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Promoter7M4D and application thereof

A DNA molecule and sequence technology, applied in recombinant DNA technology, DNA/RNA fragments, introduction of foreign genetic material using vectors, etc. It can solve the problems of TNT poisoning and high cost, and achieve the effect of good specificity and sensitivity and good component storage.

Active Publication Date: 2016-12-07
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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  • Promoter7M4D and application thereof
  • Promoter7M4D and application thereof
  • Promoter7M4D and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1. Discovery of promoter sequences

[0024] 1. Extract the chromosomal genome of Escherichia coli K-12MG1655.

[0025] 2. Using the chromosomal genome obtained in step 1 as a template, the NEB Q5MIX high-fidelity PCR system was used to amplify the promoter element.

[0026] 3. All the amplified products obtained in step 2 are respectively added with dATP through Taq enzyme polymerization reaction to form polyA, and then connected into the vector pMD18-T through DNA ligation operation to obtain a recombinant plasmid.

[0027] 4. The recombinant plasmid obtained in step 3 was double digested with restriction endonucleases Xba I and Bgl II, and a small fragment was recovered.

[0028] 5. The pET24-GFP vector was digested with restriction enzymes Xba I and Bgl II, and the vector backbone was recovered.

[0029] 6. Connect the small fragment obtained in step 4 with the vector backbone obtained in step 5 to obtain a recombinant plasmid.

[0030] 7. The recombinant pl...

Embodiment 2

[0036] Example 2, the acquisition of recombinant bacteria and control bacteria

[0037] First, the acquisition of recombinant bacteria

[0038] 1. Synthesize the double-stranded DNA molecule shown in Sequence 1 of the Sequence Listing.

[0039] 2. Double-digest the double-stranded DNA molecule obtained in step 1 with restriction endonucleases Bgl II and Xba I, and recover the digested product.

[0040] 3. The pET24-GFP vector was digested with restriction enzymes Bgl II and Xba I, and the vector backbone of about 6000 bp was recovered.

[0041] 4. Connect the enzyme-digested product of step 2 and the vector backbone of step 3 to obtain a recombinant plasmid.

[0042] 5. The recombinant plasmid obtained in step 4 was introduced into Escherichia coli BL21 (DE3) to obtain a recombinant bacteria.

[0043] Second, the acquisition of control bacteria

[0044] 1. Insert the T7 promoter (double-stranded DNA molecule shown in Sequence 3 of the Sequence Listing) between the Bgl II a...

Embodiment 3

[0056] Example 3. Functional verification of promoter

[0057] The treatment method of the recombinant bacteria TNT group: the recombinant bacteria obtained in the step 1 of Example 2 are inoculated into LB liquid culture medium, cultivated to OD 600nm When = 0.6, TNT was added and the concentration was 15 mg / L, then 30 ° C, 200 rpm shaking culture for 12 h, and then 200 μl of bacterial liquid was transferred to a 96-well polystyrene detection plate (Bio-rad), and detected on a Pekin Elmer 2300 Multilabable Reader ( GFP detection conditions are excitation / emission, 485 / 535nm).

[0058] The treatment method of the recombinant bacteria control group: inoculate the recombinant bacteria obtained in step 1 of Example 2 into LB liquid medium, and cultivate to OD 600nm = 0.6, then 30 ℃, 200rpm shaking culture for 12h, then pipette 200μl of bacterial liquid to 96-well polystyrene detection plate (Bio-rad), and detect it on Pekin Elmer 2300Multilabable Reader (GFP detection conditions...

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Abstract

The invention discloses a promoter7M4D and an application thereof. The provided promoter is named as 7M4D, and is the DNA molecule shown as 7th to 93rd nucleotide from a 5' tail end of a sequence 1 in a sequence table. The invention also provides the DNA molecule (fusion gene) which comprises the DNA molecule shown as 7th to 93rd nucleotide from a 5' tail end of the sequence 1 in the sequence table and a report gene from upstream to downstream in order. The invention also provides the recombinant plasmid containing the fusion DNA. The invention also protects recombinant bacteria obtained by introducing the recombinant plasmid to a host bacterium. The invention also protects the recombinant bacteria in an application for detecting 2,4,6-trinitrotoluene and / or 2,6-dinitrotoluene and / or toluene and / or 2,4-dinitrotoluene and / or 1,4-dinitro benzene. A biosensor provided in the invention can be used for detection and identification of position of the explosives such as land mine in wartime and after war, and also provides a novel method for detecting TNT in soil and water environment.

Description

technical field [0001] The present invention relates to the promoter 7M4D and its use. Background technique [0002] 2,4,6-Trinitrotoluene (2,4,6-Trinitrotoluene in English, 2,4,6-TNT or TNT for short) is a colorless or light yellow crystalline nitrobenzene explosive, which is a An important chemical raw material, it plays an important role in the defense industry, mining, infrastructure construction and other fields. TNT contamination has been one of the major environmental problems in explosives production, in enterprise areas where it is used, as well as in many battlefield sites and military training areas. TNT can penetrate into soil and water systems, participate in the ecological cycle for a long time, and even produce "pink water", which is very difficult and expensive to clean up. It has been reported that TNT has toxic effects on microorganisms, green algae plants and animals. Meanwhile, TNT and its degradation products can be introduced into the food chain, ther...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/11C12N15/70C12N1/21C12Q1/02C12R1/19
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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