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Method for screening and identifying stress response gene expression regulatory factors

A technology for regulating factors and gene expression, applied in the biological field, can solve the problems of identification and determination that have not been developed, and achieve the effect of reducing time and labor

Active Publication Date: 2020-11-17
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition to microbial communities, without knowledge of their genomes and TFs, a general approach has not been developed to identify and determine TFs or TFBSs required in the regulation of gene expression in response to environmental stress

Method used

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  • Method for screening and identifying stress response gene expression regulatory factors
  • Method for screening and identifying stress response gene expression regulatory factors
  • Method for screening and identifying stress response gene expression regulatory factors

Examples

Experimental program
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Embodiment 1

[0040] 1. Materials and methods

[0041] 1. Preparation of cell lysate

[0042] 1 mL of DH5α culture was centrifuged at 10,000 g for 1 min, and the pellet was resuspended in 300 μL of lysis buffer (10 mM Tris-HCl, pH 8.0, 0.1 M NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA) and 0.1% (w / v ) Polyethylene glycol octylphenyl ether (Triton X-100)). 7.5 μL of freshly made lysozyme solution (10 mg / mL in 10 mM Tris-HCl, pH 8.0, final concentration = 0.25 mg / mL) was added and mixed by tapping the tube, and the lysis mixture was incubated at room temperature for 10-20 minutes. After centrifugation, the supernatant was used for affinity screening.

[0043] 2. Affinity screening

[0044] DH5α cells were collected by centrifugation, resuspended in 200 μL lysis buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5% SDS), and treated with 20 μg / mL proteinase K at 55° C. for 2 h. Genomic DNA was extracted with phenol and chloroform. Genomic DNA was digested with Mnli,5'...CCTC(N)7...3', whic...

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Abstract

The invention discloses a method for screening and identifying stress response gene expression regulatory factors. The invention develops a method for enriching protein binding genome DNA fragments toconstruct a luciferase report library for identifying clones capable of responding to environmental stress. E.coil is used as a model system for preparing a cell lysis solution and the genome DNA fragments; the cell lysis solution is mixed with the genome DNA fragments, the protein-bound DNA fragments are enriched, are separated from non-bound DNA fragments by using a membrane rotating column andare used for making the luciferase report library, and after DH5[alpha] is transformed, resistant clones are screened on an Amp antibiotic board; mitomycin C or arsenite is used for treatment, more than two times of clones induced by luciferase are identified, and correlation is proved by further analysis; and therefore, the screening and identifying method is efficient and accurate.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for screening and identifying stress-responsive gene expression regulators. Background technique [0002] Microorganisms have strong adaptability to environmental toxic stresses such as heavy metals, pesticides, and polychlorinated biphenyls (PCBs), which is similar to their rapid response to environmental nutritional changes. Controls adaptation to changes in its environment by inducing or repressing gene expression. The binding or dissociation of transcription factors (TFs) from their DNA binding sites is a critical step in initiating the transcription of their target genes. It is critical to identify and characterize genes associated with environmental stress responses from the entire genome. This will help to understand the mechanism of gene regulation, and also help to identify the key regulators in the process of host environmental adaptation. However, m...

Claims

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Application Information

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IPC IPC(8): C12Q1/66C12N15/65
CPCC12Q1/66C12N15/65Y02A50/30
Inventor 李先强姜昕方云许玫英
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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