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Microbe-inducible gene expression control system

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

Active Publication Date: 2016-04-13
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Abstract
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  • 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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  • Microbe-inducible gene expression control system
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  • Microbe-inducible gene expression control system

Examples

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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 370bp 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 P 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 restric...

Embodiment 2

[0037] Example 2. Electrotransformation of Clostridium cellulolyticum

[0038] According to the literature (JMicrobiolMethods2012;89:201-8), the transformation method is as follows: Clostridium cellulolyticum cells are grown to OD600=0.4-0.8 in 100mLGS-2 liquid medium at 34°C, collected by centrifugation at 3000g for 10 minutes Body, using electroporation buffer (0.275mol / 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 until colon...

Embodiment 3

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

[0040] We chose PpFbFPm as a reporter gene (JMicrobiolMethods 2012;89:201-8) to demonstrate that the arabinose induction system is available in 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 has no AraR expression cassette and is used to express PpFbFPm constitutively ( figure 2 A). The ...

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Abstract

The invention aims to provide an inducible promoter, a gene induced expression control system based on the inducible promoter, and an improved microbial genetic operation tool developed based on the expression control system. L-Arabinose is used as an inducing agent. Arabinose is a carbon source common in the nature and free of inhibitory action on cell growth. The induced expression system enables gene expression level to be up-regulated up to 800 times, is better in preciseness and can be used in controlled expression of target genes in Fusiformis or other microbial cells and to optimize existing genetic modification technology and develop 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 Applications(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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