Sensing element suitable for explosive molecule detection under high-temperature condition and application of sensing element

Abstract

The invention discloses an explosive molecule detection sensing element applicable to a high-temperature condition and application of the explosive molecule detection sensing element. The promoter element is derived from a genome of pseudomonas putida, a nucleotide sequence shown in SEQ ID NO.1 is obtained through transcriptomics analysis and screening and escherichia coli high-temperature screening, and the promoter element has the obvious explosive molecule detection capacity under the high-temperature condition. In addition, the method has the advantage of adapting to high-temperature, green and efficient detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and in particular relates to a sensing element for detecting explosive molecules under high temperature conditions based on transcriptomics analysis and an application thereof. Background Art

[0002] Explosives are widely used in the military, which not only threatens the lives of people in conflict areas, but also causes serious soil and water pollution. At present, in many war-torn areas, there are many explosive remnants such as mines, and traditional mine detection methods generally have certain limitations. Therefore, explosive detection biosensors developed based on molecular biology and synthetic biology have emerged and have made a series of progress. This type of biosensor is mainly based on volatile characteristic explosive molecules, such as 2,4-dinitrotoluene (2,4-DNT), etc., for specific detection of explosives.

[0003] As wars frequently break out in the Middle East and many parts of Africa, the ambient temperature is generally high, which limits the application of most biosensors. Therefore, it is necessary to develop biosensors suitable for high temperature conditions. At present, there are many reports on biosensors that sense 2,4-DNT under normal temperature conditions. Therefore, it is feasible to further develop explosive molecular biosensors suitable for high temperature conditions on this basis.

[0004] Pseudomonas putida ( Pseudomonas putida ) is a model microorganism that can degrade aromatic compounds. It can efficiently degrade toluene, m-xylene, etc. Therefore, the promoter elements that can sense explosive molecules can be screened and analyzed through transcriptomics stimulated by explosive molecules, and further combined with reporter elements to achieve efficient detection of explosive molecules. Escherichia coli ( Escherichia coli ) is a common laboratory model strain that can survive at a high temperature of 42°C. Therefore, the selected inductive promoter can be integrated with a luminescent reporter element and transferred into Escherichia coli to construct a biosensor that can be used for explosive molecule detection under high temperature conditions. Explosive detection sensors suitable for high temperature conditions will be able to effectively realize explosive detection tasks in specific high temperature areas in the future, which is of great significance to the development of detection technology for leftover explosives. Summary of the invention

[0005] The purpose of the present invention is to provide a sensing element for explosive molecule detection under high temperature conditions and its application. The present invention explores potential sensing elements in the genome of Pseudomonas putida and further screens the target sensing elements through high temperature of Escherichia coli.

[0006] In order to achieve the above-mentioned invention object, the present invention adopts the following technical solutions: The present invention provides a sensing element suitable for explosive molecule detection and its application, wherein the promoter element has one of the following nucleotide sequences: (1) the nucleotide sequence shown in SEQ ID NO.1; (2) the nucleotide sequence shown in SEQ ID NO.2; (3) The nucleotide sequence shown in SEQ ID NO.3.

[0007] A sensing element for detecting explosive molecules under high temperature conditions, characterized in that the sensing element for detecting explosive molecules has the following nucleotide sequence: (1) the nucleotide sequence shown in SEQ ID NO.1; Furthermore, the sensing element has the ability to respond to explosive molecules under high temperature conditions.

