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Strong promoter and plasmid vector containing strong promoter and application of strong promoter and plasmid vector

A technology of strong promoter and plasmid vector, applied in the biological field, can solve the problem of limited number of efficient promoters

Active Publication Date: 2019-12-24
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, the number of highly efficient promoters available in α-proteobacteria is very limited

Method used

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  • Strong promoter and plasmid vector containing strong promoter and application of strong promoter and plasmid vector
  • Strong promoter and plasmid vector containing strong promoter and application of strong promoter and plasmid vector
  • Strong promoter and plasmid vector containing strong promoter and application of strong promoter and plasmid vector

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: The activity determination of promoter P21 in different strains

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

[0031] Taking S. meliloti as an example, the plasmid pBBR-P21-gfp in Example 1 was transferred into S. meliloti according to the three-parent method to obtain S. meliloti: SM / pBBR-P21-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 aga...

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Abstract

According to the invention, through amplifying a strong promoter in Ensifer adhaerens and carrying out bioinformatics analysis and functional verification, a strong promoter is obtained, and the strong promoter can be widely used for gene expression, genetic manipulation and strain improvement in Sinorhizobium meliloti, Zymomonas mobilis, Caulobacter crescentus, Pseudomonas denitrificans, Agrobacterium tumefaciens, Brucella abortus, Pseudomonas fluorescens, Rhizobium leguminosarum, Ensifer adhaerens and other alpha-proteobacteria and has a nucleotide sequence of SEQ ID NO: 1. The invention also relates to a plasmid vector containing the strong promoter, a method for constructing a genetic engineering strain by using the strong promoter, and an application of the corresponding strain and ahost cell in starting expression of a target 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] 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 synthesis pathway or the original p...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/74C12N15/65C12N1/21C12R1/01C12R1/37C12R1/38C12R1/39C12R1/41
CPCC07K14/195C12N15/65C12N15/74
Inventor 张大伟董会娜
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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