CN112725339B - Promoter element for improving sensitivity of explosive molecule detection and screening method and application thereof - Google Patents

Abstract

The invention discloses a novel promoter element for improving detection sensitivity of explosive molecules, and a screening method and application thereof. The novel promoter element comprises a nucleotide sequence shown in SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO. 5; or a nucleotide sequence with homology of more than 75 percent with the nucleotide sequence shown in SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO. 5. The novel promoter element obtained by the invention has the capability of remarkably improving the detection sensitivity of 2,4-DNT, and the screening method is simple, convenient to use and high in safety; the screening method of the invention can be complementary with the traditional explosive detection method, and improves the accuracy, safety and efficiency of explosive detection.

Description

Promoter element for improving sensitivity of explosive molecule detection and screening method and application thereof

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a promoter element for improving detection sensitivity of explosive molecules, and a screening method and application thereof.

Background

Explosives such as landmines (effective components such as TNT and DNT) and the like are applied in wars all the time due to low price, high damage, high concealment and high defense, and the residual unexploded landmines also become hidden troubles in the life of modern people. Explosives such as landmines and the like are environmental pollutants, can permeate into soil and water systems after being leaked out, participate in ecological cycle, have toxic and harmful effects on microorganisms, plants and animals, cause serious harm to human health, and are difficult to thoroughly remove in the environment. Therefore, the key technology for detecting explosives such as mines in a rapid, efficient and safe manner is developed, can be used for detecting explosives such as mines in military and detecting environmental pollution in an ecological system, and has important environmental protection significance.

The traditional detection methods for explosives such as landmines mainly comprise biological detection, metal detection, electromagnetic induction detection, x-ray/neutron backscattering, sound wave detection, radar detection and the like, and the traditional detection methods have certain limitations. For example, biological detection mainly utilizes the olfaction instinct of animals to realize the detection of land mines, but the detection has great uncertainty because the animal thought is uncontrollable; the metal detection is the most widely used detection means at present because of simple and convenient operation, but the method is mainly based on the detection that handheld detection equipment of an individual soldier enters a lightning field, and the danger degree is high; while other detection technologies based on resonance, acoustics and the like can achieve high accuracy for mine detection, heavy instruments and equipment are needed, and the detection is extremely inconvenient to use. In addition, based on the physicochemical properties of TNT, many TNT detection technologies, such as high performance liquid chromatography, ultraviolet detection, gas chromatography-mass spectrometry, laser surface enhanced raman spectroscopy, nuclear magnetic resonance, ion mobility spectrometry, etc., have been developed, however, these also require expensive and complicated equipment or complicated sample preparation methods, some are still in the laboratory research stage, and cannot be conveniently used for real-time detection of mines, explosives, etc. in external fields. Meanwhile, detection personnel are required to attend a mine area for field detection under most conditions, the danger coefficient is high, and the mine cannot be detected aiming at the existing mines made of novel materials such as ceramics and plastics.

The biological sensing technology is to modify the strain by means of genetic engineering, so that the microorganism can detect special compounds or specific groups of the compounds. Few biosensing techniques are currently reported for the detection of explosives. TNT-based land mines contain a variety of compounds such as 1, 3-dinitrobenzene (1,3-DNB) and 2, 4-dinitrotoluene (2, 4-DNT). Molecules of these substances will slowly leak out of the plastic package or from the fragments of the landmine explosion and can be sensed and detected by the microbiologically related sensing element. The biological sensing system for detecting the mine mainly comprises a sensing element and a reporting element, wherein the sensing element can sense a substance to be detected (TNT or DNT molecules), and is generally a promoter region of a target substance sensing gene of a cell, namely a promoter element; the reporting element can amplify the induction signal generated by the induction element due to induction mines (TNT or DNT molecules) to the extent that the induction signal can be metered, and finally the accurate position of the mines can be obtained. The commonly used reporter elements are the lacZ, gfp and lux genes which produce a color, fluorescent or bioluminescent signal.

Disclosure of Invention

The invention aims to provide a promoter element for improving detection sensitivity of explosive molecules, and a screening method and application thereof. The promoter element capable of efficiently inducing explosive molecules is screened by utilizing the photosynthetic bacterium genome library, and the promoter element has the capability of remarkably improving the detection sensitivity of 2, 4-DNT.