[0008] The present invention also provides a method for constructing the explosive molecule detection sensing element, comprising the following steps: (1) Stimulate Pseudomonas putida with explosive molecules, extract RNA from Pseudomonas putida, and perform transcriptomic analysis; (2) Using the N-pBR-C55-luxPleio plasmid to amplify the N-pBR-luxPleio large framework, in preparation for the construction of the target plasmid; (3) Extract the Pseudomonas putida genome as a template and amplify the predicted promoter elements from transcriptomics analysis to obtain PCR products; (4) Purify and recover the luxPleio large framework amplified fragment and the PCR product fragment, fuse the large framework with the fragment, and transform it into competent cells to obtain a recombinant strain; (5) Single colonies were selected to activate the recombinant strains, and different concentrations of 2,4-DNT were used to induce the strains for spontaneous luminescence detection at room temperature. The recombinant plasmids with obvious luminescence effects were then transferred into Escherichia coli BW25113 and the strains were spontaneously detected at high temperature under the same induction conditions to screen the strains that can sense 2,4-DNT under high temperature conditions. The promoter sequence in the strains is the target element.

[0009] Furthermore, the stimulating concentration of the explosive in step (1) is 0.15 mM-0.45 mM.

[0010] Preferably, the stimulating concentration of the explosive in step (1) is 0.22 mM.

[0011] Furthermore, the amplification primer sequences in step (2) are: lux-F, lux-R.

[0012] Furthermore, the nucleotide sequence of the amplified sequence in step (3) is shown as SEQ ID NO.1-NO.3.

[0013] The invention also provides the application of the sensing element to detect explosive molecules in real time under high temperature conditions.

[0014] Furthermore, the sensitivity of the detection sensing element for the explosive molecules under high temperature conditions is ≥ 0.1 mg / L.

[0015] Furthermore, the explosive molecules include 1,3-DNB, 2,4-DNT, HMX cyclotetramethylene tetranitramine, and methyl benzoquinone.

[0016] The present invention has the advantages that: The present invention obtains a promoter sensing element that responds to explosive molecules under high temperature conditions through explosive molecule stimulation and transcriptomics analysis, and then obtains the final target sensing element through high temperature screening. This element realizes the detection of explosive molecules under high temperature conditions for the first time. This method is green and pollution-free, has high detection efficiency, and can realize detection under high temperature conditions, which increases the possibility of on-site detection and can also be applied to the detection of contaminated wastewater, land, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the recombinant plasmid map for constructing p1-luxPleio.

[0018] Figure 2 This is the recombinant plasmid map for constructing p2-luxPleio.

[0019] Figure 3 To construct the recombinant plasmid map of p3-luxPleio.

[0020] Figure 4 This is the result of induced spontaneous luminescence detection of the constructed p1 engineering strain.

[0021] Figure 5 The results of induced spontaneous luminescence detection of the constructed p2 engineering strain.

[0022] Figure 6 The results of induced spontaneous luminescence detection of the constructed p3 engineering strain.

[0023] Figure 7 This is the result of the self-luminescence detection induced by the negative control BW25113.

[0024] Figure 8 This is the result of high temperature induced self-luminescence detection of the constructed p1 engineering strain.

[0025] Fig. 9 This is the result of high temperature induced self-luminescence detection of the constructed p2 engineering strain.

[0026] Fig.10 This is the result of high temperature induced self-luminescence detection of the constructed p3 engineering strain.

[0027] Fig.11 This is the result of high temperature induced self-luminescence detection of negative control BW25113.

[0028] Fig.12 The high-temperature self-luminescence detection results of the sensitivity test of the constructed p1 engineering strain.

[0029] Fig.13 This is the result of high-temperature self-luminescence detection of the substrate specificity of the constructed p1 engineering strain. Fig.14 This is the Ratio result of the substrate-specific high-temperature self-luminescence detection of the constructed p1 engineering strain. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments.

[0031] Example 1 1. Pseudomonas putida ( Pseudomonas putida ) and total RNA extraction (1) Prepare LB medium for Pseudomonas putida, the formula is as follows (1 L liquid medium): Aliquot into 20 mL culture medium and sterilize at 121°C for 20 min.

[0032] LB solid plate (1L solid culture medium): (2) Inoculation: Take out the tube of Pseudomonas putida from the -80 ℃ refrigerator, use the tip of a gun to take a small amount of bacteria and streak it on an LB solid plate. After placing the plate in a 37 ℃ incubator for 12 h, take a single colony on the plate and activate it in LB liquid culture medium at 37 ℃ overnight. The culture medium volume is 20 mL and then transferred to new LB liquid culture medium at a rate of 1%.

