CN114410556B

CN114410556B - Biosensor for responding to explosive molecules by using transcription regulatory protein, and preparation method and application thereof

Info

Publication number: CN114410556B
Application number: CN202210026960.XA
Authority: CN
Inventors: 王兆宝; 杨建明; 马冉; 于晓彤; 汤若昊; 李美洁
Original assignee: Qingdao Agricultural University
Current assignee: Qingdao Agricultural University
Priority date: 2022-01-11
Filing date: 2022-01-11
Publication date: 2023-12-19
Anticipated expiration: 2042-01-11
Also published as: CN114410556A

Abstract

The invention discloses a biosensor for responding to explosive molecules by using transcription regulatory proteins, a preparation method and application thereof. The biosensor comprises self-luminescenceluxABCDEOperon, mexT regulatory protein inducing explosive molecules and promoter P thereof _mexT Downstream protein-regulated promoter P _MDR2 . The invention is verified by experiments that the MexT protein and the promoter P _MDR2 Has a strong interaction with it, which is able to activate promoter P _MDR2 Thereby improving the transcription of the self-luminous operator genes, realizing the signal cascade amplification effect, improving the sensitivity of detecting the 2,4-DNT of the explosive molecules, and being also used for detecting the explosive molecules in real time, and having simple preparation method and wide application.

Description

Biosensor for responding to explosive molecules by using transcription regulatory protein, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a biosensor for responding to explosive molecules by using transcription regulatory proteins, and a preparation method and application thereof.

Background

The biological induction technology is to modify bacterial strain by genetic engineering means, so that after the induction element in the microorganism induces specific compounds or metabolites of the specific compounds in the microorganism, the microorganism report element generates detectable changes, such as various fluorescence and self-luminescence induction signals which can be detected, thereby achieving the purpose of detecting the specific compounds. For biosensors, the sense element is important, and mainly includes a promoter for gene transcription, a ribosome binding site, a terminator, a transcription regulator, and the like.

Residual explosives (such as mines) in a war zone can cause irreparable damage to life safety and ecological environment, so that the safety and effective detection of the mines has important strategic and ecological significance. 2, 4-Dinitrotoluene (DNT) is the major component of the gas phase generated by explosive TNT and is often used as a characteristic chemical for detecting the presence of explosives because of its relatively high volatility. The conventional explosive detection technology cannot realize remote detection, and has great danger and low sensitivity in the detection process, and the residual mine is detected by the biological induction detection technology. Burland et al therefore propose a biological solution to disseminate genetically engineered microbial bioreactors in a target detection zone, allowing for remote location of the location of explosives. Numerous research and development efforts have been conducted to develop sensing 2,4-DNT biosensors, including in vivo systems with whole cell biosensors and in vitro systems using purified proteins/peptides. Shimshon Belkin et al screen for a 2,4-DNT sensor yqjF promoter from E.coli and optimize it to increase the sensitivity of the detection.

Pseudomonas aeruginosa (Pseudomonas aeruginosa) belongs to Pythium gracile and is a model environmental microorganism strain with the function of degrading harmful aromatic and aliphatic compounds in the environment. A LysR global transcription regulator MexT in Pseudomonas aeruginosa consists of an N-terminal DNA binding domain and a C-terminal Regulatory Domain (RD). MexT has an arginine-rich region and a hydrophobic end arranged by a variable ring, both of which are putative ligand binding sites capable of binding to both hydrophobic and negatively charged ligands, and thus molecules featuring polar aldehyde moieties and hydrophobic benzene rings are capable of affecting MexT and altering its association with DNA, demonstrating that transcriptional modulator-protein MexT has the ability to function as an element of an effective aromatic molecule. However, the use of transcriptional regulatory proteins to respond to explosive molecules to produce biosensors has not been reported.

Disclosure of Invention

The invention aims to provide a biosensor responding to explosive molecules by using transcription regulatory proteins, a preparation method and application thereof, wherein the biosensor has the capability of obviously sensing and detecting 2,4-DNT.

In order to achieve the aim of the invention, the invention is realized by adopting the following technical scheme:

the invention provides a biosensor for responding to explosive molecules by using transcription regulatory proteins, wherein the biosensor comprises a self-luminous operator, the transcription regulatory proteins and a promoter.

Furthermore, the self-luminous operon is luxABCDE operon from the luminous bacillus of the fish, and the nucleotide sequence of the luxABCDE operon is shown as SEQ ID NO. 1.

Further, the promoter is a promoter P derived from pseudomonas aeruginosa _MDR2 And promoter P _mexT The method comprises the steps of carrying out a first treatment on the surface of the The promoter P _MDR2 The nucleotide sequence of (2) is shown as SEQ ID NO. 4.

Further, the transcription regulatory protein is a MexT protein derived from Pseudomonas aeruginosa, and the MexT protein and a promoter P thereof _mexT The nucleotide sequence of (2) is shown as SEQ ID NO. 7.

