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A recombinant vector for constructing Saccharomyces cerevisiae lysis engineering bacteria and its application

A technology of Saccharomyces cerevisiae and recombinant vector, which is applied in genetic engineering, recombinant DNA technology, and the introduction of foreign genetic material using vectors, etc. It can solve the problems of limiting foreign gene cloning, expression and activity identification, high instrument requirements, and low wall breaking efficiency. problems, achieve the effects of short cracking time, simple operation, and simplified experimental procedures

Active Publication Date: 2021-07-20
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these methods have been continuously improved, there are still problems such as cumbersome operation, low efficiency of wall breaking, long time and high requirements for instruments.
[0004] Saccharomyces cerevisiae is more and more widely used in the field of biological research, but the existing wall-breaking methods severely limit the cloning, expression and activity identification of foreign genes in Saccharomyces cerevisiae. It is particularly important to effectively realize the extraction and preparation of various active substances such as nucleic acids, proteins, enzymes, and polypeptides derived from Saccharomyces cerevisiae cells, as well as the activity identification, extraction, and production of genetically engineered yeast expression products

Method used

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  • A recombinant vector for constructing Saccharomyces cerevisiae lysis engineering bacteria and its application
  • A recombinant vector for constructing Saccharomyces cerevisiae lysis engineering bacteria and its application
  • A recombinant vector for constructing Saccharomyces cerevisiae lysis engineering bacteria and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Construction of Saccharomyces cerevisiae recombinant vector and donor DNA

[0055] To construct the Saccharomyces cerevisiae recombinant vector p426-Cas9-gSED1 and donor DNA, the specific construction method is as follows:

[0056] 1. Construction of recombinant vector p426-gSED1

[0057] Using the p426 vector as a template and using P1 / P2 and P3 / P4 as primer pairs, SED1-1 and SED1-2 fragments were respectively amplified. use II Recombination Cloning Kit, recombine the two fragments of SED1-1 and SED1-2 to obtain the recombinant vector p426-gSED1 carrying the SED1 guide RNA sequence.

[0058] P1: 5'-TAATAATGGTTTCTTAGTATGA-3';

[0059] P2: 5'-CCGATGTCACTTCCTCCTCTGATCATTTATCTTTCACTGC-3';

[0060] P3: 5'-AGAGGAGGAAGTGACATCGGGTTTTAGAGCTAGAAATA-3';

[0061] P4: 5'-ACTAAGAAACCATTATTATCAT-3'.

[0062] 2. Construction of recombinant vector p426-Cas9-gSED1

[0063] The p414 vector was used as a template, and the fragment containing the promoter TEF1p, the gene ...

Embodiment 3

[0088] Application of Example 3 Saccharomyces cerevisiae Rapid Cleavage Method in Colony PCR

[0089] 1. Saccharomyces cerevisiae colony PCR

[0090] (1) Pick a single colony of the recombinant wild strain and recombinant mutant strain of S. cerevisiae BY4741 obtained according to Example 6, draw a patch on the SD (Ura) plate, and culture at 30°C.

[0091] (2) Pick a little bacteria from the patch plate and put it into 20 μL Tris-HCl (pH 7.5) buffer, shake to make it even.

[0092] (3) Add 1U Zymolyase respectively, incubate at 30°C for 30 minutes, and inactivate at 60°C for 5 minutes.

[0093] (4) Centrifuge at 14,000 rpm for 2 minutes, take 0.5 μL of the supernatant as a template for PCR, and use the primers P13 / P14.

[0094] (5) Identify by agarose gel electrophoresis.

[0095] 2. The experimental results show that both the recombinant mutant strain and the recombinant wild strain have amplified the target band, but the band amplified by the recombinant mutant strain is ...

Embodiment 4

[0096] Example 4 Application of Saccharomyces cerevisiae Rapid Cleavage Method in Plasmid Extraction

[0097] 1. Culture of strains

[0098] (1) Resuscitate and activate the recombinant mutant strain and recombinant wild strain of S. cerevisiae BY4741 obtained according to Example 6 from -80°C, and cultivate them at 30°C.

[0099] (2) Pick a single colony and insert it into SD (Ura) liquid medium, and culture at 30°C for 36h.

[0100] 2. Cell Lysis

[0101] (1) Centrifuge the bacterial solution at 3000×g for 5 minutes to collect the bacterial cells, and collect 5 OD of bacterial cells in each tube.

[0102] (2) The recombinant mutant strain was resuspended in 0.5 mL Tris-HCl (pH 7.5) lysis buffer into a 2 mL centrifuge tube, and then 25 U Zymolyase lyase was added to lyse the cells at 30° C. and 250 rpm for 0.5 h.

[0103] (3) Recombinant wild strains Cells were lysed according to the method of Omega and Sangon Yeast Plasmid Extraction Kit.

[0104] 3. Purification treatme...

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Abstract

The invention discloses a recombinant vector for constructing Saccharomyces cerevisiae lysing engineering bacteria and application thereof, belonging to the field of biotechnology. The recombinant vector of the present invention can knock out the SED1 gene in the Saccharomyces cerevisiae genome, and the obtained mutant strain can rapidly lyse the cells under the action of the lyase Zymolyase, and release the intracellular substances to the extracellular. The object of operation is Saccharomyces cerevisiae, which has wide applicability. There is no need to add additional reagents during the lysis process, and the cost is low. The lysis time is short, and the cells can be completely lysed within 2 hours, which greatly shortens the time for Saccharomyces cerevisiae cell lysis in the past. It is easy to operate, does not need cumbersome steps such as glass beads and the preparation of complex lysates, and only needs a simple buffer solution, which greatly simplifies the experimental process. Therefore, the Saccharomyces cerevisiae cracking method of the present invention can be used for enzyme activity detection, colony PCR, plasmid extraction, protein identification and high-throughput screening, etc., which has the advantages of simplicity, quickness and low cost, and has broad application prospects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular provides a recombinant vector and a donor DNA for constructing rapid lysing engineered bacteria of Saccharomyces cerevisiae, as well as the extraction of active substances in yeast cells of Saccharomyces cerevisiae engineered bacteria obtained therefrom, and the expression of genetically engineered yeast expression system The extraction of the product and the application of rapid detection of recombinant protein (enzyme) expressed in yeast cells. Background technique [0002] Saccharomyces cerevisiae is a widely studied unicellular eukaryotic microorganism with a complex cell wall structure. The thickness of the cell wall of Saccharomyces cerevisiae is about 100-300nm, similar to the "sandwich" structure, mainly composed of β-D-glucan (β-D-glucan), α-D-mannose (α-D-mannose) and a small amount of few Ding quality (chitin) composition. Among them, dextran is the most important struc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/81C12N1/19C12N15/90C12N1/06C12R1/865
CPCC07K14/395C12N1/06C12N15/81C12N15/905
Inventor 李爽察亚平卓敏朱晁谊
Owner SOUTH CHINA UNIV OF TECH
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