[0033] (3) Genome extraction: The well-cultured strains were stimulated with 0.22 mM 1,3-DNB and the genome was extracted from the stimulated bacterial solution.

[0034] (4) The total RNA of Pseudomonas putida was extracted using the Trizol method, and transcriptomics processing was performed by Meiji Biotechnology.

[0035] The steps for extracting RNA are as follows: 1) Cultivate the strain to an appropriate growth period, and collect 2 mL of bacterial solution by centrifugation at 12,000 rpm for 1 min; 2) Add 1 mL of Trizol reagent to the collected bacteria, mix well, shake for 1 min, centrifuge at 12,000 rpm for 5 min, and transfer the supernatant to a new centrifuge tube; 3) Add 200 µL of chloroform to the supernatant, shake well, let stand at room temperature for 2 min, and then centrifuge at 13,300 rpm in a 4°C centrifuge for 5-10 min; 4) Transfer 400 µL of the supernatant from the aqueous phase to a new RNase-free centrifuge tube, then add an equal volume of isopropanol, mix well, and centrifuge at 13,300 rpm in a 4 °C centrifuge for 5-10 min; 5) After discarding the supernatant, add 500 µL of 75% alcohol to the centrifuge tube, gently invert to mix, centrifuge at 13,300 rpm for 5 min at 4 °C, discard the supernatant, and place the centrifuge tube in a clean bench for 5-7 min to allow the alcohol remaining on the wall of the centrifuge tube to completely evaporate; 6) Finally, dissolve the extracted RNA with 50 µL of DEPC water. If necessary, gently blow with a pipette tip to completely dissolve the RNA. Then take 1 µL of RNA and run it on a 1% agarose gel electrophoresis to check whether the RNA is degraded or contaminated with proteins. At the same time, take 1 µL of RNA and test the purity and concentration of RNA under NanoDrop 2000. The remaining RNA can be used for reverse transcription immediately or stored at -80 °C for later use.

[0036] (5) Data table showing that transcriptome was significantly upregulated after stimulation with 2,4-DNT. Three groups of significantly upregulated genes, TX55_RS95352, TX55_RS11647, and TX55_RS83451, were selected and their promoters were predicted and named p1, p2, and p3, respectively.

[0037] 2. Extract the genome of Rhodopseudomonas palustris: The genomic DNA from Pseudomonas putida KT2440 was extracted using the Vazyme genomic bacterial extraction kit. The extraction steps are as follows: 1) Take 1-5 mL of bacterial culture medium, centrifuge at 10,000 rpm for 1 min, and discard the culture medium; 2) Add 230 µL Buffer GA and shake until the bacteria are completely suspended; 3) Add 20 µL Proteinase K and vortex to mix; 4) Add 250 µL Buffer GB, vortex to mix, and incubate in a 70°C water bath for 10 min; 5) If the residual RNA has a significant impact on subsequent experiments, add 4 µL RNase A to the digestion solution, shake for 15 sec, and leave at room temperature for 5-15 min; 6) Add 180 µL of anhydrous ethanol and vortex to mix. Flocculent precipitation may appear. Centrifuge briefly to collect the liquid on the inner wall of the tube cap. 7) Transfer the above mixed solution to the FastPure gDNA Mini Columns |II adsorption column (the adsorption column has been placed in the collection tube), centrifuge at 12,000 rpm for 1 min, and discard the filtrate; 8) Add 500 µL Buffer PB (please check whether anhydrous ethanol has been added before use) to the adsorption column, centrifuge at 12,000 rpm for 1 min, and discard the filtrate; 9) Add 600 µL Buffer PW (please check whether anhydrous ethanol has been added before use) to the adsorption column, centrifuge at 12,000 rpm for 1 min, and discard the filtrate; 10) Repeat step 4; 11) Place the adsorption column back into the collection tube and centrifuge the empty tube at 12,000 rpm for 2 min; (After centrifugation of the empty column, the lid can be opened and left for 2-5 minutes to allow the residual ethanol to evaporate completely.) 12) Transfer the adsorption column to a new 1.5 ml centrifuge tube (self-prepared), add 50-100 µL Elution Buffer to the center of the adsorption column membrane, leave at room temperature for 2-5 min, and centrifuge at 12,000 rpm for 1 min; 13) Discard the adsorption column and store the DNA product at -30 ~ -15°C to prevent degradation.