Further, the MexT protein and promoter P _MDR2 Has strong interaction; the MexT protein is capable of activating promoter P _MDR2 Thereby enhancing transcription of the self-luminescent operator gene.

The invention also provides a preparation method of the biosensor, which comprises the following steps:

(1) Amplifying self-luminescent luxABCDE operon and P _MDR2 After the promoter gene fragment and purification recovery, the promoter gene fragment and the promoter gene fragment are connected to pACYCDuet-1 plasmid which is subjected to double enzyme digestion, competent cells are transformed by the product, positive clones are screened on LB solid plates containing antibiotics, and recombinant plasmid P-P is obtained _MDR2 -luxPleio；

(2) Amplification of MexT-P _mexT After the gene fragment is purified and recovered, the gene fragment is connected to P-P subjected to single enzyme digestion _MDR2 On the luxPLeio plasmid, the product is transformed into competent cells, and positive clones are screened on LB solid plates containing antibiotics to obtain recombinant plasmid P-P _mexT -MexT-P _MDR2 -luxPleio；

(3) Recombinant plasmid P-P _mexT -MexT-P _MDR2 The luxPleio is transformed into competent cells to obtain engineering strain MEXT, and the engineering strain MEXT is cultured and activated in LB liquid medium containing antibiotics and then transferred into M9 medium for culture to obtain the biosensor.

The invention also provides application of the biosensor in real-time detection of explosive molecules.

Further, the use method of the biosensor comprises the following steps: and uniformly mixing the biosensor with a sample to be detected in a volume ratio of 9:1, adding a pure white ELISA plate, sealing, and monitoring the self-luminous intensity at a constant temperature of 30 ℃ by using an ELISA meter in real time, wherein the self-luminous intensity is monitored once every 10min, and the total monitoring time is 12h.

Further, the concentration range of the explosive molecules detected by the biosensor is as follows: 1 mug/L-10 mg/L.

Further, the explosive molecules include 2,4-DNT, 1,3-DNB, catechol, hydroquinone, and methyl benzoquinone.

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

the invention screens out a protein regulatory element MexT capable of inducing explosive molecules from pseudomonas aeruginosa for degrading various aromatic compounds, and uses the protein regulatory element MexT to downstream P _MDR2 Activation of the promoterA novel biological sensor. Unlike the traditional explosive detection biosensor which utilizes a promoter element to sense 2,4-DNT, the biosensor obtained by the method utilizes regulatory protein to sense 2,4-DNT, can realize cascade amplification of detection signals through regulating and controlling a downstream promoter, and remarkably improves the capability of detecting sensitivity of explosive molecules 2, 4-DNT; the invention verifies the MexT protein and the promoter P through experiments _MDR2 Has a strong interaction with it, which is able to activate promoter P _MDR2 Thereby enhancing transcription of the self-luminescent operator gene. Therefore, the biological sensor screened by the invention can be used for detecting explosives such as mines on military and the like, can also be used for detecting environmental pollutants in an ecological system, and has important significance for military, environmental protection and the like.

Drawings

FIG. 1 shows the constructed vector P-P _MDR2 Plasmid map of luxpleio.

FIG. 2 is a constructed vector P-P _mexT -MexT-P _MDR2 Plasmid map of luxpleio.

FIG. 3 is a plasmid map of the constructed vector pET28 a-MexT.

FIG. 4 is a diagram showing the verification of MexT protein and P _MDR2 Results of EMSA experiments of interactions.

FIG. 5 shows the results of self-luminescence gene LuxC RT-qPCR.

Fig. 6 shows the detection results of the self-luminescence microplate reader of the engineering strains MEXT1 and MEXT 2.

Detailed Description

The technical scheme of the invention is further described in detail below with reference to specific embodiments.

Example 1: gene acquisition and vector construction

1. Gene acquisition

A luxABCDE operon derived from the A.mori (Photobacterium leiognathi) is chemically synthesized on pUC-57 vector by Huada genes company to obtain pUC-luxPleio vector, wherein the nucleotide sequence of the luxABCDE operon is shown as SEQ ID NO. 1.

2. Construction of vectors

The PCR is performed by using pUC-luxPleio as template, primer luxPleio-F and primer luxPleio-R, and the fragment of luxPleio is amplified as follows:

the PCR procedure was: 3min at 95 ℃;30 cycles X (95 ℃ C. 15s,58 ℃ C. 15s,72 ℃ C. 6 min); 72 ℃ for 5min;16 ℃ infinity. The primer sequences are shown below:

luxPleio-F：

5’-ATGATTAAGAAGATCCCAATG-3’(SEQ ID NO.2)；

luxPleio-R：

5’-TACCAGACTCGAGGGTACCTTACGTATAGCTAAATGCATCA-3’(SEQ ID NO.3)。

p was amplified using P.aeruginosa genome as template _MDR2 The nucleotide sequence of the promoter sequence is shown as SEQ ID NO.4, and the PCR amplification system is shown as follows:

the PCR procedure was: 3min at 95 ℃;30 cycles X (95 ℃ C. 15s,58 ℃ C. 15s,72 ℃ C. 2 min); 72 ℃ for 5min;16 ℃ infinity. The primer sequences are shown below:

P _MDR2 -F：5’-AGGTCGACAAGCTTGCGGCCGCTCGTTTCGATTCATCGG TG-3’(SEQ ID NO.5)；

P _MDR2 -R：5’-CATTGGGATCTTCTTAATCATCGCAAACCTCGCCTTTGTG AACC-3’(SEQ ID NO.6)。

the PCR product was recovered and purified using a gel recovery purification kit (Vazyme, cat. DC 301-01).

