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Preparation method for synthesizing microbial self-luminous biosensor by using self-luminous operon, corresponding biosensor and application thereof

A biosensor and self-luminous technology, applied in the fields of genetic engineering and molecular biology, can solve problems such as complexity, interference signals, and unfriendly applications, and achieve the effects of simple operation, high sensitivity, and good application prospects

Active Publication Date: 2021-06-22
QINGDAO AGRI UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This biosensor uses GFP as the reporting element. When detecting explosives in the field, it is necessary to use a special instrument for ultraviolet wavelength excitation and green fluorescence collection, which is not friendly to practical applications.
In addition, the field situation is more complicated, and many non-GFP substances can emit green fluorescence under the excitation of ultraviolet light, thereby generating interference signals

Method used

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  • Preparation method for synthesizing microbial self-luminous biosensor by using self-luminous operon, corresponding biosensor and application thereof
  • Preparation method for synthesizing microbial self-luminous biosensor by using self-luminous operon, corresponding biosensor and application thereof
  • Preparation method for synthesizing microbial self-luminous biosensor by using self-luminous operon, corresponding biosensor and application thereof

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Effect test

Embodiment 1

[0039] Embodiment 1: the acquisition of gene and the construction of vector

[0040] 1. Acquisition of genes

[0041]A luxABCDE operon derived from Photobacterium leiognathi (Photobacterium leiognathi), whose nucleotide sequence is shown in SEQ ID NO.1, was chemically synthesized onto the pUC-57 vector by BGI Corporation to obtain the pUC-lumPleio vector .

[0042] A luxABCDE operon derived from Aliivibrio fischeri, whose nucleotide sequence is shown in SEQ ID NO.2, was chemically synthesized by Huada Gene Company onto the pUC-57 vector to obtain the pUC-lumVf vector .

[0043] The 114 promoter gene, whose nucleotide sequence is shown in SEQ ID NO.3, was chemically synthesized by Huada Gene Company into pUC-57 vector to obtain pUC-114 vector.

[0044] 2. Construction of p-lumPleio and p-lumVf expression vectors

[0045] (1) Using pUC-lumPleio as a template, primer LuxC-F and primer LuxG-R, perform polymerase chain reaction (PCR) to amplify the lumPleio fragment. The PCR am...

Embodiment 2

[0105] Embodiment 2: the construction of biosensor

[0106] The p-114-lumPleio recombinant plasmid was transformed into Escherichia coli BW25113 competent cells (purchased from Weidi Biotechnology, product number DL2050), spread on the LB solid plate containing 34 mg / L chloramphenicol, and obtained positive clones by PCR screening, thus The engineering strain XLUM1 containing the vector p-114-lumPleio was obtained.

[0107] The p-114-lumVf recombinant plasmid was transformed into Escherichia coli BW25113 competent cells (purchased from Weidi Biotechnology, product number DL2050), spread on the LB solid plate containing 4 mg / L chloramphenicol, and obtained positive clones by PCR screening, thus The engineering strain XLUM2 containing vector p-114-lumVf was obtained.

Embodiment 3

[0108] Embodiment 3: The application of microbial self-luminescence biosensor to detect explosive molecules

[0109] 1. Strain activation and cultivation

[0110] The engineering strains XLUM1 and XLUM2 with correct sequencing were respectively transferred to LB liquid medium containing 34 mg / L chloramphenicol, and cultured overnight at 37°C to obtain bacterial liquid.

[0111] Add 330 μL 60% glucose, 10 μL 1 M magnesium sulfate stock solution, and 200 μL bacterial solution to 10 mL of M9 liquid medium containing 34 mg / L chloramphenicol, culture on a shaker at 37 ° C, and grow to OD 600 = about 0.2.

[0112] 2. Preparation of 2,4-DNT solution

[0113] Prepare 20mg / mL mother solution 2,4-DNT (100mg2,4-DNT dissolved in 5mL absolute ethanol);

[0114] Prepare the diluted 2,4-DNT solution in the following proportions:

[0115] 100mg / L: 5μL stock solution + 980μL M9 medium + 15μL absolute ethanol;

[0116] 50mg / L: 2.5μL mother solution + 980μL M9 medium + 17.5μL absolute ethan...

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Abstract

The invention discloses a preparation method for synthesizing a microbial self-luminous biosensor by using a self-luminous operon, and a corresponding biosensor and application thereof. The microbial self-luminous biosensor comprises a reporting element containing a self-luminous operon luxABCDE operon and a sensing element containing a promoter 114. The microbial self-luminous biosensor can sense explosive molecules with different concentrations to generate self-luminescence with different RLU values, and the concentration of the explosive molecules and the self-luminous RLU values are coupled, so that real-time fluorescence detection of the explosive molecules is realized, and the defect that luciferase is required to generate fluorescence for detection can be overcome. Compared with other detection methods, the microbial self-luminous biosensor has the advantages that the operation of detecting the explosive molecules is simpler and more convenient, the signal collection is easier, and the explosive molecules with a wider concentration range can be detected, so that the microbial self-luminous biosensor has a very good application prospect.

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering and molecular biology, and in particular relates to a preparation method for synthesizing a microbial self-luminescence biosensor by using a self-luminescence operon, a corresponding biosensor and an application thereof. Background technique [0002] Using the idea of ​​synthetic biology, by mining the biological recognition elements sensitive to specific compounds in nature, connecting them with reporter elements to form gene circuits, and transforming them into model microorganisms with clear genetic background and simple operation (such as Escherichia coli), to construct and obtain microorganisms sensor. The microbial sensor can be used in environmental pollution detection, food detection and other fields. Among them, biological recognition elements include promoters responsible for gene transcription, ribosome binding sites, terminators, transcriptional regulatory proteins, etc.,...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/113C12N15/31C12N15/66C12Q1/02G01N21/64C12R1/19
CPCC07K14/195C07K14/28C12N15/113C12N15/66C12N15/70C12Q1/02G01N21/6428
Inventor 杨建明李美洁吕书喆王兆宝
Owner QINGDAO AGRI UNIV
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