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Method for replacing essential gene promoter in yeast genome

An essential gene and promoter technology is applied in the field of replacing essential gene promoters in yeast genome, which can solve the problems of difficult removal and ineffective regulation of essential genes, and achieves the effects of avoiding residues, facilitating the construction of strains, and realizing repeated use.

Inactive Publication Date: 2018-01-09
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] (2) pop-in / pop-out method: construct the expression vector of "repeat sequence-URA3-repeat sequence-target sequence", integrate the target sequence into the genome through two stages of pop-in and pop-out, and at the same time URA3 Pop-up realizes the reuse of marker genes, but at the same time leaves long fragments of repetitive sequences in the genome, which are not easy to remove
However, in this method, there are other sequences between the constitutive promoter and the essential gene, which cannot effectively regulate the expression of the essential gene

Method used

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  • Method for replacing essential gene promoter in yeast genome
  • Method for replacing essential gene promoter in yeast genome
  • Method for replacing essential gene promoter in yeast genome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1DDI2

[0056] Example 1 DDI2 promoter seamlessly replaces yeast essential gene CDC6 promoter

[0057] 1. Construction of "DDI2-URA3-DDI2" repeat sequence plasmid

[0058] 1. Digest the plasmid pBlueScript-URA3 with restriction endonucleases BamHI and EcoRI, and recover the digested product of the plasmid;

[0059] 2. Amplify the promoter P with Phusion enzyme DDI2-U , recovering the PCR product;

[0060] 3. Ligate the vector backbone of step 1 and the recovered product of step 2 with Quick-Fusion enzyme to obtain recombinant plasmid pBlueScript-P DDI2-U -URA3, for sequencing verification.

[0061] 4. Then digest the plasmid pBlueScript-P with restriction enzymes HindIII and SalI DDI2-U -URA3, recovery of plasmid digestion products;

[0062] 5. Amplify the promoter P with Phusion enzyme DDI2-D ; Recover the PCR product;

[0063] 6. Ligate the vector backbone of step 4 and the recovered product of step 5 with Quick-Fusion enzyme to obtain recombinant plasmid pBlueScript-P DDI2-...

Embodiment 2

[0081] Example 2 Coated plate verification P DDI2 Replacement yeast essential gene CDC6 promoter strain

[0082] 1. Cultivate the strains obtained above in YPD-5mM cyanamide medium at 30°C overnight, and then transfer them to YPD / YPD-5mM medium the next day for 4 hours;

[0083] 2. Take the bacterial solution to measure the OD value. After calculation, take 107 bacterial solution, and after gradient dilution to 104 / 105, take 100 μL and 50 μL to smear the plate respectively, such as Figure 4 Shown, respectively 104, 1000, 500 bacteria;

[0084] 3. After coating on YPD / YPD-5mM plates, culture at 30°C for 2-4 days. Such as Figure 5 As shown, no plaques grew on the medium without cyanamide, indicating that P DDI2 The promoters of essential genes in yeast can be replaced by the above methods, and the expression of essential genes can be strictly regulated by inducers.

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Abstract

The invention relates to the field of genetic engineering, in particular to a method for replacing an essential gene promoter in a yeast genome. The method comprises steps as follows: S1, a DDI2-URA3-DDI2 fragment is prepared; S2, the DDI2-URA3-DDI2 fragment is transformed into the yeast genome, and a transformant is obtained; S3, the transformant is cultured in a YPD culture medium containing cyanamide, pop-out URA3 and a DDI2 promoter are obtained through screening by 5-FOA, and the essential gene promoter is replaced with a yeast strain of the DDI2 promoter. On the basis of a pop-in / pop-outmethod, a new genome integration strategy is developed for replacement of the essential gene promoter in yeast, so that recycling of marker genes is realized, and no traces of repetitive sequences remain in the yeast genome.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for replacing the essential gene promoter of yeast genome, Background technique [0002] Saccharomyces cerevisiae, also known as budding yeast, is an important model organism for eukaryotic molecular biology research. Saccharomyces cerevisiae has many advantages, such as easy cultivation, fast reproduction speed, strong environmental adaptability, small variation, production Stable and safe, easy to carry out molecular level transformation, etc. The transformation of Saccharomyces cerevisiae has become a research hotspot in this field. In the process of strain construction, in order to ensure the expression of essential genes in the yeast genome and to achieve efficient regulation of the expression of essential genes, a compensation plasmid is often used to transfer a copy of the essential gene, and then the method of genome integration is used to replace the essentia...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N1/19C12N15/113
Inventor 付钰张恺宁
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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