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Method for screening environmental stress response promoters

A promoter and environment technology, applied in the field of screening environmental stress response promoters, can solve the problems of inability to mine potential unknown conditional responses and low efficiency of promoter research, and achieve the effect of meeting the needs of engineering strain construction

Inactive Publication Date: 2017-03-22
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Traditionally, the characterization of promoters is mostly based on the conditional setting of the functional information of their related genes, and it is impossible to mine potential unknown conditional responses, and the research efficiency of individual promoters is low, and it is impossible to systematically characterize various environments at the global level of cells. Promoter Responses to Stress Conditions

Method used

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  • Method for screening environmental stress response promoters
  • Method for screening environmental stress response promoters
  • Method for screening environmental stress response promoters

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Construction of promoter library.

[0037]Saccharomyces cerevisiae S288c Chromosome V DNA sequence, refer to http: / / www.ncbi.nlm.nih.gov / nuccore / NC_001137.3 for the reference sequence, take the spacer sequence between two adjacent protein-coding genes in the same direction as the promoter region, and find out whether Containing a BsaI restriction site (GGTCTC or GAGACC) or a BsmBI restriction site (CGTCTC or GAGACG), design primers containing a BsaI or BsmBI restriction site according to its sequence. For the promoter sequence containing both BsaI and BsmBI restriction sites, the characterization vector was constructed by the circular polymerase extension method (CPEC), and the 5' and 3' of each 20bp were cut according to the sequence and introduced into the homologous fragment of the expression vector as a primer.

[0038] The genome of Saccharomyces cerevisiae BY4741 was extracted by the phenol / chloroform method, and the promoter fragment was amplified by P...

Embodiment 2

[0039] Example 2 Characterization of the response of the promoter library under various stress conditions

[0040] The media used in various stress conditions are as follows:

[0041] Different carbon source media are: Sc-Leu medium, sucrose medium, galactose medium, glycerol medium, ethanol medium.

[0042] High-salt stress condition medium: NaCl medium.

[0043] Medium for hyperosmotic stress conditions: Sorbitol medium.

[0044] Amino acid deficient medium: Sc-Leu amino acid deficient medium, galactose carbon source amino acid deficient medium.

[0045] The medium used in the high temperature culture condition is: Sc-Leu medium.

[0046] The seeds of the promoter library were activated first, and 5 μL of each preserved recombinant Saccharomyces cerevisiae seeds were taken and connected to each well of a 96-well cell culture plate, each well containing 200 μL of Sc-Leu medium, and the sealed cell culture plate was placed on a microplate shaker for 30 Cultivate overnight ...

Embodiment 3

[0048] Example 3 Data processing to mine stress-responsive promoters.

[0049] Under the culture condition of Sc-Leu medium at 30°C, the measured results of the promoter strength of chromosome V are as follows: figure 1 As shown in a, the results are reordered from large to small, and numbered 1-279, the result is as follows figure 1 as shown in b.

[0050] According to the sequence of rearranged promoters, the promoters and their strengths under other environmental stress culture conditions were sorted. Under the culture condition of sucrose medium, the results were as follows: figure 2 As shown in a, the results under the culture condition of galactose medium are as follows figure 2 As shown in b, the results under the culture condition of glycerol medium are as follows figure 2 As shown in c, the result under the ethanol medium culture condition is as follows figure 2 Shown in d; under the culture condition of Sc-AA medium, the results are as follows figure 2 Sho...

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Abstract

The invention belongs to the technical field of biology and provides a method for screening environmental stress response promoters. The method comprises the steps that a promoter library is established, representation strains of the promoter library are activated and then cultivated under different stress conditions, OD600 and the fluorescence intensities are measured, the fluorescence intensities of different component culture media and under different culture conditions are analyzed, and the environmental response promoters are excavated. The method is based on the natural promoter library of saccharomyces cerevisiae chromosomes V, quantitative presentation and excavation of promoters of a whole chromosome size under specific culture conditions are achieved, and the promoters giving response to different environment conditions are obtained through screening. The method provided by the invention can be further applied to screening of promoters, giving to response to different environment conditions, of other promoter libraries, and screening of promoters giving response to other conditions, and the target stress response promoters can be excavated systematically, conveniently, effectively and visually.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method and application for screening environmental stress-responsive promoters. Background technique [0002] With the in-depth research and wide application of synthetic biology, the demand for various chassis, components and modules is increasing. Among them, components are the basic units for building various functional modules and complex systems, and the characterization and mining of components is a higher level. The foundation of engineered construction. Promoters, as commonly used gene expression regulatory elements, are mainly divided into two types according to their different regulatory types: inducible promoters and constitutive promoters. Constitutive promoter refers to that under the control of this type of promoter, the expression of structural genes is generally constant at a certain level, and there is no obvious difference in the expression level in d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02G01N21/64
CPCC12Q1/02G01N21/6486
Inventor 李霞李飞飞李炳志元英进
Owner TIANJIN UNIV
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