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Xylose-induced promoter and plasmid vector and application thereof

A technology of plasmid vector and promoter, applied in the direction of vector, using vector to introduce foreign genetic material, virus/phage, etc., can solve problems such as poor expression effect

Active Publication Date: 2019-12-20
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

P rhaR The expression effect is the worst (Mostafavi,Lewis et al.2014)

Method used

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  • Xylose-induced promoter and plasmid vector and application thereof
  • Xylose-induced promoter and plasmid vector and application thereof
  • Xylose-induced promoter and plasmid vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Construction of a promoter-containing plasmid vector

[0037] 1. Preparation of vector containing reporter gene

[0038] 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

[0049] Example 2: Activity determination of xylose-inducible promoter in Escherichia coli

[0050] LB medium components: 1% tryptone, 0.5% yeast extract, 1% sodium chloride.

[0051] Escherichia coli bacterial strain E.coli / pBBR-P among the embodiment 1 A -gfp, E.coli / pBBR-P B -gfp and E.coli / pBBR-gfp (Control), after streaking on the LB solid plate containing 50mg / L kanamycin for activation, pick a single colony and inoculate it into 5mL LB containing 50mg / L kanamycin In liquid culture medium, culture at 200r / min, 37°C for 16h. Transfer the above-mentioned bacterial solution to a 24-well plate containing 1.8 mL of LB liquid medium containing 50 mg / L kanamycin with an inoculum amount of 1%, and place it in a well plate shaker at 37 ° C, 700 rpm, 80% Humidity shaking culture, wait for OD 600 When it reaches 0.4-0.6, correspondingly add a certain amount of xylose inducer to induce, so that the final concentration of the inducer is 0% and 1%, respectively, and regularly take ...

Embodiment 3

[0052] Example 3: Determination of the activity of arabinose and xylose-induced promoters in Sinorhizobium meliloti CGMCCNO.9638

[0053] 1. Transformation—three-parent transformation method

[0054] With 3 plasmids pBBR-P in embodiment 1 araA -gfp, pBBR-P A -gfp, pBBR-P B -gfp was transferred into Sinorhizobium meliloti CGMCC NO.9638 according to the following method. Get S. meliloti: SM / pBBR-P araA -gfp, SM / pBBR-P A -gfp, SM / pBBR-P B -gfp.

[0055] (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;

[0056] (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.

[0057] (3) Discard the supernatant under aseptic conditions, and susp...

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Abstract

The invention discloses a xylose-induced promoter. By amplifying a xylose-induced promoter in sinorhizobium meliloti, bioinformatics analysis and function verification are carried out, the xylose-induced promoter which can be widely applied to gene expression, genetic gene operation and strain improvement in alpha-proteobacteria such as Sinorhizobium meliloti, Zymomonas mobilis, Caulobacter crescentus, Pseudomonas denitrificans, Agrobacterium tumefaciens, Brucella abortus, Pseudomonas fluorescens, Rhizobium leguminosarum and Sinorhizobium adhaerens is obtained, and the nucleotide sequence of the promoter is SEQ ID NO: 1 or SEQ ID NO: 2. The invention also relates to a plasmid vector containing the xylose-induced promoter, a method for constructing a genetic engineering strain by using thepromoter, a corresponding strain, and application of a host cell to induction and starting of expression of a target gene under xylose inducible conditions.

Description

[0001] Technical field: the invention belongs to the field of biotechnology, and relates to a xylose-inducible promoter and its plasmid vector and application Background technique: [0002] Promoters are divided into constitutive promoters (Constitutive promoter) and specific promoters (Specific promoter). Constitutive promoters can be transcribed in all cells at any time; specific promoters can be further divided into tissue-specific promoters and inducible promoters. Inducible promoters can specifically induce the expression of target genes, and produce a large number of specific products in organisms, so as to make regulatory responses, adapt to the external environment or increase the production of metabolites. [0003] S. meliloti is a non-type strain. At present, there are few studies on the inducible promoter of S. meliloti. Mostafavi et al. studied the P araA ,P tauA ,P rhaR and P melA 4 promoters and compared their functions, P araA The promoter region is locat...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/74C12N1/21C12R1/01
CPCC07K14/195C12N15/74C12N2830/34
Inventor 张大伟董会娜吴香莹刘振权
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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