In order to realize the purpose of the invention, the invention adopts the following technical scheme to realize:

the invention provides a promoter element for improving the detection sensitivity of explosive molecules, which has one of the following nucleotide sequences:

(1) a nucleotide sequence shown as SEQ ID NO. 3;

(2) a nucleotide sequence shown as SEQ ID NO. 4;

(3) a nucleotide sequence shown as SEQ ID NO. 5;

(4) a nucleotide sequence with over 75 percent of homology with the nucleotide sequence shown in SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO. 5.

The invention also provides a promoter element for improving the sensitivity of detecting explosive molecules, wherein the promoter element has one of the following nucleotide sequences:

(1) a nucleotide sequence shown as SEQ ID NO. 3;

(2) a nucleotide sequence shown as SEQ ID NO. 4;

(3) a nucleotide sequence shown as SEQ ID NO. 5;

(4) a nucleotide sequence with homology of more than 95 percent with the nucleotide sequence shown in SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO. 5.

Further, the promoter element has the ability to significantly improve the sensitivity of 2,4-DNT detection.

The invention also provides a screening method of the promoter element for improving the detection sensitivity of the explosive molecules, which comprises the following steps:

(1) constructing a recombinant plasmid containing yqjF gene: after Sal I and BamH I enzyme cutting sites are introduced into two ends of the yqjF gene, the gene and a plasmid pEGFP-1 are subjected to double enzyme cutting by using restriction enzymes Sal I and BamH I, and are incubated in water bath at 22 ℃ by using T4 ligase for 2h for connection to obtain a recombinant plasmid pEGFP-yqjF;

(2) extracting genome DNA of Rhodopseudomonas palustris (Rhodopseudomonas palustris), carrying out incomplete enzyme digestion on the Rhodopseudomonas palustris by utilizing Sau3AI enzyme with the enzyme amount of 1.5-6U, carrying out water bath at 37 ℃ for 1 hour, and recovering to obtain a genome enzyme digestion product with the length of less than 2000 bp;

(3) carrying out site-directed mutagenesis on the recombinant plasmid pEGFP-yqjF by adopting a QuikChange method, mutating a middle 4-site base TCGA of a SalI site of a yqjF gene in the recombinant plasmid pEGFP-yqjF into a GATC of a BamHI site, carrying out PCR amplification by taking the site-directed mutagenesis plasmid as a template, introducing new BamH I enzyme digestion sites at two ends of an obtained mutagenesis gene sequence, carrying out single enzyme digestion treatment on the site-directed mutagenesis plasmid, carrying out water bath at 37 ℃ for 3h, and carrying out gel recovery to obtain a single enzyme digestion product;

(4) and (3) connecting the genome enzyme digestion product in the step (2) with the single enzyme digestion product in the step (3), transforming the obtained connection product into escherichia coli DH5 alpha competent cells, primarily screening the obtained recombinant strain by using a flat fluorescence observation method, and secondarily screening by using a microplate reader fluorescence photometry method to obtain a single colony with an obvious fluorescence signal, namely the promoter element.

Further, the primer sequence of the PCR in the step (3) is as follows:

2-F：5‘-CTTCGAATTCTGCAGGATCCCGGTTTTGGCGTAT-3’；

2-R：5‘-ATACGCCAAAACCGGGATCCTGCAGAATTCGAAG-3’。

further, the library was initially screened using flat panel fluorescence observation: both the 2,4-DNT concentration and the incubation time can be determined according to the sensitivity of the promoter element to sense 2, 4-DNT. 2,4-DNT enters bacteria on a plate, a promoter element contained in the bacteria induces the 2,4-DNT, a reporter element, namely the expression of gfp gene is started, and an expression product shows green fluorescence which is visible to naked eyes. The primary screening by using a flat fluorescence observation method comprises the following steps: the recombinant strain is coated on a solid plate containing nutrient substances after being subjected to antibiotic resistance screening, the plate with good strain growth vigor is subjected to nitrocellulose microporous membrane conversion, the cellulose microporous membrane is transferred to the solid plate containing 2,4-DNT and the nutrient substances, the solid plate is placed in a constant temperature incubator, when an obvious bacterial colony grows, fluorescence observation is carried out, and a single bacterial colony with obvious fluorescence is obtained preliminarily.

