temperature, and discard the supernatant;
[0042] (2) Add 600 μL of sorbitol Buffer to the cell pellet, add 100 μL of 100 mg / mL helicase, mix well, place in a water bath shaker at 30°C at 220 rpm for 90 minutes;
[0043] (3) YeastDNA Kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) was used to extract the genome of Saccharomyces cerevisiae.
[0044] 1.2 Cloning of ARO8 gene
[0045] Using Saccharomyces cerevisiae S288c genomic DNA as a template, specific primers were designed to amplify the ARO8 gene fragment by PCR.
[0046] ARO8 gene primer sequence:
[0047] Upstream primer: 5'-CGGGAT...
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