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Novel yeast inducible promoter and application thereof

A promoter and inducible technology, applied in the field of molecular biology, can solve the problems of time-consuming and labor-intensive culture medium, constitutive promoters do not show time-space specificity, and gene expression cannot be regulated at any time.

Active Publication Date: 2017-10-10
青岛明德生物科技研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Among the above two yeast promoters, the constitutive promoter does not show spatiotemporal specificity, is not regulated by inducers, and cannot regulate gene expression at any time; the most commonly used inducible promoter in yeast is P GAL1 , it can be induced by galactose, but it needs to be induced by galactose after raffinose starvation treatment in the culture, and it is time-consuming and labor-intensive to replace the medium

Method used

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  • Novel yeast inducible promoter and application thereof
  • Novel yeast inducible promoter and application thereof
  • Novel yeast inducible promoter and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1, promoter strength comparison

[0058] 1. Construction of recombinant expression vector

[0059] 1. Digest plasmid Ycplac111-sfGFP-His6-T with restriction enzymes BamHI and SphI CYC1 , recovering the plasmid digestion product;

[0060] 2. Amplify the promoter P with Phusion enzyme ADH1 ,P GAL1 ,P DDI2 ; Recover the PCR product;

[0061] 3. Ligate the vector backbone of step 1 and the recovery product of step 2 with Quick-Fusion enzyme to obtain three recombinant plasmids (Ycplac111-P ADH1 -sfGFP-His6-T CYC1 , Ycplac111-P GAL1 -sfGFP-His6-T CYC1 , Ycplac111-P DDI2 -sfGFP-His6-T CYC1 ), for sequencing verification, the sequencing results showed that three kinds of target plasmids were obtained, such as figure 1 , figure 2 , image 3 shown.

[0062] 2. Observation of fluorescence intensity by laser confocal microscope

[0063] 1. Transform the plasmids of the three promoters constructed above into yeast strain W303;

[0064] 2. Cultivate strai...

Embodiment 2

[0072] Embodiment 2, Western detection cyanamide induces P DDI2 Regulation of sfGFP expression

[0073] 1. Put P DDI2 After the strain was cultured in SD-Leu medium for 3 hours, it was induced by adding different concentrations (0-5mM) of cyanamide for 4 hours, and the W303 wild strain was also cultured (with 5mM of cyanamide) as a control;

[0074] 2. Collect the above-mentioned 5OD cultured bacterial liquid, and extract protein according to the method of yeast small amount of total protein. The obtained protein samples were used for Western detection (GFP antibody: 1:2000).

[0075] Summary: by Image 6 , 7 It can be seen that the inducible activity of this promoter increases with the increase of the concentration of cyanamide, and has stronger activity at the concentration of 5-8mM.

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Abstract

The invention provides a novel yeast inducible promoter and application thereof and belongs to the technical field of molecular biology. According to the technical scheme, the DNA (deoxyribonucleic acid) sequence of the novel yeast inducible promoter is shown as a sequence 1 in a sequence table, and the length of the novel yeast inducible promoter is 673 bp. The novel yeast inducible promoter has the advantage that the novel yeast inducible promoter DDI2 can be effectively induced by cyanamide; the strength of the novel yeast inducible promoter DDI2 is close to that of a known inducible promoter GAL1 and higher than that of a constitutive promoter ADH1; during induction, the novel yeast inducible promoter can save change of mediums and control the expression quantity of genes through inducing time and amount of inducing agent, thereby having advantages over the common GAL1 in terms of time cost and price cost. Detecting results of a flow cytometer shows that the strength of the promoter DDI2 is higher than that of the promoter ADH1 and close to that of the promoter GAL1, the required inducing time of the promoter DDI2 is shorter than that of the promoter GAL1, so that change of the mediums can be saved; according to the detecting results of Western and the flow cytometer, the strength of the promoter DDI2 can reach a maximum when the concentration of the inducing agent is 5-8 mM.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a novel yeast inducible promoter and its application. Background technique [0002] The expression of genes in cells is affected by many factors, such as cis-acting elements, trans-acting factors, etc. During the cell growth stage, the expression level of transcription factors associated with RNA polymerase and the regulation of other gene levels are related. Among them, the promoter is a DNA sequence that initiates the transcription of a specific gene, is an important cis-element for gene expression regulation, and is also an important component of a genetic engineering expression vector. The strength of the promoter function is crucial for the expression of the target gene. [0003] Promoters contain specific DNA sequences that, for example, provide reliable initial binding sites for RNA polymerase and transcription factors that recruit RNA polymerase. Usually, incr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/10
CPCC07K14/395C12N15/10C12Q2531/113
Inventor 傅钰张恺宁郝智慧王苹苹
Owner 青岛明德生物科技研究院有限公司