[0038] Example 2 1. Construction of recombinant strains containing p1 sensing elements (1) luxPleio framework expansion Using N-pBR-C55-luxPleio as a template, lux-F and lux-R were used for polymerase chain reaction (PCR) to amplify the luxPleio fragment. The PCR amplification system is as follows: The PCR program was: 35 cycles × (98°C for 10 s, 60°C for 15 s, 72°C for 2 min); 68°C for 5 min; 16°C for 10 min.

[0039] The PCR products were purified using the Vazyme Liquid Recovery Purification Kit.

[0040] Liquid recovery and purification kit (Vazyme, Cat. No.: DC301-01) Steps: 1) Add 5 volumes of Buffer GDP to the centrifuge tube containing the amplified product and vortex to mix well.

[0041] 2) After a brief centrifugation, place the Fast Pure DNA Mini Columns-G adsorption column in the Collection Tubes 2 mL collection tube, transfer ≤700 µL of the sol to the adsorption column, and centrifuge at 12,000 rpm for 1 min.

[0042] 3) Discard the waste liquid, add 700 µL Buffer GW (with ethanol) to the adsorption column, and centrifuge at 12,000 rpm for 1 min.

[0043] 4) Repeat step 3).

[0044] 5) Discard the waste liquid and leave it in the air for 2 minutes.

[0045] 6) Place the adsorption column in a 1.5 mL centrifuge tube, dry it in a 55°C metal bath for 7 min, add 50 µL of ddH2O, let it stand for 2 min, centrifuge for 2 min, elute the DNA and store it at -20°C. Label the name and date.

[0046] (2) Using the genome of Pseudomonas putida as a template, primers p1-F and primers p1-R were used to perform polymerase chain reaction (PCR) to amplify the p1 fragment, the nucleotide sequence of which is shown in SEQ ID NO. 1. The PCR amplification system is as follows: The PCR program was: 98°C 40 s; 35 cycles × (98°C 10 s, 60°C 15 s, 72°C 30 s); 72°C 5 min; 16°C 10 min.

[0047] The PCR product was purified by liquid recovery using the Vazyme liquid recovery purification kit, and the eluted DNA was stored at -20°C and labeled with the name and date.

[0048] (3) Using Vazyme's 2×ClonExpress Mix, the purified luxPleio vector and the sensor element gene were mixed in a certain ratio in 10 μL fusion reaction system and incubated in a 50°C water bath for 30 min; The fusion system is: (4) Transform the fusion system into DH5ɑ, spread it on an ampicillin-resistant LB plate, grow it at 37°C overnight, pick a single colony on the plate for bacterial P verification, and inoculate bacteria after successful verification. Identify the gene sequence of the recombinant plasmid by sequencing. The vector map is shown in Figure 1 shown.

[0049] (5) The bacteria that were successfully sequenced were streaked, grown on LB resistance plates at 37°C, and the single bacteria were extracted and grown overnight at 37°C to extract the plasmid, which was then transferred into competent E. coli BW25113, coated on LB resistance plates, and grown overnight at 37°C. Finally, a recombinant strain containing the p1 sensing element was obtained.

[0050] Example 3 1. Construction of recombinant strains containing p2 sensing elements (1) LuxPleio vector amplification The steps are the same as in Example 2.

