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Self-induced protein expression vector based on AI-2 quorum sensing and application

A quorum sensing and AI-2 technology, applied in the field of molecular biology, can solve the problems of inhibiting cell viability, high expression of exogenous genes, and affecting the viability of host strains, and achieve the effect of less inclusion bodies and high protein expression

Pending Publication Date: 2020-12-01
ANHUI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although many protein expression vectors exist at present, these vectors have the following disadvantages more or less: 1. Multiple copy numbers and strong promoters; own vitality
At the same time, the overexpression of the corresponding key enzymes causes the synthetic metabolic pathway of the product to form a congestion at the position of the key enzymes, which affects the vitality of the host strain itself.
2. IPTG induction is required; on the one hand, IPTG is expensive, and the cost of inducing expression is high. It is only suitable for small-scale sample preparation on a laboratory scale, and the cost of applying large-scale fermentation to produce recombinant genetic engineering drugs is high; on the other hand, IPTG It is difficult to completely remove in subsequent protein products, which limits its application in the production of food and pharmaceutical products
3. The selection range of host bacteria is narrow

Method used

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  • Self-induced protein expression vector based on AI-2 quorum sensing and application
  • Self-induced protein expression vector based on AI-2 quorum sensing and application
  • Self-induced protein expression vector based on AI-2 quorum sensing and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Construction of vector pXWZ-1

[0033] The primers were designed according to the sequence of the Escherichia coli lsrR promoter gene reported on NCBI, and the genome of the Escherichia coli model strain MG1655 with the lsr operon was used as a template. Use plsrR-Xba I-f, plsrR-Xba I-r: as primers to amplify the lsrR promoter sequence.

[0034] Upstream primer: plsrR-Xba I-f:5'-GC TCTAGA AATTCATTCTTCACTTTGAA-3' (the underline is the restriction site of XbaI, SEQ ID NO.3)

[0035] Downstream primer: plsrR-Xba I-r:5'-GC TCTAGA ATTTCCCCCGTTCAGTTTTG-3' (the underline is the restriction site of XbaI, SEQ ID NO.4)

[0036] The amplified lsrR promoter fragment and the pET28(a)+ plasmid were respectively digested with restriction endonuclease Xba I, incubated at 37°C for 2 hours for agarose gel electrophoresis and gel recovery, and the recovered DNA fragment was named as A1, A2. Mix A1 and A2 in a certain system, connect overnight with DNA ligase, and then tra...

Embodiment 2

[0037] Example 2 Construction of vectors pET28a-GFPuv and pXWZ-1-GFPuv

[0038] Extract the pGFPuv plasmid, use the pGFPuv plasmid as a template, gfp-EcoR I-f, gfp-Hind III-r as primers, PCR amplify the GFPuv fragment, connect it to the vector pET28(a)+ and pXWZ-1 respectively, and construct the expression vector pET28a- GFPuv and pXWZ-1-GFPuv.

[0039] Upstream primer: gfp-EcoR I-f:5'-CCG GAATTC ATGAGTAAAGGAGAAGAACT-3' (underlined is the restriction site of EcoR I)

[0040] Downstream primer: gfp-Hind III-r:5'-CCC AAGCTT TTATTTGTATAGTTCATCCA-3' (the restriction site of Hind III is underlined)

[0041] The amplified GFPuv fragment, pET28(a)+ plasmid, and pXWZ-1 plasmid were respectively digested with restriction endonucleases EcoR I and Hind III, incubated at 37°C for 2 hours for agarose gel electrophoresis and gel recovery. The recovered DNA fragments were named B1, B2, and B3, respectively. B1 and B2, B1 and B3 were mixed in a certain system, connected overnight with ...

Embodiment 3

[0042] Embodiment 3 expresses green fluorescent protein

[0043] 1. Construction of engineered bacteria

[0044] The expression vectors pET28a-GFPuv and pXWZ-1-GFPuv were respectively transformed into Escherichia coli BL21(DE3) by chemical transformation, and spread on LB solid plates containing 50 μg / mL kanamycin. Incubate overnight in a 37°C incubator. Single clonal colonies were selected for PCR identification.

[0045] 2. Using a fluorescence microscope to monitor the fluorescence intensity of GFPuv

[0046] The BL21(DE3) bacterial solution containing the expression vectors pET-GFPuv and pXWZ-1-GFPuv was transferred to 100 mL fresh LB medium containing 50 μg / mL kanamycin at a ratio of 1:100 (v / v). Marked as ①, ②, ③ respectively. Among them, ① sample is BL21 / pET28-GFPuv, ② sample is BL21 / pET28-GFPuv induced by adding final concentration of 0.5mM IPTG, and ③ sample is BL21 / pXWZ-1-GFPuv. Cultivate at 37°C with shaking at 150rpm until OD600=0.3, add 0.5mM IPTG to ②, and p...

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Abstract

The invention discloses a self-induced protein expression vector based on AI-2 quorum sensing and application. A promoter used by the self-induced expression vector is an LsrR promoter which can be activated by an AI-2 quorum sensing system of escherichia coli, and can be activated by AI-2 signal molecules generated by bacteria to automatically start expression of a target gene when the escherichia coli grows to a logarithmic phase. The promoter sequence of the LsrR is shown as SEQ ID NO.1. The invention provides a construction method of the novel expression vector and application of the novelexpression vector in protein expression. Compared with a pET expression vector needing IPTG induction, the self-induction pXWZ-1 expression vector constructed by introducing the LsrR promoter has theadvantages that an inducer does not need to be added, economy and safety are achieved, the protein expression quantity is high, few inclusion bodies are formed, and low-temperature induction is not needed.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and specifically relates to the cloning of an autoinducible promoter, and the construction and application of an autoinducible protein expression vector based on the above-mentioned promoter. Background technique [0002] The traditional E. coli protein expression system needs to select a suitable expression vector to promote efficient and high-quality protein expression. Although many protein expression vectors exist at present, these vectors have the following disadvantages more or less: 1. Multiple copy numbers and strong promoters; its own vitality. At the same time, the overexpression of the corresponding key enzymes causes the synthetic metabolic pathway of the product to form a congestion at the position of the key enzymes, which affects the vitality of the host strain itself. 2. IPTG induction is required; on the one hand, IPTG is expensive, and the cost of inducing expression...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/113C12N15/66C12N15/65C12N1/21C12R1/19
CPCC12N15/70C12N15/66C12N15/65C07K14/245C12N2800/60
Inventor 薛挺汪晖汪瑾王文辉张丹
Owner ANHUI AGRICULTURAL UNIVERSITY
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