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Explosive molecular biosensor synthesized by utilizing regulatory element as well as preparation method and application of explosive molecular biosensor

A molecular biology and regulatory element technology, applied in the fields of genetic engineering and molecular biology, can solve the problems of limited application and poor sensor specificity, and achieve the effects of simple use, accurate detection results, and great application prospects.

Active Publication Date: 2021-11-05
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the specificity of this sensor is poor, which greatly limits its application in complex environments.

Method used

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  • Explosive molecular biosensor synthesized by utilizing regulatory element as well as preparation method and application of explosive molecular biosensor
  • Explosive molecular biosensor synthesized by utilizing regulatory element as well as preparation method and application of explosive molecular biosensor
  • Explosive molecular biosensor synthesized by utilizing regulatory element as well as preparation method and application of explosive molecular biosensor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] The acquisition of embodiment 1 gene and the construction of carrier

[0046] 1. Acquisition of genes

[0047] The luxABCDE operon derived from Photobacterium leiognathi, whose nucleotide sequence is shown in SEQ ID No.1, was chemically synthesized by Huada Genomics Co., Ltd. onto the pUC-57 vector to obtain the pUC-lumPleio vector.

[0048] The nahR promoter gene derived from Pseudomonas putida, the nucleotide sequence of which is shown in SEQ ID No.2, was chemically synthesized by Huada Gene Co., Ltd. onto the pUC-57 vector to obtain pUC-nahR carrier.

[0049] The nucleotide sequence of the TetH promoter of the RsrR regulatory protein derived from Acidithiobacillus caldus MTH-04 is shown in SEQ ID No. 3, which was chemically synthesized by Huada Gene Company onto the pUC-57 vector , to obtain pUC-RsrR-P tetH carrier.

[0050] Among them, SEQ ID No.1:

[0051]

[0052] SEQ ID No.2:

[0053] CGCATACCTCGCCTTTGTGAACCTCTAATTACCACTGACTCTATCCGGGCTTTGCCCGTTAGACCATCAGTGGG...

Embodiment 2

[0105] The construction of embodiment 2 biosensors

[0106] Combine p-nahR-luxpleio and p-nahR-RsrR-P tetH The two recombinant plasmids of -luxpleio were transformed into Escherichia coli BW25113 competent cells (purchased from Weidi Biotechnology, product number DL2050), spread onto LB solid plates containing 34 mg / L chloramphenicol, and obtained positive clones by PCR screening, thus obtaining Engineering strain MR-11 containing vector p-nahR-luxpleio and containing vector p-nahR-RsrR-P tetH -Engineered strain MR-12 of luxpleio.

Embodiment 3

[0107] Example 3 Application of Biosensors to Detect Explosives Molecules

[0108] 1. Strain activation and cultivation

[0109] The engineered strains MR-11 and MR-12 with correct sequencing were respectively transferred to LB liquid medium containing 34 mg / L chloramphenicol, and cultured overnight at 37°C. Pipette 200 μL of the overnight activated bacterial solution into 10 mL of M9 liquid medium, add 10 μL of 34 mg / mL chloramphenicol, 330 μL of 60% glucose and 10 μL of 1M magnesium sulfate stock solution to the M9 liquid medium, and culture in a shaker at 37°C until OD 600 = about 0.2.

[0110] 2. Preparation of 2,4-DNT solution

[0111] Prepare 20mg / mL mother solution 2,4-DNT (100mg 2,4-DNT dissolved in 5mL absolute ethanol);

[0112] Prepare the diluted 2,4-DNT solution in the following proportions:

[0113] 50mg / L: 2.5μL mother solution + 980μL M9 medium + 17.5μL absolute ethanol;

[0114] 10mg / L: 0.5μL mother solution + 980μL M9 medium + 19.5μL absolute ethanol; ...

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Abstract

The invention discloses an explosive molecular biosensor synthesized by utilizing a regulatory element as well as a preparation method and application of the explosive molecular biosensor. The explosive molecular biosensor comprises a report element of a self-luminous operon luxABCDE operon, a sensing element of a promoter nahR, RsrR regulatory protein and a tetH promoter regulated by the RsrR regulatory protein at the downstream, according to the explosive molecule biosensor, a signal of a nahR promoter for sensing explosive molecules is subjected to cascade amplification through the RsrR-PtetH regulation and control element, so that a self-luminous signal generated after sensing the explosive molecules is enhanced, and the detection sensitivity is improved; the method and the biosensor for sensing molecular signals of explosives by regulating the protein regulation element cascade amplification promoter are established, and the sensor is wide in detection range, high in sensitivity, simple and convenient in method, low in cost and high in safety, and can be applied to the fields of environmental protection, national safety maintenance and the like.

Description

technical field [0001] The invention relates to the technical fields of genetic engineering and molecular biology, in particular to an explosive molecular biosensor synthesized by using regulatory elements and its preparation method and application. Background technique [0002] Biosensing technology is the use of genetic engineering to modify strains, so that after the microorganism senses a specific compound or a metabolite of a specific compound in the microorganism, the microorganism undergoes detectable changes, so as to achieve the purpose of detecting specific compounds. This biosensor is mainly composed of two parts: a sensing element and a reporter element. The sensing element can specifically sense the target compound. The sensing element includes a gene transcription promoter, ribosome binding site, terminator, transcriptional regulatory factor, etc.; The reporter element can generate a sensing signal under the action of the sensing element. Commonly used reporter...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/66C12N15/65C12N1/21C12Q1/02C12Q1/6897C12R1/19
CPCC12N15/70C12N15/65C12Q1/025C12Q1/6897C12N2830/002G01N2333/245Y02A50/30
Inventor 杨建明王兆宝马冉汤若昊李美洁梁波罗明
Owner QINGDAO AGRI UNIV
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