[0051] (2) Using the genome of Pseudomonas putida as a template, primers p2-F and primers p2-R were used to perform polymerase chain reaction (PCR) to amplify the p2 fragment, the nucleotide sequence of which is shown in SEQ ID NO. 2. The PCR amplification system is as follows: The PCR program was: 98°C 40 s; 35 cycles × (98°C 10 s, 60°C 15 s, 72°C 16 s); 72°C 5 min; 16°C 10 min.

[0052] The PCR product was recovered using the Vazyme liquid recovery purification kit, and the eluted DNA was stored at -20°C and labeled with the name and date.

[0053] (3) Using Vazyme's 2×ClonExpress Mix, the purified luxPleio vector and the sensor element gene were mixed in a certain ratio in 10 μL fusion reaction system and incubated in a 50°C water bath for 30 min; The fusion system is: (4) Transform the fusion system into DH5ɑ, spread it on an ampicillin-resistant LB plate, grow it at 37°C overnight, pick a single colony on the plate for bacterial P verification, and inoculate bacteria after successful verification. Identify the gene sequence of the recombinant plasmid by sequencing. The vector map is shown in Figure 2 shown.

[0054] (5) The bacteria that were successfully sequenced were streaked, grown on LB resistance plates at 37°C, and the single bacteria were extracted and grown overnight at 37°C to extract the plasmid, which was then transferred into competent E. coli BW25113, coated on LB resistance plates, and grown overnight at 37°C. Finally, a recombinant strain containing the p2 sensing element was obtained.

[0055] Example 4 1. Construction of recombinant strains containing p3 sensing elements (1) LuxPleio vector amplification The steps are the same as in Example 2.

[0056] (2) Using the genome of Pseudomonas putida as a template, primers p3-F and primers p3-R were used to perform polymerase chain reaction (PCR) to amplify the P3 fragment, the nucleotide sequence of which is shown in SEQ ID NO. 3. The PCR amplification system is as follows: The PCR program was: 98°C 40 s; 35 cycles × (98°C 10 s, 62°C 15 s, 72°C 16 s); 72°C 5 min; 16°C 10 min.

[0057] The PCR product was purified by liquid recovery using the Vazyme liquid recovery purification kit, and the eluted DNA was stored at -20°C and labeled with the name and date.

[0058] (3) Using Vazyme's 2×ClonExpress Mix, the purified luxPleio vector and the sensor element gene were mixed in a certain ratio in 10 μL fusion reaction system and incubated in a 50°C water bath for 30 min; The fusion system is: (4) Transform the fusion system into DH5ɑ, spread it on an ampicillin-resistant LB plate, grow it at 37°C overnight, pick a single colony on the plate for bacterial P verification, and inoculate bacteria after successful verification. Identify the gene sequence of the recombinant plasmid by sequencing. The vector map is shown in Figure 3 shown.

[0059] (5) The bacteria that were successfully sequenced were streaked, grown on LB resistance plates at 37°C, and the single bacteria were extracted and grown overnight at 37°C to extract the plasmid, which was then transferred into competent E. coli BW25113, coated on LB resistance plates, and grown overnight at 37°C. Finally, a recombinant strain containing the p3 sensor element was obtained.

[0060] Example 5 1. Self-luminescence detection of explosives by recombinant strains at room temperature (1) Single colonies of the three recombinant strains p1, p2, and p3 were picked and placed in 20 mL LB medium, activated at 37°C overnight, and transferred to M9 liquid medium with a final concentration of 2% glucose and 1 mM magnesium sulfate solution at a 2% inoculum. The culture was continued at 37°C in a shaking incubator until the OD 600 Between 0.2-0.25, a certain concentration of 2,4-DNT was added for induction.