Further, the library is rescreened by a fluorescence photometry method of a 96 microplate reader: both the 2,4-DNT concentration and the incubation time can be determined according to the sensitivity of the promoter element to sense 2, 4-DNT. 2,4-DNT enters a bacterial body in a culture medium, a promoter element contained in the bacterial body senses the 2,4-DNT, a reporter element, namely expression of a gfp gene is started, an expression product has green fluorescence, and the fluorescence intensity is detected by an enzyme-labeling instrument, so that the sensitivity of sensing the 2,4-DNT by the promoter element is determined. The method for re-screening by using the fluorescence photometry method of the microplate reader comprises the following steps: placing the single colony obtained by primary screening in a microplate containing a culture medium containing antibiotics and nutrients, activating and culturing, transferring the culture bacteria liquid to a new 96 microplate containing a culture medium containing nutrients, and culturing to OD₆₀₀When the fluorescence intensity is 0.2, 2,4-DNT is added, and the mixture is left to stand for culture, and a strain with high fluorescence intensity is further selected, namely, a promoter element.

The invention also provides application of the promoter element in preparing a biological agent for improving detection sensitivity of explosive molecules.

Further, the biological agent comprises an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing the promoter element.

Compared with the prior art, the invention has the following advantages and beneficial effects:

according to the invention, the photosynthetic bacteria genome library is constructed to screen the promoter element for sensing explosive molecules, the obtained promoter element has the capability of remarkably improving the detection sensitivity of 2,4-DNT, and the screening method is simple, convenient to use and high in safety; the method subverts the traditional detection technology, can develop a key technology for quickly, efficiently and safely detecting explosives such as land mines and the like through large-scale screening of inductive elements which can be induced by the explosives in photosynthetic bacterial genomes, can be used for detecting explosives such as the land mines in military and detecting environmental pollutants in an ecological system, can supplement the traditional explosive detection method, improves the accuracy, safety and efficiency of explosive detection, and has important significance for military, counter-terrorism, environmental protection and the like.

Drawings

FIG. 1 is a schematic diagram of the construction of random genomic library of Rhodopseudomonas palustris CGA009 according to the present invention.

FIG. 2 is a gel electrophoresis of restriction enzyme digestion verification of Rhodopseudomonas palustris CGA009 genome in the present invention.

FIG. 3 is a schematic flow chart of large-scale screening of random promoter libraries for induction of 2,4-DNT in the present invention.

FIG. 4 is a fluorescence detection image of large-scale plate screening of random starter library in the present invention, wherein the left side image is a fluorescence colony image observed under the irradiation of a fluorescence light source, the right side image is a colony observed by the same plate under visible light, and the circle part is a target fluorescence colony.

FIG. 5 is a fluorescence detection map of microplates from the large-scale plate screening of random starter libraries in the present invention.

FIG. 6 is a graph comparing the fluorescence values of different promoter elements for detecting explosives in the present invention.

FIG. 7 is a solid plate fluorescence detection of sensor gas 2,4-DNT molecules.

Detailed Description

The technical solution of the present invention will be described in further detail with reference to specific examples.

Rhodopseudomonas palustris (Rhodopseudomonas palustris) belongs to rhodobacter of photosynthetic bacterium purple nonsulfur flora, can perform light energy heterotrophic growth under the condition of anaerobic illumination, can perform aerobic heterotrophic growth under the condition of aerobic darkness, and almost extends over soil, peat, marsh, fresh water, seawater and aquatic plant root systems, so the Rhodopseudomonas palustris strain has superiority compared with other microbial materials. The rhodopseudomonas palustris can degrade compounds such as benzene rings, the degradation efficiency of the rhodopseudomonas palustris on TNT can reach 95 percent at most, and the genome of the rhodopseudomonas palustris contains related promoter elements which can effectively sense TNT or DNT molecules. However, there is no report on the use of Rhodopseudomonas palustris for screening promoter elements that are highly sensitive to TNT or DNT molecules.

Example 1

As shown in FIG. 1, the specific steps of constructing the random library by using the Rhodopseudomonas palustris Rhoudomonas palustris CGA009 genome are as follows:

1. construction of recombinant plasmid pEGFP-yqjF

1) yqjF gene (GenBank No. AAC76136.1) was synthesized by Biotech, and the gene sequence was obtained by PCR amplification, with Sal I (G) introduced at both ends of the geneTCGAC) And BamH I (G)GATCC) A restriction enzyme site;

2) recovering a PCR product of the yqjF gene by glue, and performing double enzyme digestion by using restriction enzymes Sal I and BamH I to obtain a gene fragment after enzyme digestion;

3) meanwhile, plasmid pEGFP-1 (purchased from vast forest plasmid platform http:// www.miaolingbio.com) without promoter is subjected to the same enzyme double-enzyme digestion treatment, and the product is subjected to dephosphorylation treatment to obtain plasmid pEGFP-1 after enzyme digestion;

4) using a T4 DNA ligase kit, incubating the purified pEGFP-1 enzyme digestion product and the yqjF gene enzyme digestion product in 22 ℃ water bath for 2h for connection to obtain a T4 connection product;

5) and transforming the T4 ligation product into escherichia coli DH5 alpha competent cells, coating the cells on an LB plate containing kanamycin to grow overnight, screening kanamycin resistance, selecting a single colony growing well on the plate to perform sequencing verification to obtain a recombinant plasmid pEGFP-yqjF, and sequencing to identify the success of preparation of the recombinant plasmid.

