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The strong promoter derived from the cohesin bacteria and its plasmid vector and application

A plasmid vector, strong promoter technology, used in vectors, viruses/phages, microorganism-based methods, etc.

Active Publication Date: 2021-09-07
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

[0004] However, due to the fact that there are very few studies on S. adhesica, the development and research of strong promoters in S. adhesica is rarely reported.

Method used

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  • The strong promoter derived from the cohesin bacteria and its plasmid vector and application
  • The strong promoter derived from the cohesin bacteria and its plasmid vector and application
  • The strong promoter derived from the cohesin bacteria and its plasmid vector and application

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] Utilize the primers gfp-EcoRI-F and gfp-XhoI-R in Table 1, and use the ECE164 plasmid as a template to amplify by PCR and introduce EcoRI and XhoI 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 pBBR1MCS2 plasmid were double-digested with EcoRI and XhoI respectively, and the two double-digested products were ligated overnight at 4°C with T4 ligase. The ligation product was transformed into Escherichia coli DH5α, spread on the LB solid plate containing 50mg / L kanamycin, cultured for 16 hours, then carried out colony PCR detection, and sent to Jinweizhi for sequencing. After the sequence was correct, the obtained positive bacteria were name...

Embodiment 2

[0029] Embodiment 2: Activity determination of promoter P18 in different strains

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

[0031] Taking S. meliloti as an example, the plasmid pBBR-P18-gfp in Example 1 was transferred into S. meliloti according to the three-parent method to obtain S. meliloti: SM / pBBR-P18-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 shake culture in the incubator at 30°C and 37°C respectively 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) Centrifuge again a...

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Abstract

The invention discloses a strong promoter derived from the cohesin bacterium and its plasmid carrier and application. In the present invention, by amplifying a strong promoter in S. cohescens and performing functional verification, the obtained promoter can be widely used in Sinorhizobium meliloti, Zymomonas mobilis, Caulobacter crescentus, Pseudomonas denitrification, tumorigenic A strong promoter for gene expression, genetic manipulation and strain improvement in α-proteobacteria such as Agrobacterium abortus, Brucella abortus, Pseudomonas fluorescens, Rhizobium leguminosae and Sword bacteria adherent, its nucleotide sequence is SEQ ID NO: 1. The present invention also relates to a plasmid vector containing the strong promoter, a method for constructing a genetically engineered bacterial strain using the promoter, a corresponding bacterial strain, and the application of host cells in promoting the expression of an objective gene.

Description

[0001] Technical field: the present 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, and to a plasmid containing the strong promoter and a transformant containing the plasmid vector , and their application in heterologous or homologous protein expression. Background technique: [0002] 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 synthesis pathway or the original pathway, and then affects the metabolic function of the strain. Promoters are divided into constitutive promoters and specific promoters. Constitutive promoters can be transcribed in all cells at any time, the regulation of constitutive promoters is not aff...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/74C12N1/21C12R1/19
CPCC07K14/195C12N2830/34
Inventor 张大伟董会娜
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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