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Novel photosynthetic bacteria for detecting explosives as well as preparation method and application of novel photosynthetic bacteria

A technology of photosynthetic bacteria, explosives, applied in the fields of genetic engineering and molecular biology

Active Publication Date: 2021-09-10
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the use of biosensing technology to detect landmines has made some progress, the unit of detection limit is still micrograms / liter, but the concentration of 2,4-DNT in volatile gas is only nanograms / liter, so it is urgent to improve the sensitivity to meet the actual detection needs

Method used

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  • Novel photosynthetic bacteria for detecting explosives as well as preparation method and application of novel photosynthetic bacteria
  • Novel photosynthetic bacteria for detecting explosives as well as preparation method and application of novel photosynthetic bacteria
  • Novel photosynthetic bacteria for detecting explosives as well as preparation method and application of novel photosynthetic bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: the construction of gene expression vector

[0038] The amino acid sequences of the transcriptional regulatory proteins of Rhodopseudomonas palustris are shown in SED ID NO.1 and SED ID NO.2 respectively, and the upstream promoters are found according to the positive two amino acid sequences:

[0039] 1-4 The promoter gene fragment, whose nucleotide sequence is shown in SED ID NO.3, was chemically synthesized by Suzhou Jinweizhi Co., Ltd. onto the pUC-GW-Kan vector to obtain pUC- 1-4 carrier.

[0040] The 1-11 promoter gene fragment, whose nucleotide sequence is shown in SED ID NO.4, was chemically synthesized by Suzhou Jinweizhi Co., Ltd. onto the pUC-GW-Kan vector to obtain pUC- 1-11 carrier.

[0041] Green Fluorescent Protein Encoding Gene Fragment gfp , whose nucleotide sequence is shown in SED ID NO.5, was chemically synthesized by Suzhou Jinweizhi Company and subcloned into pUC- 1-4 and pUC- 1-11 On the vector, obtain pUC- 1-4 - gfp and pUC- ...

Embodiment 2

[0081] Example 2. Construction of Rhodopseudomonas palustris biosensor

[0082] 1. Preparation of Rhodopseudomonas palustris Competent

[0083] One night in advance, add 200 μL of bacterial solution into 10 mL of PM medium, put it in a constant temperature light incubator, measure OD the next morning, and culture until OD≈0.5 to stop the culture.

[0084] The volume of PM medium is 2.5 L. According to the formula of PM medium, Na 2 HPO 4 4.43625g, KH 2 PO 4 4.2528125g, (NH 4 ) 2 SO 4 2.5g, Na 2 S 2 o 3 5H 2 O 0.0622g, p-aminobenzoic acid 0.005g.

[0085] Prepare a bottle of sterilized water, a bottle of sterilized glycerin, two 50 mL round-bottom centrifuge tubes, one 15 mL centrifuge tube, and some sterile 10 mL gun tips.

[0086] Cool all preparations (except for pipette tips, including bacteria) on ice while pre-chilling the refrigerated centrifuge at 4°C.

[0087] Take 4.5 mL of sterile water and 500 μL of glycerol into a 15 mL centrifuge tube, blow and aspi...

Embodiment 3

[0095] Example 3. Detection of 2,4-DNT by Rhodopseudomonas palustris biosensor

[0096] 1. Inoculate Rhodopseudomonas palustris (containing pBBR1MCS-5-1-4- gfp and pBBR1MCS-5-1-11- gfp Plasmid vector), the OD was measured at about 0.3 the next day, and 2,4-DNT was added, and the final concentration was divided into 0 mg / L, 0.05 mg / L, 0.01 mg / L, 0.005 mg / L, 0.001 mg / L, each Each gradient has 3 parallels. Bacteria were taken at 0 h, 2 h, 4 h, 6 h, 8 h, and 10 h to measure the fluorescence. The excitation light was 485 nm, and the emission light was 525 nm.

[0097] The result is as figure 2 and 3 As shown, the fluorescence value gradually increased with the prolongation of culture time. When the concentration of 2,4-DNT is 0.05 mg / L, 0.01 mg / L, 0.005 mg / L, 0.001 mg / L, the fluorescence value is significantly higher than that of the bacterial solution without 2,4-DNT, and with the concentration of DNT With increasing, the fluorescence value also gradually increased.

[0...

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Abstract

The invention provides novel photosynthetic bacteria for detecting explosives as well as a preparation method and application of the novel photosynthetic bacteria. The preparation method of the novel photosynthetic bacteria is simple, an exogenous reporter gene is introduced into rhodopseudomonas palustris, an original biological sensing element in a strain body is used for recognizing explosive molecules, and the detection on the explosive molecules is realized through a signal generated by a reporter gene expression product. The novel photosynthetic bacteria are finally used for preparing a rhodopseudomonas palustris biosensor, and the rhodopseudomonas palustris biosensor contains a promoter gene and a transcriptional regulation protein gene, a green fluorescent protein gene or a luciferase gene. The rhodopseudomonas palustris biosensor is wide in detection range, high in sensitivity, simple and convenient in method, low in cost, high in safety and high in adaptability, is environmentally friendly and has a broad 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 novel photosynthetic bacterium for detecting explosives and its preparation method and application. Background technique [0002] Traditional detection methods for explosives such as landmines mainly include biological detection, metal detection, electromagnetic induction detection, x-ray / neutron backscattering, acoustic wave detection, radar detection, etc. These traditional detection methods have certain limitations, such as biological detection It is mainly based on the animal's sensory response to the smell. Since the animal's thoughts are uncontrollable, the detection has great uncertainty; metal, sound wave and radar detection are mainly based on the subjective judgment of the detector, while electromagnetic induction detection, X-ray / neutron back Although the detection method of scattering has high sensitivity, it requires b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/31C12N15/65C07K14/21G01N21/64C12R1/38
CPCC07K14/21C12N15/65G01N21/6428G01N21/643Y02A50/30
Inventor 杨建明梁波张鑫平汤若昊王兆宝李美洁
Owner QINGDAO AGRI UNIV
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