2. Construction of genomic libraries

2.1: bacterial genomic DNA extraction

Obtaining Rhodopseudomonas palustris CGA009, culturing, extracting genome DNA from the obtained bacterial liquid by using a bacterial genome extraction kit:

1) 2.5mL of fresh bacterial liquid is added into a 1.5mL centrifuge tube, and the supernatant is removed by centrifugation. Adding 200 mu L of Buffer digest and 2 mu L of beta-mercaptoethanol, shaking and uniformly mixing, and carrying out water bath at 80 ℃ for 5min until the cells are completely lysed;

2) to obtain RNA-free DNA, 2 μ L of RNase A (10mg/mL) can be added after water bath, and the mixture is placed at 37 ℃ for 1h, and is inverted and mixed uniformly every 10min in the water bath process;

3) adding 20 μ L of protease K solution, shaking, mixing, and water bath at 55 deg.C for 2h, wherein the sample can be cracked by mixing once every 10min during the water bath process;

4) cooling to room temperature, adding 100 μ L Buffer PF, fully reversing, mixing, and standing in refrigerator at-20 deg.C for 5 min;

5) centrifuging at room temperature at 10,000rpm for 5min, and transferring the supernatant to a new 1.5mL centrifuge tube;

6) adding 200 mu L of Buffer BD, fully reversing and uniformly mixing; adding Buffer BD, and water-bathing at 70 deg.C for 10min if precipitate exists;

7) adding 200 μ L of anhydrous ethanol, and fully reversing and mixing; semi-transparent fibrous suspended matters can be generated after the absolute ethyl alcohol is added, and the extraction and the application of DNA are not influenced;

8) putting the adsorption column into a collecting pipe, adding the solution and the semitransparent fibrous suspended matters into the adsorption column by a liquid transfer device, standing for 2min, centrifuging at 10,000rpm at room temperature for 1min, and pouring off waste liquid in the collecting pipe;

9) putting the adsorption column back to the collecting tube, adding 500 μ L PW Solution, centrifuging at 10,000rpm for 30s, and pouring off the waste liquid in the collecting tube; before use, whether isopropanol is added into the solution according to the proportion or not is noticed;

10) putting the adsorption column back to the collecting pipe, adding 500 μ L Wash Solution, centrifuging at 10,000rpm for 30s, pouring off the waste liquid in the collecting pipe, and paying attention to whether absolute ethyl alcohol is added into the Solution in proportion before use; if the yield of the DNA is required to be improved, the step can be repeated;

11) putting the adsorption column back into the collection tube again, centrifuging at 12,000rpm for 2min at room temperature, and removing the residual Wash Solution; opening a cover of the adsorption column, and standing at room temperature for several minutes to thoroughly dry the Wash Solution remaining in the adsorption material, wherein the residue of the Wash Solution can influence the yield of the genome DNA and subsequent experiments;

12) the adsorption column was removed and placed in a new 1.5mL centrifuge tube, 50. mu.L ddH was added₂O stands for 3min, is centrifuged at 12,000rpm for 2min at room temperature, and the DNA solution is collected.

2.2: incomplete digestion of bacterial genomic DNA

The optimum enzyme amount of the Sau3AI enzyme for incomplete digestion of the bacterial genomic DNA obtained above was explored to better break the genomic DNA into small promoter fragments. The bacterial genome DNA is subjected to enzyme digestion under the enzyme dosage of 1.5U, 3U and 6U of Sau3AI enzyme respectively, water bath at 37 ℃ is carried out for 1 hour, the specific enzyme digestion reaction system is shown in Table 1, and agarose gel electrophoresis detection is carried out, so that better dispersion bands are obtained under the enzyme dosage of 3 types, wherein the dispersion bands are all below 2000bp as shown in figure 2. Therefore, 3U was selected as an appropriate enzyme amount for the enzyme digestion. In order to ensure the completeness of the construction of the promoter library, simultaneously, a band below 2000bp is recovered through gel recovery to obtain an enzyme digestion recovery product of a promoter small fragment for the construction of the library.

