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Novel MR-1 gel bead for detecting explosive molecules as well as preparation method and application of novel MR-1 gel bead

An MR-1, explosive technology, applied in the fields of genetic engineering and molecular biology, can solve problems such as difficulties, high activity of sensor strains cannot be guaranteed, and real-time detection of explosives increases.

Active Publication Date: 2021-09-14
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, they also immobilized the strain carrying the promoter in the beads, but it took a long time for the beads to sense the explosive molecules to generate fluorescent signals, and the high activity of the sensor strain in the beads could not be guaranteed during this period. Furthermore, when eGFP is used as a reporter gene for detection, the fluorescence of the strain needs to be irradiated by an exciting light source, which undoubtedly adds difficulties to the outdoor real-time detection of explosives.

Method used

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  • Novel MR-1 gel bead for detecting explosive molecules as well as preparation method and application of novel MR-1 gel bead
  • Novel MR-1 gel bead for detecting explosive molecules as well as preparation method and application of novel MR-1 gel bead
  • Novel MR-1 gel bead for detecting explosive molecules as well as preparation method and application of novel MR-1 gel bead

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: the acquisition of gene and the construction of vector

[0036] 1. Acquisition of genes

[0037] One from Qinghai Vibrio Q67 ( Vibrio qinghaiensis Q67) luxABCDE operon, the nucleotide sequence of which is shown in SEQ ID NO.1, was chemically synthesized by BGI Corporation into pUC-57 vector to obtain pUC-luxQ67 vector.

[0038] The Pdnt-2 promoter gene, whose nucleotide sequence is shown in SEQ ID NO.2, was chemically synthesized by Huada Gene Co., Ltd. into the pUC-57 vector to obtain the pUC-Pdnt-2 vector.

[0039] 2. Construction of p-luxQ67 expression vector

[0040]Using pUC-luxQ67 as a template, primer luxQ67-F and primer luxQ67-R, perform polymerase chain reaction (PCR) to amplify the luxQ67 fragment. The PCR amplification system is as follows:

[0041]

[0042] The PCR program was: 95ºC 3 min; 30 cycles × (95ºC 15 s, 58ºC 15 s, 72ºC 4 min); 72ºC 5min; 16ºC ∞.

[0043] The primer sequences are as follows:

[0044] luxQ67-F:

[0045] 5'-...

Embodiment 2

[0071] Embodiment 2: the construction of biosensor

[0072] Transform the p-Pdnt-2-LuxQ67 recombinant plasmid Escherichia coli BW25113 competent cells (purchased from Weidi Biotechnology, product number DL2050), were spread on LB solid plates containing 34 mg / L chloramphenicol, and positive clones were obtained by PCR screening, thereby obtaining vector p-Pdnt-2-luxQ67 The engineering strain MR-1.

Embodiment 3

[0073] Embodiment 3: The preparation of the novel condensed beads of detection engineering strain MR-1

[0074] 1. Activation and cultivation of strains

[0075] The engineered strain MR-1 with correct sequencing was inoculated into 10 mL LB medium supplemented with 10 µL of Kanna, cultured overnight at 37 °C with shaking (200 rpm), and then the culture was transferred to a new 10 mL LB medium at 2%. medium and grown under the same conditions to an optical density of 600 nm (OD 600 ) is 0.8.

[0076] 2. Preparation of related solutions for immobilized bacteria

[0077] (1) Preparation of 2.2 % (w / v) sodium alginate solution: Weigh 1.1 g of sodium alginate solid, dissolve it in deionized water to 50 mL, stir the solution until all the sodium alginate is dissolved, and then Add agar powder to the mixed sodium alginate at 1.1%, and sterilize by high pressure steam.

[0078] (2) 9% polyacrylic acid (PAA) solution (35% polyacrylic acid, molecular weight 250000): Take 9 mL of 35...

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Abstract

The invention discloses a novel MR-1 gel bead for detecting explosive molecules as well as a preparation method and application of the novel MR-1 gel bead. The novel MR-1 gel bead is prepared by the following steps of amplifying and recombining a report element of a self-luminous operon luxABCDE operon and a sensing element of a promoter Pdnt-2, transfecting competent cells, and stirring with a sodium alginate-agar-polyacrylic acid solution and a calcium chloride solution until complete gelation. The novel MR-1 gel bead can sense explosive molecules 2, 4-dinitrotoluene (2, 4-DNT) with different concentrations and generate fluorescence with different intensities, the concentration of the explosive molecules can be obtained simply through the fluorescence intensity, and then real-time fluorescence detection of the explosive molecules is achieved. The novel MR-1 gel bead is simple in construction method, convenient to operate and store, high in safety and capable of prolonging the activity maintaining time of engineering strains and improving the safety and efficiency of explosive molecule detection.

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering and molecular biology, and in particular relates to a novel MR-1 bead for detecting explosive molecules and its preparation method and application. Background technique [0002] Rapid, efficient, and safe detection of DL and other explosives in the environment is of great strategic significance to my country's national defense security and social stability. The current traditional landmine detection methods cannot realize out-of-position detection, and have certain limitations in terms of safety and accuracy. Among other things, the main limiting factor hampering global demining efforts is not actually removing buried mines, but determining their exact location. Most of the current landmine detection methods require inspectors to enter the minefield for detection, which is very dangerous. Therefore, biosensors are proposed as a possible alternative. Biosensors can sense explosive mo...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/31C12N15/113C12N15/70C12N15/66C12N11/10C12N11/082G01N21/64C12R1/19
CPCC07K14/28C07K14/245C12N15/70C12N15/66C12N11/10C12N11/082G01N21/6428G01N21/643
Inventor 杨建明王兆宝马冉李美洁梁波汤若昊
Owner QINGDAO AGRI UNIV
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