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A microbial inducible gene expression regulation system

A technology of microorganisms and genes, applied in the introduction of foreign genetic material, DNA/RNA fragments, recombinant DNA technology, etc. using vectors, can solve the problems of short recognition sequences, affecting applications, and high off-target rate of non-specific recognition frequency

Active Publication Date: 2018-07-27
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the specificity of the type II intron gene targeting operation technology depends on the recognition of specific sequences by the RNP complex, and the recognition sequences are short, usually several to more than ten bases, resulting in non-specific recognition frequency (i.e. off-target rate) is high, seriously affecting its application in precise gene targeting

Method used

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  • A microbial inducible gene expression regulation system
  • A microbial inducible gene expression regulation system
  • A microbial inducible gene expression regulation system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1. Construction of an arabinose induction system

[0031] We chose Clostridium acetobutylicum phosphoketolase promoter and AraR repressor expression cassette to construct the arabinose induction system. The 370 bp upstream of the phosphoketolase gene was selected as the promoter to ensure the inclusion of the AraR binding region. The araR expression cassette uses its own predicted promoter and terminator and is self-regulating.

[0032] The construction of the arabinose induction system includes:

[0033] 1) The structural gene (SEQ ID NO: 1) and its promoter region of the feedback inhibitory protein AraR were obtained by PCR amplification araR (SEQ ID NO: 3) and terminator region T araR (SEQ ID NO: 4);

[0034] 2) The promoter element P with feedback inhibitor protein binding site was obtained by PCR amplification ara (SEQ ID NO: 2);

[0035] 3) The above elements are integrated into a plasmid backbone with a resistance gene by gene cloning, and a restr...

Embodiment 2

[0037] Example 2. Electrotransformation of Clostridium cellulolyticum

[0038] According to the literature (J Microbiol Methods 2012;89:201-8), the transformation method is as follows: Clostridium cellulolyticum cells are grown in 100mL GS-2 liquid medium at 34°C to OD600=0.4-0.8, 3000g, 10 Minutes of centrifugation to collect the bacteria, using electroporation buffer (0.275 mol / L sucrose, 5mmol / LK 2 HPO 4 ) after washing twice, and set the volume to 1 mL, take 200 μL of bacterial liquid and 1 μg of plasmid, mix them into a 0.2 cm electro-cup for electric shock. The parameters of the electrorotator were set as follows: voltage 1200, frequency 2000, duty cycle 10%, and number of electric shocks 40. The bacteria solution after electrotransformation was added to 5 mL of fresh GS-2 medium, and incubated at 34°C for 6 hours. The revived cells were centrifuged and spread on GS-2 solid plates containing erythromycin antibiotics, and cultured under anaerobic conditions at 34°C unt...

Embodiment 3

[0039] Example 3. Induced expression of anaerobic fluorescent protein PpFbFPm in Clostridium cellulolyticum

[0040] We chose PpFbFPm as a reporter gene (J Microbiol Methods 2012;89:201-8) to demonstrate that the arabinose induction system is available in the Clostridium cellulolyticum H10 strain.

[0041] PpFbFPm is connected to the P of pARA plasmid through NheI and SalI restriction sites ara Downstream, the plasmid pARA-PpFbFPm was obtained. By the method of gene cloning, the structural gene of AraR on the plasmid pARA-PpFbFPm and its promoter region P araR and terminator region T araR If removed, the plasmid pPTK-PpFbFPm is obtained. Contains P ara - The pARA-PpFbFPm plasmid of the PpFbFPm and AraR expression cassettes was used to demonstrate the expression of PpFbFPm regulated by the arabinose-inducible system in Clostridium cellulolyticum. The control plasmid pPTK-PpFbFPm does not have the AraR expression cassette and is used to constitutively express PpFbFPm (Fig....

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Abstract

The object of the present invention is to provide an inducible promoter, a gene induction expression control system based on the promoter, and an improved microbial genetic manipulation tool developed based on the expression control system. Using L-arabinose as the inducer, arabinose is a common carbon source in nature and has no inhibitory effect on cell growth. The inducible expression system can up-regulate the gene expression level up to 800 times, and has good stringency, and can be used for the controllable expression of the target gene in Clostridium or other microbial cells, as well as for the existing genetic modification technology Optimization and development of new tools.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for regulating and controlling microbial gene expression. Background technique [0002] The regulation of genes at the transcriptional level is the main way of regulating gene expression in microorganisms, and it is also a research hotspot in microbial metabolic engineering. Transcriptional regulation is controlled by a variety of cis-acting elements and trans-acting factors, among which the promoter is an important cis-acting element, which basically determines whether, when and where a gene is expressed. The promoter is usually located upstream of the 5' end of the functional gene, and can combine with trans elements such as RNA polymerase and other protein cofactors to control the initiation and efficiency of gene transcription. Microbial promoters can be divided into constitutive promoters and inducible promoters. Constitutive promoters can initiate transcr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/63
Inventor 崔球张杰刘亚君崔古贞
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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