[0061] The specific steps are as follows: first prepare 10 mg / L of 2,4-DNT as a mother solution, and use the mother solution to prepare and dilute 2,4-DNT with different concentration gradients, and finally form final concentrations of 0 mg / L, 1 mg / L, 10 mg / L, and 50 mg / L. Take a black 96-well ELISA plate, add 98 µL of bacterial solution to each well, add 2 µL of 2,4-DNT of different concentrations, 3 parallel experimental groups, add a negative control, and then seal the plate with a transparent sealing film. Use an ELISA instrument to perform continuous measurements for several hours, keep a constant temperature of 30°C, and detect the RLU value every 10 min. According to the values ​​of the ELISA instrument, process the experimental data and draw images, such as Figure 4-Figure 6 This indicates that the three recombinant strains have the ability to respond to 2,4-DNT at room temperature.

[0062] 2. Self-luminescence detection of explosives by recombinant strains under high temperature conditions (1) Single colonies of the three recombinant strains p1, p2, and p3 were picked and placed in 20 mL LB medium, activated at 37°C overnight, and transferred to M9 liquid medium with a final concentration of 2% glucose and 1 mM magnesium sulfate solution at a 2% inoculum. The culture was continued at 37°C in a shaking incubator until the OD 600 Between 0.2-0.25, a certain concentration of 2,4-DNT was added for induction.

[0063] The specific steps are as follows: first prepare 10 mg / L of 2,4-DNT as a mother solution, and use the mother solution to prepare and dilute to obtain different concentration gradients of 2,4-DNT, and finally form final concentrations of 0 mg / L, 1 mg / L, 10 mg / L, and 50 mg / L. Take a black 96-well ELISA plate, add 98 µL of bacterial solution to each well, add 2 µL of different concentrations of 2,4-DNT, 3 parallel experimental groups, add a negative control, and seal the plate with a transparent sealing film. Use an ELISA instrument to perform continuous measurements for several hours, keep a constant temperature of 42°C, and detect the RLU value every 10 min. According to the values ​​of the ELISA instrument, process the experimental data and draw images, such as Figure 8-Figure 10 This indicates that the recombinant strain containing the p1 sensing element significantly responds to 2,4-DNT under high temperature conditions.

[0064] 3. Sensitivity test A single colony of the recombinant strain containing the p1 sensing element was picked and placed in 20 mL LB medium, activated overnight at 37°C, cultured in a shaking incubator at 37°C for 8-10 hours, and transferred to M9 liquid medium with a final concentration of 2% glucose and 1 mM magnesium sulfate solution according to a 2% inoculation volume to set the sensitivity of the 2,4-DNT detection strain to sense 2,4-DNT at different final concentrations.

[0065] The specific steps are as follows: first prepare 10 mg / L of 2,4-DNT as a mother solution, and use the mother solution to prepare and dilute 2,4-DNT with different concentration gradients, and finally form final concentrations of 0 mg / L, 0.001 mg / L, 0.01 mg / L, 0.05 mg / L, 0.1 mg / L, 0.5 mg / L, and 1 mg / L 2,4-DNT. Take a black 96-well ELISA plate, add 98 µL of bacterial solution to each well, add 2 µL of 2,4-DNT of different concentrations, 3 parallel experimental groups, add a negative control, and then seal the plate with a transparent sealing film. Use an ELISA reader to perform continuous measurements for several hours, keep a constant temperature of 42°C, and detect the RLU value every 10 min. According to the values ​​of the ELISA reader, process the experimental data and draw images, such as Fig.12 This indicates that the sensitivity of the recombinant strain containing the P1 sensing element to the detection of explosive molecules reaches 0.01 mg / L.

[0066] 3. Substrate specificity test Pick a single colony of the recombinant strain containing the p1 sensor element and place it in 20 mL LB medium, activate it overnight at 37°C, and culture it in a shaking incubator at 37°C for 8-10 hours. Then, transfer it to M9 liquid medium with a final concentration of 2% glucose and 1 mM magnesium sulfate solution according to a 2% inoculum, and continue to culture it in a shaking incubator at 37°C until the OD reaches 0. 600 The pH value is between 0.2 and 0.25, and 1,3-DNB, 2,4-DNT, HMX cyclotetramethylene tetranitramine and methyl benzoquinone are added respectively.