TABLE 1 genome digestion reaction System Components Table

2.3 site-directed mutagenesis of recombinant plasmid pEGFP-yqjF

1) Site-directed mutagenesis was performed by QuikChange method, and the middle 4 bases G of SalI site of yqjF gene on recombinant plasmid pEGFP-yqjFTCGAG with C mutated to BamHI siteGATCAnd C, taking the point mutation plasmid pEGFP-yqjF as a template, designing and synthesizing primers 2-F and 2-R according to the site, carrying out PCR amplification to obtain a mutation gene sequence, and introducing BamH I enzyme cutting sites into two ends of the gene.

Wherein, the sequence of the primer is as follows:

primer 2-F: 5'-CTTCGAATTCTGCAGGATCCCGGTTTTGGCGTAT-3' (SEQ ID NO. 1);

primers 2-R: 5'-ATACGCCAAAACCGGGATCCTGCAGAATTCGAAG-3' (SEQ ID NO. 2).

The PCR reaction system is shown in Table 2:

TABLE 2 composition of point mutation PCR reaction system

Point mutation PCR reaction is carried out according to the system, and the reaction conditions are as follows: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 30s, and repeating for 30 cycles; extension was continued for 5min at 72 ℃.

2) The PCR product after the completion of the reaction was directly purified in accordance with 1:25(Dpn I enzyme: PCR product, V: v) adding Dpn I enzyme according to the proportion, and carrying out enzyme digestion at 37 ℃ for 1 hour to obtain an enzyme digestion product;

3) directly taking the enzyme digestion product and transforming the enzyme digestion product into escherichia coli DH5 alpha competent cells;

4) a single transformant is picked and cultured in an LB (peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L) liquid medium, and then sequencing test is carried out, and the result shows that the point mutation of the plasmid pEGFP-yqjF is successful.

2.4: single enzyme digestion of point mutation plasmid pEGFP-yqjF

1) Carrying out single enzyme digestion treatment on the point mutation plasmid pEGFP-yqjF, adding 1 mu L of BamH I endonuclease, 2 mu L of plasmid pEGFP-yqjF (the total mass is 1 mu g), 2 mu L of buffer solution, 15 mu L of distilled water, a reaction system with the total volume of 20 mu L, and carrying out water bath at 37 ℃ for 3h to obtain a single enzyme digestion product;

2) in order to prevent the self-ligation of the single enzyme digestion product, the single enzyme digestion product needs to be dephosphorized, and the specific operation method comprises the following steps: after the gel imaging of the single enzyme digestion product verifies that the size is correct, 1 mu L of dephosphorylation enzyme Taq DNA Polymerase is added into each tube of product, the product is inactivated in water bath at 37 ℃ for 30min and 75 ℃ for 10min, and the gel is recovered to obtain a purified single enzyme digestion product;

3) using a T4 DNA ligase kit, incubating a single enzyme digestion product of the purified point mutation plasmid pEGFP-yqjF and the enzyme digestion recovery product of the promoter small fragment with the genome of less than 2000bp obtained by recovery in a water bath at 16 ℃ for 10h for connection to obtain a T4 connection product;

4) the T4 ligation product was transformed into E.coli DH 5. alpha. competent cells, incubated at 37 ℃ for about 20 hours, and the color change of colonies on the plates was observed by kanamycin resistance screening on LB solid plates. Each transformation plating resulted in more than 60 LB solid plates, 1 plate had approximately 150 clones, and a total of 9000 transformants all grew well.

3. Large-scale screening of random promoter libraries for induction 2,4-DNT

3.1 preliminary screening of libraries by Flat fluorescence Observation

The 60 well-developed plates obtained above were subjected to membrane transfer with a microporous membrane of nitric acid, transferred to LB solid plates containing 10mg/L of 2,4-DNT, and left in a constant temperature incubator at 37 ℃ for 12 hours. When the LB solid plate with the transferred membrane has obvious colony growth, the whole plate is subjected to fluorescence observation, and a single colony with obvious fluorescence is obtained by screening, namely the promoter element. As shown in FIG. 4, the present inventors initially screened 19 promoter elements in 9000 clones.

