Recombinant saccharomyces cerevisiae for production of ambrein and construction method
A technology of Saccharomyces cerevisiae and ambroxol, applied in the field of recombinant Saccharomyces cerevisiae and its construction
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0037] Example 1. Construction method of plasmid PXP320-ΔSHC
[0038] Table 1 Primer sequences
[0039]
[0040] According to Table 1, the artificially synthesized ΔSHC gene fragment (SEQ ID No. 2, site-directed mutagenic hopene cyclase encoding gene ΔSHC) was used as the template, Spe1-SHC-F (SEQ ID No. 6) was used as the front primer, SHC -Xho1-R (SEQ ID No. 7) is the back primer, and the fragment ΔSHC is amplified.
[0041] The empty plasmid PXP320 (purchased from Addgene) stored in E. coli was extracted with a plasmid mini-kit, and the extraction steps were as follows:
[0042] 1. Inoculate E. coli containing the empty plasmid PXP320 into LB liquid medium containing antibiotics (ampicillin) for 12 hours, collect 3 mL of bacterial liquid in a centrifuge tube, centrifuge at 12,000 rpm for 1 min, discard the supernatant (the supernatant should be as much as possible);
[0043] 2. Equilibrate the adsorption column with 500 μL of BL equilibration solution, centrifuge at 1...
Embodiment 2
[0056] Embodiment 2, the construction method of plasmid PXP218-BmeTC
[0057] The construction method of plasmid PXP218-BmeTC is the same as that of Example 1. The artificially synthesized BmeTC gene fragment (SEQ ID No. 3) is used as a template, and Spe1-BmeTC-F is used as a front primer (SEQ ID No. R (SEQ ID No. 9) is the back primer, and the fragment BmeTC was amplified by PCR. The empty plasmid PXP218 (purchased from Addgene) stored in E. coli was extracted.
[0058] The amplified fragment BmeTC and the extracted plasmid PXP218 were double digested with restriction enzymes SpeI and XhoI, prepared according to the enzyme digestion system in Example 1, and the prepared enzyme digestion system was placed in a 37°C water bath. Enzyme digestion was carried out in the pot for 1 h, and the digested plasmids and fragments were purified and recovered, and ligation reaction was carried out. The prepared ligation system was placed at 22 °C for 1 h. According to the method in Examp...
Embodiment 3
[0059] Embodiment 3, the construction method of Saccharomyces cerevisiae recombinant bacteria 1
[0060] Saccharomyces cerevisiae W303-1a was introduced into Saccharomyces cerevisiae W303-1a with site-directed mutagenesis of the hopene cyclase encoding gene ΔSHC and the tetraphenyl-β-curcumene synthase encoding gene BmeTC to obtain recombinant strain 1. The nucleotide sequence of the site-directed mutagenic hopene cyclase encoding gene ΔSHC is shown in SEQ ID No. 2; the nucleotide sequence of the tetraphenyl-β-curcumene synthase encoding gene BmeTC is shown in SEQ ID No. 2 No.3 shown.
[0061] ΔSHC (SEQ ID No.2) is a site-directed mutation of hopene cyclase obtained by mutating the 377th amino acid of hopene cyclase SHC (SEQ ID No.1) from aspartic acid to cysteine, The hopene cyclase (SHC) was derived from Alicyclobacillus acidocaldarius, and the tetraphenyl-β-curcumene synthase (BmeTC) was derived from Bacillus megaterium, by Wuhan Jinkarui Bioengineering Co., Ltd. synthesi...
PUM
| Property | Measurement | Unit |
|---|---|---|
| Diameter | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to view more - R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap