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Arac mutant AraCmt1 for sensing explosive molecules as well as screening method and application of Arac mutant AraCmt1

A screening method and mutant technology, which is applied in the fields of genetic engineering and molecular biology, can solve the problems of low sensitivity of the sensing system and inability to meet field detection, and achieve great application prospects and simple methods

Active Publication Date: 2021-10-22
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this study, 2,4-DNT and its metabolites can directly induce the yqjF promoter, and after the yqjF promoter is induced, the downstream GFP gene is transcribed. This direct induction induction system has low sensitivity and cannot meet the requirements of field detection.

Method used

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  • Arac mutant AraCmt1 for sensing explosive molecules as well as screening method and application of Arac mutant AraCmt1
  • Arac mutant AraCmt1 for sensing explosive molecules as well as screening method and application of Arac mutant AraCmt1
  • Arac mutant AraCmt1 for sensing explosive molecules as well as screening method and application of Arac mutant AraCmt1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Screening of AraC mutants

[0039] 1. Basic carrier p-AraC-P BAD - Construction of eGFP and its corresponding strain

[0040] Using plasmid pEGFP-1 (Clontech, Cat. No. 6086_1) as a template, primers pBAD-EGFP-F and primers pBAD-EGFP-R, polymerase chain reaction (PCR) was carried out to amplify the eGFP fragment. The PCR amplification system is as follows Show:

[0041]

[0042] The PCR program is: 95ºC 3 min; 30 × (95ºC 15 s, 55ºC 15 s, 72ºC 1 min); 72ºC 5min; 16ºC ∞.

[0043] The primer sequences are as follows:

[0044] PBAD-EGFP-F: 5'-TTGGGCTAGCAGGAGGAATTCTTTGTTTAACTTTAAGAAGGAGATATACAAATGG-3' (SEQ ID NO.1);

[0045] PBAD-EGFP-R: 5'-CTCTAGAGGATCCCCGGGTACCTTACTTGTACAGCTCGTCCATG-3' (SEQ ID NO. 2).

[0046]PCR products were purified by gel recovery and purification kit (Vazyme, Cat. No. DC301-01) ( figure 1 A).

[0047] restriction endonuclease 1 EcoR І (TaKaRa, Cat. No. 1611) and restriction enzyme 2 Kpn І (TaKaRa, Cat. No. 1618) double enzyme d...

Embodiment 2

[0090] Example 2: Application of AraC Mutant in Detecting Explosive Molecule 2,4-DNT

[0091]A single colony containing the AraCmt1 mutant was picked with a sterile toothpick, placed in 10 mL of M9Y medium containing 100 mg / L ampicillin for activation culture, and cultured overnight on a shaker at 37 °C. According to the 1% inoculum amount, transfer to the 96-microwell plate of the same medium, and when cultured to OD600=0.2, add 2,4-DNT at concentrations of 0 mg / L, 5 mg / L, and 10 mg / L respectively, and set In a microplate reader (Biotek), monitor the fluorescence intensity (RFU) at 30°C, and the results are as follows: Figure 5 As shown, the RFU values ​​of the 5 mg / L and 10 mg / L groups were significantly higher than the 0 mg / L group, and the higher the concentration of 2,4-DNT, the higher the RFU value, indicating that the AraCmt1 mutation screened by the present invention The body has the effect of obviously sensing the explosive molecule 2,4-DNT, and the sensitivity is g...

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Abstract

The invention discloses an Arac mutant AraCmt1 for sensing explosive molecules as well as a screening method and an application of the Arac mutant AraCmt1. The amino acid sequence of the Arac mutant AraCmt1 is as shown in SEQ ID NO.13, and the nucleotide sequence of the coding gene of the Arac mutant AraCmt1 is as shown in SEQ ID NO.12, or a nucleotide sequence which has more than 95% of homology with the nucleotide sequence as shown in SEQ ID NO.12 and can code the amino acid sequence as shown in SEQ ID NO.13. The mutant AraCmt1 can be specifically combined with explosive molecule 2, 4-dinitrotoluene (2, 4-DNT), and is induced by the explosive molecule to activate a PBAD promoter, so that a downstream reporter gene is expressed, trace 2, 4-DNT is detected through fluorescence intensity or color reaction, and therefore, the mutant AraCmt1 is suitable for preparing a microbial sensor for sensing 2, 4-DNT.

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering and molecular biology, and in particular relates to an Arac mutant AraCmt1 capable of sensing explosive molecules, a screening method and application thereof. Background technique [0002] Residual explosives in theaters (such as landmines) have caused irreparable damage to life safety and ecosystems, and safe and effective detection of explosives is of great strategic significance. The active ingredient of the explosive is TNT, which can be decomposed into various compounds, such as 1,3-dinitrobenzene (1,3-DNB) and 2,4-dinitrotoluene (2,4-DNT). [0003] The explosive molecule 2,4-DNT has a CAS number of 121-14-2, and its structural formula is as follows: [0004] [0005] Israeli scientist Shimshon Belkin reported the sensing element for explosive molecule 2,4-DNT, that is, the yqjF promoter, and used the GFP gene as a reporter element to construct a microbial sensor for detectin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C12N15/12C12N15/70C12N1/21G01N33/68C12R1/19
CPCC07K14/47C12N15/70G01N33/68
Inventor 杨建明李美洁吕书喆汤若昊梁波王兆宝
Owner QINGDAO AGRI UNIV
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