Promoter recea4 and its application

A DNA molecule and sequence technology, applied in recombinant DNA technology, DNA/RNA fragments, introduction of foreign genetic material using vectors, etc., can solve problems such as high cost and TNT poisoning

Active Publication Date: 2019-04-02
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Due to the obvious toxic effect of TNT, many TNT detection techniques have been developed based on the physical and chemical properties of TNT, such as high performance liquid chromatography (HPLC), ultraviolet, gas chromatography-mass spectrometry (GC-MS), laser surface enhanced Raman Spectroscopy (SERS), nuclear magnetic resonance, ion mobility spectrometry and other methods, however, they all require expensive and complicated instruments or complicated sample preparation methods

Method used

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  • Promoter recea4 and its application
  • Promoter recea4 and its application
  • Promoter recea4 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1, discovery of promoter sequence

[0023] 1. Extract the chromosomal genome of Escherichia coli K-12MG1655.

[0024] 2. Take the chromosomal genome obtained in step 1, partially digest it with restriction endonuclease Sau3AI (37° C., 2 hours), and then perform agarose gel electrophoresis to recover and purify DNA fragments below 2 kb.

[0025] 3. Take the pTJ629 vector (containing the luxCDABE gene with promoter deletion, and containing the broad-host replicon oriV and the stringent replicon pSC101), and perform single-digestion with the restriction endonuclease BamHI (BamHI can produce the same sticky end as Sau3AI ), recovering the vector backbone.

[0026] 4. Ligate the DNA fragment obtained in step 2 with the vector backbone obtained in step 3 to obtain a recombinant plasmid.

[0027] 5. Transform the recombinant plasmid obtained in step 4 into Escherichia coli DH5ɑ to obtain recombinant bacteria.

[0028] 6. Take the recombinant bacteria obtained in ...

Embodiment 2

[0030] Example 2, functional verification of the recEA4 promoter

[0031] 1. Acquisition of recombinant bacteria

[0032] 1. Synthesize the double-stranded DNA molecule shown in sequence 1 of the sequence listing.

[0033] 2. Using the double-stranded DNA molecule synthesized in step 1 as a template, a primer pair composed of F1 and R1 is used for PCR amplification to obtain a PCR amplification product.

[0034] F1: 5'-CG GGATCC ATCATTCACTGAACAA-3';

[0035] R1: 3'-GG GGTACCGGATCAGGTAGTCCAGAGTGG-5'.

[0036] 3. Digest the PCR amplified product obtained in step 2 with restriction endonucleases BamHI and KpnI, and recover the digested product.

[0037] 4. The vector pPROBE-TT was double-digested with restriction enzymes BamHI and KpnI, and the vector backbone of about 6.8 kb was recovered.

[0038] 5. Ligate the digested product of step 3 with the vector backbone of step 4 to obtain a recombinant plasmid. According to the sequencing results, the structure of the recombi...

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Abstract

The invention discloses a promoter recEA4 and applications of the promoter recEA4. The promoter provided by the invention is derived from Escherichia coli K-12 MG1655, is named as recEA4, and is DNA molecules shown by the sequence 1 of the sequence table. The invention also protects the DNA molecules (fusion DNA), sequentially comprising the DNA molecules shown by the sequence 1 of the sequence table and reporter genes from the upstream to the downstream. The invention also protects recombinant plasmids comprising the fusion DNA. The invention further protects recombinant bacteria obtained by importing the recombinant plasmids into host bacteria. The recombinant bacteria are a biosensor for detecting 2,4,6-trinitrotoluene. The biosensor provided by the invention can be used for detecting and identifying the position of the explosive, such as the mine during the wartime and after the war, and moreover, a novel method is provided for detecting TNT in the soil and the water environment.

Description

technical field [0001] The present invention relates to promoter recEA4 and its application. Background technique [0002] 2,4,6-Trinitrotoluene (English name is 2,4,6-Trinitrotoluene, referred to as 2,4,6-TNT or TNT) is an important chemical raw material with a wide range of applications and is the most commonly used Explosive materials are widely used in military, civil and other fields. While TNT exerts its good chemical and explosive properties, it is also causing harm to people's lives as a toxic pollutant. As an important environmental pollutant, TNT is distributed in air, water and soil, and has strong toxicity. Long-term contact with TNT can cause serious adverse effects on human health. Many military test bases and soils in regions that have experienced TNT production process Both groundwater and groundwater are polluted and even produce "pink water", which is difficult and expensive to clean up. TNT and its degradation products are mutagenic and carcinogenic, an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/65C12N15/70C12N1/21C12Q1/6897C12Q1/02
Inventor 刘刚谭俊杰阚乃鹏陈惠鹏王微凌静怡曲国龙
Owner ACADEMY OF MILITARY MEDICAL SCI
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