[0067] The specific steps are as follows: first prepare a mother solution concentration of 10 mg / mL, and prepare different concentration gradients of 1,3-DNB, 2,4-DNT, HMX cyclotetramethylene tetranitramine, and methyl benzoquinone to obtain final concentrations of 0 mg / L and 1 mg / L, respectively. Add 98 µL of bacterial solution to each well, and then add 2 µL of 1,3-DNB, 2,4-DNT, HMX cyclotetramethylene tetranitramine, and methyl benzoquinone, respectively. There are three parallel experimental groups. Add a negative control and seal the plate with a transparent sealing film. Seal with a transparent sealing film. Use an ELISA reader to perform continuous measurements for several hours, keep a constant temperature of 42°C, and detect the RLU value every 10 min. According to the values ​​of the ELISA reader, process the experimental data and draw images, such as Fig.13 This indicates that the recombinant strain containing the p1 sensing element has a significant response to explosive molecules under high temperature conditions.

[0068] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A sensing element for explosive molecule detection and its application, characterized in that: The explosive molecule detection sensing element has one of the following nucleotide sequences: (1) the nucleotide sequence shown in SEQ ID NO.1; (2) the nucleotide sequence shown in SEQ ID NO.2; (3) The nucleotide sequence shown in SEQ ID NO.

3.

2. A sensing element for detecting explosive molecules under high temperature conditions and its application, characterized in that: The explosive molecule detection sensing element has the following nucleotide sequence: (1) The nucleotide sequence shown in SEQ ID NO.

1.

3. The method for constructing the explosive molecule-sensing high temperature promoter element according to claim 1, characterized in that: The following steps are involved: (1) Pseudomonas putida was stimulated with the explosive molecule 2,4-DNT, and RNA of Pseudomonas putida was extracted for transcriptomic analysis; (2) using N-pBR-C55-luxPleio plasmid to amplify the N-pBR-luxPleio large framework to prepare for the construction of the target plasmid; (3) extracting the Pseudomonas putida genome as a template, amplifying the predicted promoter elements from transcriptomic analysis to obtain PCR products; (4) purifying and recovering the luxPleio large framework amplified fragment and the PCR product fragment, fusing the large framework with the fragment, and transforming into competent cells to obtain a recombinant strain; (5) Single colonies were selected to activate the recombinant strains, and different concentrations of 2,4-DNT were used to induce the strains for spontaneous luminescence detection at room temperature. The recombinant plasmids with obvious luminescence effects were then transferred into Escherichia coli BW25113 and the strains were spontaneously detected at high temperature under the same induction conditions to screen the strains that can sense 2,4-DNT under high temperature conditions. The promoter sequence in the strains is the target element.

4. The construction method as claimed in claim 2, characterized in that The method comprises the following steps: the explosive molecule in step (1) has a stimulation concentration of 0.15 mM to 0.45 mM in 20 mL of culture medium.

5. The screening according to claim 3, characterized in that The large framework sequences amplified in step (2) are: lux-F, lux-R.

6. The construction method according to claim 2, characterized in that: The nucleotide sequences of the amplification primers in step (3) are shown in SEQ ID NO.4-NO.

11.

7. The promoter element according to claim 1 senses 2,4-DNT under high temperature conditions and responds to the signal of the recombinant strain.

8. The use according to claim 6, characterized in that: The detection concentration of the explosive molecules is not less than 1 mg / L under high temperature conditions.

9. The promoter element of claim 1 is used in a biosensor for detecting explosive molecules under high temperature conditions.

10. The use according to claim 8, characterized in that: The explosive molecules mainly include 1,3-DNB, 2,4-DNT, HMX cyclotetramethylene tetranitramine, and methyl benzoquinone.

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