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Saccharomyces cerevisiae integrated expression vector

A technology of Saccharomyces cerevisiae and chromosome, applied in the field of integrated expression vectors of Saccharomyces cerevisiae, which can solve the problems of increased operation complexity, unstable production performance of engineering bacteria, easy loss of plasmids, etc.

Inactive Publication Date: 2014-07-09
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If the plasmid-type expression vector is transferred into Saccharomyces cerevisiae using in vivo assembly technology, although the original DNA of prokaryotes can be removed, the complexity of the operation will be increased
In addition, the construction of engineering bacteria with plasmid-type expression vectors has the problem of easy loss of plasmids, resulting in unstable production performance of engineering bacteria

Method used

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  • Saccharomyces cerevisiae integrated expression vector
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  • Saccharomyces cerevisiae integrated expression vector

Examples

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preparation example Construction

[0033] The preparation method of the plasmid pSK-mCherry is as follows: the DNA molecule shown in the 1568th to 2296th nucleotides from the 5' end of the sequence 1 of the artificially synthesized sequence listing (wherein the 1568th to 1573rd nucleotides are recognized by ClaI digestion) The sequence "ATCGAT", the 1574th to 1579th nucleotides are the NdeI enzyme digestion recognition sequence "CATATG", the 1580th to 2296th nucleotides are the coding gene of mCherry protein, and the 2297th to 2302th nucleotides are EcoRI digestion The recognition sequence "GAATTC") was inserted between the ClaI and EcoRI restriction sites of the pBlueScriptIISK(-) vector to obtain the plasmid pSK-mCherry.

[0034] pBlueScriptIISK(-) vector: purchased from Agilent Technologies Genomics, Catalog No. 212206.

[0035]Saccharomyces cerevisiae w303 strain (strain attributes: MATa; ura3-52; trp1Δ2; leu2-3,112; hi s3-11; ade2-1; can1-100): purchased from EUROSCARF, number 20000A.

Embodiment 1

[0036] Embodiment 1, the preparation of Saccharomyces cerevisiae expression vector

[0037] 1. Construction of recombinant plasmid pEXILV5-mCherry

[0038] 1. Extract the genomic DNA of Saccharomyces cerevisiae (the Latin name is Saccharomyces cerevisiae; China Center for General Microorganism Culture Collection and Management Center, the strain number is 2.1882).

[0039] 2. Using the genomic DNA extracted in step 1 as a template, use a primer pair (target sequence 554bp) consisting of ILV5P-S (underlined SalI recognition sequence) and ILV5P-AS (underlined ClaI recognition sequence) to perform PCR amplification to obtain PCR amplification product with ILV5 gene promoter of Saccharomyces cerevisiae.

[0040] ILV5P-S: 5'-ACGC GTC GAC CCCTATCTGTTCTTCCGCTCTACC-3';

[0041] ILV5P-AS: 5'-CC ATCGAT GTTTTATTTTTACTTATATTGCTGGTAGG-3'.

[0042] 3. Double-digest the PCR amplified product in step 2 with restriction enzymes SalI and ClaI, and recover the digested product.

[0043] ...

Embodiment 2

[0087] Example 2, Detection of the expression level of the Saccharomyces cerevisiae expression vector of the present invention

[0088] 1. Construction of Saccharomyces cerevisiae recombinant bacteria expressing mCherry

[0089] 1. Digest the recombinant plasmid pEXILV5-mCherry with restriction endonucleases XhoI and BstXI, and recover the digested product (with the upstream homology arm of 18SrDNA, the ILV5 gene promoter, the coding gene of mCherry protein, IRES sequence, URA3 gene, URA3 gene transcription termination sequence and downstream homology arm of 18S rDNA). The digested product was passed the lithium acetate method (Ito, H., Fukuda, Y., Murata, K., and Kimura, A.1983. Transformation of intact yeast cells treated with alkaline cations. J. Bacteriol. 53: 163-168 .) Transform Saccharomyces cerevisiae w303 to obtain a recombinant strain, which is named recombinant strain I-rfp.

[0090] 2. Digest the recombinant plasmid pEXTDH3-mCherry with restriction endonucleases ...

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Abstract

The present invention discloses a saccharomyces cerevisiae integrated expression vector, and provides a double-stranded DNA fragment, which sequentially comprises elements such as a saccharomyces cerevisiae chromosome upstream homology arm, an eukaryotic promoter, a saccharomyces cerevisiae IRES sequence, an eukaryotic screening gene, an eukaryotic transcription termination sequence and a saccharomyces cerevisiae chromosome downstream homology arm from upstream to downstream. The invention further relates to plasmid containing the double-stranded DNA fragment, wherein the plasmid is a saccharomyces cerevisiae integrated expression vector, can be provided for making exogenous or endogenous genes express in saccharomyces cerevisiae without induction, can adopt saccharomyces cerevisiae to produce the target protein, or can be used for metabolic pathway construction of genetic engineering strains. The saccharomyces cerevisiae integrated expression vector provides a good tool for protein expression and metabolic pathway construction of saccharomyces cerevisiae.

Description

technical field [0001] The invention relates to a brewing yeast integrative expression vector. Background technique [0002] Saccharomyces cerevisiae, also known as baker's yeast or budding yeast. Saccharomyces cerevisiae is the yeast most closely related to humans. Saccharomyces cerevisiae is a traditional microorganism for making food such as bread and steamed buns and brewing wine. It is by far the most biologically safe microorganism. Meanwhile, in modern molecular and cell biology, Saccharomyces cerevisiae is used as a eukaryotic model organism, and its role is equivalent to the prokaryotic model organism Escherichia coli. Saccharomyces cerevisiae is the most commonly used biological species in fermentation. [0003] The cells of Saccharomyces cerevisiae are spherical or ovoid, 5–10 μm in diameter. Its method of reproduction is budding. [0004] Saccharomyces cerevisiae is a microorganism commonly used in modern industrial biotechnology, health care products indust...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/81C12R1/865
Inventor 贺鹏张新杰陶勇
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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