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Promoter 6m3b and its application

A technology of DNA molecules and sequences, applied in the direction of using vectors to introduce foreign genetic material, bacteria, biochemical equipment and methods, etc., can solve the problems of expensive, TNT poisoning, etc., and achieve the effect of good component reserve, good specificity and sensitivity

Active Publication Date: 2019-05-21
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 6m3b and its application
  • Promoter 6m3b and its application
  • Promoter 6m3b and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1, discovery of promoter sequence

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

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

[0026] 3. Add dATP to all the amplified products obtained in step 2 respectively through Taq enzyme polymerization reaction to form polyA, and then connect them into vector pMD18-T through DNA ligation operation to obtain recombinant plasmids.

[0027] 4. Digest the recombinant plasmid obtained in step 3 with restriction endonucleases XbaI and BglII to recover small fragments.

[0028]5. Digest the pET24-GFP vector with restriction endonucleases XbaI and BglII to recover the vector skeleton.

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

[0030] 7. Introduce the recombinant plasmid obtained in step 6 into Esche...

Embodiment 2

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

[0037] 1. 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 BglII and XbaI, and recover the digested product.

[0040] 3. Digest the pET24-GFP vector with restriction endonucleases BglII and XbaI, and recover the vector backbone of about 6000 bp.

[0041] 4. Ligate the digested product of step 2 with the vector backbone of step 3 to obtain a recombinant plasmid.

[0042] 5. Introduce the recombinant plasmid obtained in step 4 into Escherichia coli BL21(DE3) to obtain recombinant bacteria.

[0043] Second, the acquisition of control bacteria

[0044] 1. Insert the T7 promoter (the double-stranded DNA molecule shown in Sequence 3 of the Sequence Listing) between the BglII and XbaI restriction sites o...

Embodiment 3

[0056] Example 3, functional verification of the promoter

[0057] The treatment method of the recombinant bacteria TNT group: inoculate the recombinant bacteria obtained in step 1 of Example 2 into LB liquid medium, and cultivate to OD 600nm When =0.6, add TNT and make its concentration 15mg / L, then 30 ℃, 200rpm shaking culture 12h, then draw 200 μ l bacterial liquid to 96 hole polystyrene detection plate (Bio-rad), detect in Pekin Elmer 2300MultilablelReader (GFP The 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°C, 200rpm shaking culture for 12h, then pipette 200 μl of bacterial liquid to a 96-well polystyrene detection plate (Bio-rad), and detect it in Pekin Elmer 2300 Multilablel Reader (GFP detection conditions are excitation / emission, 485 / 535nm ).

[0059] ...

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Abstract

The invention discloses a promoter 6M3B and applications of the promoter 6M3B. The promoter provided by the invention is named as 6M3B, and is DNA molecules shown by the nucleotide from the 7th site to the 94th site of the 5' terminal in the sequence 1 of the sequence table. The invention also protects the DNA molecules (fusion genes), sequentially comprising the DNA molecules shown by the nucleotide from the 7th site to the 94th site of the 5' terminal in the sequence 1 of the sequence table and reporter genes from the upstream to the downstream. The invention also protects recombinant plasmids containing the fusion DNA. The invention further protects recombinant bacteria obtained by importing the recombinant plasmids into host bacteria. The invention also protects the applications of the recombinant bacteria in detecting 2,4,6-trinitrotoluene and / or 2,6-dinitrotoluene and / or methylbenzene. A 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 6M3B 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 a colorless or light yellow crystalline nitrobenzene explosive. An important chemical raw material, it plays an important role in national defense industry, mining, infrastructure construction and other fields. TNT pollution has always been one of the main environmental problems in explosives production and use enterprise areas, as well as 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, and TNT and its degradation products can be introduced into the food chain, thereby causing s...

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