3.2 rescreening the library by a 96 microplate reader fluorescence photometry method

As shown in FIG. 3, 19 single colonies of promoter elements obtained by primary screening were picked with sterile toothpicks and cultured in a 96-well plate containing 1mL LB medium for activation with kanamycin sulfate (final concentration 50. mu.g/mL), and wild type strains containing yqjF primary sensor elements were used as controls and cultured overnight in a shaker at 37 ℃. Transferred to a new 96-well plate containing LB medium at an inoculum size of 1%, and cultured to OD₆₀₀When the concentration is 0.2, 2,4-DNT is added to a concentration of 10mg/L, the mixture is left at 37 ℃ (time is 0), samples are taken at intervals of 1 hour, green fluorescence intensity is directly measured by using a microplate reader, excitation light is 485nm, emission light is 528nm, three replicates in each group are measured, and fluorescence signal values are recorded. As shown in FIG. 5, the number 7, 11, and 14 of the 19 strains showed significant fluorescent signals, and the numbers of the three strains were YY007, YY011, and YY014, respectively.

3.3 determination of the Activity of the promoter element for detecting explosive liquids

Performing 10mL LB vial culture on 3 strains obtained by re-screening 96 microwell plates, transferring into a triangular flask according to 1% inoculum size, and culturing to OD₆₀₀When the concentration was 0.2, 2,4-DNT (10 mg/L) was added as an inducer, and the mixture was left at 37 ℃ for 0 hour, and samples were taken every 1 hour to allow the mixture to standThe green fluorescence intensity was measured with a microplate reader, while the wild type strain containing the original promoter element yqjF was used as a positive control, with three in each group being in parallel. As shown in FIG. 6, the fluorescence signal values of the YY011, YY014 and YY007 strains were 4.6, 3.12 and 2.4 times as high as that of the yqjF wild-type strain at 300 minutes of induction.

3.4 determination of the Activity of the promoter element for detecting explosive gas molecules

An activated strain comprising: coli DH5 α (negative control), strain containing yqjF promoter element, YY011, YY014, YY007 strain. The above strains were inoculated in 10mL of LB medium at 1% inoculum size, respectively, and cultured at 37 ℃ and 200 rpm. 10mL of the bacterial liquid is collected, centrifuged for 5min at 4 ℃ and 8000Xg, the supernatant is discarded, 4mL of M9 liquid culture medium is added for washing, and the suspension is resuspended in 350 mu L M9 culture medium. Agar (final concentration 1.5%) was added to M9 broth, heated, added to 6-well plates, 3mL per well, and filters added after coagulation. 400 μ L of the thawed 1.5% M9 solid medium was mixed with the resuspended pellet solution, three drops of 20 μ L were added to each filter. 50mg of 2,4-DNT powder are weighed out in a closed container and dispersed as far as possible. The prepared 6-well plate was fixed to the lid of the vessel with a rubber band, the lid was closed, and the vessel was cultured by standing at 37 ℃. M9 medium containing 6g/L disodium hydrogen phosphate, 3g/L potassium dihydrogen phosphate, 1g/L ammonium chloride, and 0.5g/L sodium chloride, glucose (final concentration is 2%) and MgSO 2 are added before use₄(final concentration is 1 mM). As shown in fig. 7, the detection results showed that the YY011, YY014, and YY007 strains had significant fluorescence intensities compared to the strains of escherichia coli DH5 α (negative control) and yqjF promoter element, and the highest fluorescence intensity of YY011 among them, indicating that 3 strains of YY011, YY014, and YY007 contained promoter elements with significantly improved sensitivity for 2,4-DNT detection.

Therefore, the invention obtains 3 promoter elements with obviously improved 2,4-DNT detection sensitivity by large-scale screening of Rhodopseudomonas palustris genome library, the nucleotide sequences of the promoter elements are respectively SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, and the promoter elements obtained by the invention can be prepared into expression cassettes, recombinant vectors, recombinant microorganisms or transgenic cell lines and the like so as to achieve wider application scenes.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.

Sequence listing

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<120> novel promoter element for improving sensitivity of detection of explosive molecules, screening method and application thereof

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Claims (2)

1. A promoter element for improving sensitivity of detection of an explosive molecule, wherein the promoter element has a nucleotide sequence selected from one of the following sequences:

(1) a nucleotide sequence shown as SEQ ID NO. 3;

(2) a nucleotide sequence shown as SEQ ID NO. 4;

(3) the nucleotide sequence shown as SEQ ID NO. 5.

2. Use of the promoter element of claim 1 in the preparation of a biological agent for increasing sensitivity of detection of an explosive molecule.

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