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ERG1 gene-deficient yeast engineering bacteria and construction method and utilization thereof

A gene defect and yeast engineering technology, which is applied in the field of ERG1 gene defect yeast engineering bacteria, its construction and application, can solve the problems of false positives, difficulty, and incomplete knockout of ERG1 gene, etc.

Active Publication Date: 2018-12-18
CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the later stage, the squalene cyclooxygenase gene of plants is generally verified by KLN1, which is constructed by inserting a screening marker into the coding region of the ERG1 gene and then integrating it into the yeast chromosome by homologous recombination. , the disadvantage is that the ERG1 gene is not completely knocked out, and there may be false positives when verifying the function of the exogenous se gene
There are also individual strains using another modified strain RXY6 (MATaerg7::HIS3 erg1::KanMX4 hem1::TRP1 ura3-52 trp1-△63 leu2–3,112 his3-△200ade2 Gal + ), the main advantage of this strain is that Hem1 is also knocked out on the premise of knocking out ERG1. Knocking out Hem1 can make yeast absorb ergosterol from the medium under aerobic conditions, but it is difficult to knock out ERG1 and Hem1 at the same time Large, and Hem1 is very important for the synthesis of hematin, which is involved in various metabolic reactions in the body, so the yeast knocked out of the Hem1 gene needs to supplement hematin in the medium

Method used

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  • ERG1 gene-deficient yeast engineering bacteria and construction method and utilization thereof
  • ERG1 gene-deficient yeast engineering bacteria and construction method and utilization thereof
  • ERG1 gene-deficient yeast engineering bacteria and construction method and utilization thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Implementation example 1, the construction of Erg1 yeast-defective bacteria

[0050] 1. Use yeast CRISPR / Cas9gRNA to design website http: / / yeastriction.tnw.tudelft.nl / #! / Perform gRNA site screening on the ERG1 gene, and the gRNA sequence of the ERG1 gene is shown in the table below.

[0051] Table 1 ERG1 gene gRNA sequence

[0052]

[0053] 2. Construction of gRNA vector

[0054] The gRNA sequence was added to the front of the gRNA backbone by the method of primer design, and the linear vector sequence was cloned using the p426-SNR52p-gRNA eukaryotic expression vector (purchased from Addgene official website) as a template by RF cloning, and blunt-ended by T4 ligase After ligation, the gRNA vector containing the site specifically recognized by the yeast ERG1 gene can be obtained. The primer information is as follows:

[0055] Table 2 Primers for gRNA vector construction

[0056]

[0057] 1. RF clone

[0058] system:

[0059]

[0060] Reaction condition...

Embodiment 2

[0148] Implementation example 2, full-length cloning of Tripterygium wilfordii epoxy squalene cyclase gene

[0149] 1. Extraction of total RNA from Tripterygium wilfordii suspension cells

[0150] 1. Culture and induction of Tripterygium wilfordii suspension cells

[0151] (1) Preparation of MS culture (including hormone): 4.43g L -1 MS, 0.5mg.L -1 2,4-D, 0.1mg L -1 KT, 0.5mg.L -1 IBA, 30g·L -1 Sucrose, adjust pH=5.8. Then sterilize in a high-pressure steamer at 121°C for 20 minutes.

[0152] (2) Preparation of elicitor methyl jasmonate (MeJA) solution: 115 μL of 95% MeJA was dissolved in 885 μL of DMSO to prepare a 0.5M MeJA stock solution. Both MeJA and DMSO used in the preparation were filtered through a 0.22 μm microporous membrane and mixed in a sterile environment.

[0153] (3) Take the suspension cells of Tripterygium wilfordii preserved in MS liquid culture, inoculate about 2 g of suspension cells in a 100 mL Erlenmeyer flask containing 25 mL of MS liquid medium...

Embodiment 3

[0254] Implementation Example 3, Tripterygium wilfordii squalene cyclooxygenase gene functional verification

[0255] 1. Competent preparation of Erg1-deficient yeast

[0256] According to Frozen-EZ Yeast Transformation II TM Kit instructions for the preparation of yeast chemically competent cells, the steps are as follows:

[0257] (1) Pick a single erg1 colony that has been successfully constructed and culture it in 20mL YPD+ergosterol (20ug / mL) at 30°C under anaerobic conditions until OD 600 = 0.8-1.0.

[0258] (2) Centrifuge at 500×g for 4 minutes to precipitate the bacteria, discard the supernatant;

[0259] (3) Resuspend and wash the cells in 10 mL of EZ1, centrifuge at 500×g for 4 min, and discard the supernatant;

[0260] (4) 1 mL of EZ2 resuspended bacteria, each 50 μ L into a tube, set aside.

[0261] Note: The prepared competent cells can be directly transformed, or slowly cooled and frozen to -80°C (4°C, 1h; -20°C, 1h; -80°C).

[0262] 2. Functional complemen...

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Abstract

The invention relates to ERG1 gene-deficient yeast engineering bacteria and a construction method thereof, wherein the ERG1 gene-deficient yeast engineering bacteria transforms yeast by a CRISPR / Cas9gene editing technology, an Erg1 gene sequence can be completely deleted, back mutation phenomena produced by yeast self-repair can be avoided, and engineering bacteria that can be used to verify other squalene-derived epoxidase genes can be obtained.

Description

technical field [0001] The invention relates to an ERG1 gene-deficient yeast engineering bacterium, and a method for knocking out the ERG1 gene in the yeast. The Saccharomyces cerevisiae is transformed by the CRISPR / Cas9 gene editing technology, and the squalene cyclooxygenase in the yeast chromosome is transformed. (Squalene epoxidase, ERG1) gene was knocked out, making it a defective bacterium suitable for verifying the function of squalene epoxidase gene from other sources, and the squalene cloned from Tripterygium wilfordii was successfully verified by this yeast-defective bacterium The cyclooxygenase gene (Twse) belongs to the field of genetic engineering. Background technique [0002] The acquisition of active natural products from traditional Chinese medicine or other sources mainly depends on the extraction of the original species, but the natural products obtained by this method are far from meeting the demand, and it is easy to cause damage to the environment and r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/90C12R1/865
Inventor 高伟周家伟胡添源
Owner CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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