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Strong promoter and application thereof in vitamin B<12>-producing strains

A technology of strong promoter and promoter, applied in the application of vitamin B12-producing strains, plasmids and transformants containing the plasmid vector, in the field of strong promoters, can solve the problem of limited number of efficient promoters and the like

Active Publication Date: 2020-04-21
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, vitamin B 12 Very limited number of high-efficiency promoters available in strains

Method used

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  • Strong promoter and application thereof in vitamin B&lt;12&gt;-producing strains
  • Strong promoter and application thereof in vitamin B&lt;12&gt;-producing strains
  • Strong promoter and application thereof in vitamin B&lt;12&gt;-producing strains

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Construction of a promoter-containing plasmid vector

[0022] 1. Preparation of vector containing reporter gene

[0023] Using the primers gfp-EcoRI-F and gfp-KpnI-R in Table 1, the ECE164 plasmid (Yafeng Song Jonas M. Nikoloff Gang Fu, Jingqi Chen, Qinggang Li, Nengzhong Xie, Ping Zheng, Jibin Sun, Dawei Zhang*. Promoter screening from Bacillus subtilis in various conditions hunting for synthetic biology and industrial applications. PLoS One .2016Jul 5; 11(7):e0158447.doi:10.1371 / journal.pone.0158447.) was used as a template, amplified by PCR, and introduced EcoRI and KpnI restriction sites to obtain the gfp fragment of the green fluorescent protein gene. After verification by electrophoresis, DpnI enzymatic treatment, and electrophoresis gel recovery, the purified gfp fragment was obtained. The purified gfp fragment and the pBBR1MCS2 plasmid were double-digested with EcoRI and KpnI, respectively, and the two double-digested products were ligated ove...

Embodiment 2

[0029] Example 2: Promoter P29 produces vitamin B in different 12 Activity assays in strains

[0030] 1. Transformation—three-parent transformation method

[0031] Taking S. meliloti as an example, the plasmid pBBR-P29-gfp in Example 1 was transferred into S. meliloti according to the three-parent method to obtain S. meliloti: SM / pBBR-P29-gfp. Specific steps are as follows:

[0032] (1) Inoculate the newly activated Sinorhizobium meliloti CGMCC NO.9638, Escherichia coli (containing the corresponding plasmid) and the helper vector MT616, and culture them in an incubator at 30°C and 37°C with shaking until the OD value is about 1.0;

[0033] (2) Under aseptic conditions, transfer 500 μL each of Sinorhizobium meliloti CGMCC NO.9638, MT616 and Escherichia coli to 1.5 mL sterile EP tubes and centrifuge at 12,000 rpm for 1 min at 4°C.

[0034] (3) Discard the supernatant under aseptic conditions, and suspend the precipitate with 1 mL of 0.85% sterile saline.

[0035] (4) Centrifug...

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Abstract

The invention provides a strong promoter and application thereof in vitamin B<12>-producing strains. According to the invention, a strong promoter in Ensifer adhaerens is amplified; bioinformatics analysis and functional verification are carried out to obtain the strong promoter which can be widely applied to the vitamin B<12>-producing strains such as Sinorhizobium meliloti, Pseudomonas denitrificans and Ensifer adhaerens for gene expression, genetic gene operation and strain improvement, and the nucleotide sequence of the obtained strong promoter is SEQ ID NO: 1. The invention also relates to a plasmid vector containing the strong promoter, a method for constructing a gene engineering strain by using the promoter and a corresponding strain thereof, and application of a host cell in promoting expression of a target gene.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a strong promoter, in particular to a strong promoter isolated and cloned from S. cohescens, a plasmid containing the strong promoter and a transformant containing the plasmid vector, and their In terms of heterologous or homologous protein expression and vitamin B production 12 Application of strains. Background technique [0002] The promoter is a part of the gene that can be recognized by RNA polymerase and initiate the transcription of the gene behind the promoter. The strength of the promoter has a direct impact on the expression efficiency of foreign genes in gene manipulation. Metabolic engineering often needs to express exogenous genes or regulate the expression of endogenous genes, and the choice of promoter is crucial to the regulation of gene expression. The promoter affects the transcription level of the gene, affects the coordination between the genes in the artificial s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/78C12N15/74C12N15/65C12N1/21C12P19/42C12R1/38C12R1/01
CPCC07K14/195C12N15/78C12N15/74C12N15/65C12P19/42
Inventor 张大伟董会娜
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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