The pACYCDuet-1 plasmid was digested simultaneously with restriction enzyme 1Not I (TaKaRa, cat# 1623) and restriction enzyme 2Kpn I (TaKaRa, cat# 1618), and the digestion system is as follows:

the enzyme digestion system is incubated for 1h at 37 ℃ for gel recovery and purification.

P Using seamless cloning _MDR2 And the luxpleio fragment was cloned into pACYCDuet-1 plasmid, the system of which is as follows:

the ligation system was incubated at 50℃for 30min. E.coli DH5 alpha competence is converted by the connection product, the mixture is coated on an LB solid plate containing 34mg/L chloramphenicol, positive clones are screened by PCR, and recombinant plasmid P-P is extracted from the positive clones _MDR2 Luxpleio (fig. 1), and then identified by restriction and sequencing.

The genome of pseudomonas aeruginosa is used as a template to amplify the MexT protein and the promoter sequence thereof, the nucleotide sequence is shown as SEQ ID NO.7, and the PCR amplification system is shown as follows:

P _mexT -F：5’-TCGACAAGCTTGCGGCCGCTCGCAAACCTCTGCAGTGCAT-3’ (SEQ ID NO.8)；

MexT-R：5’-ATGAATCGAAACGAGCGGCCGCTCAGAGACTGTCCGGA TCG-3’(SEQ ID NO.9)。

the PCR product was gel-recovered and purified using gel recovery purification kit (Vazyme, cat. DC 301-01). At the same time, P-P is digested with restriction enzyme Not I (TaKaRa, cat 1623) _MDR2 The cleavage system of the luxpleio plasmid is:

MexT protein and promoter P thereof by using seamless cloning _mexT Cloning of fragments intop-P _MDR2 -on the luxpleio plasmid. The ligation system was incubated at 50℃for 30min. E.coli DH5 alpha competence is converted by the connection product, the mixture is coated on an LB solid plate containing 34mg/L chloramphenicol, positive clones are screened by PCR, and recombinant plasmid P-P is extracted from the positive clones _mexT -MexT-P _MDR2 Luxpleio (fig. 2), and then identified by restriction and sequencing.

Example 2: gel migration assay (EMSA) to verify MexT protein and P _MDR2 Interaction between promoters

1. Construction of pET28a-MexT plasmid

The genome of pseudomonas aeruginosa is used as a template, the primer pET28a-F and the primer pET28a-R are subjected to Polymerase Chain Reaction (PCR), and the MexT gene fragment is amplified, and the PCR amplification system is as follows:

pET28a-F：5’-TGCCGCGCGGCAGCCATATGCCTGTCAGTGATCCTATG-3’ (SEQ ID NO.10)；

pET28a-R：5’-TCGAGTGCGGCCGCAAGCTTTCAGAGACTGTCCGGAT-3’ (SEQ ID NO.11)。

the PCR product was gel-recovered and purified using gel recovery purification kit (Vazyme, cat. DC 301-01).

The pET28a plasmid was double digested with restriction enzyme 1 HindIII (TaKaRa, cat. 1615) and restriction enzyme 2 NdeI (TaKaRa, cat. 1621), the system was:

The MexT fragment was cloned into pET28a plasmid using T4 ligase as follows:

the ligation system was incubated at 22℃for 2h. BL21 was competent for transformation of the ligation product, plated on LB solid plates containing 50mg/L kanamycin, positive clones were PCR-screened, recombinant plasmid pET28a-MexT was extracted from the positive clones (FIG. 3), and identified by restriction enzyme digestion and sequencing.

2. Purification of MexT protein

(1) The strain was activated overnight at 37℃and transferred to 50mL of LB liquid medium containing kanamycin resistance, and cultured to OD ₆₀₀ ＝0.6。

(2) To the medium, 20. Mu.L of IPTG (final concentration: 0.2 mM) was added at a concentration of 500mM to the stock solution for induction.

(3) All bacteria were harvested by centrifugation at 8000rpm at 4℃for 3min, washed twice with PBS buffer and finally the pellet was resuspended in 5mL PBS.

(4) The bacterial liquid was sonicated, centrifuged at 13000rpm at 4℃for 5min, and the supernatant was collected.

(5) The supernatant was poured into His-tag protein purification resin to bind to it, gradient eluted with imidazole at different concentrations (50 mM, 150mM, 300mM, 500 mM), the protein was eluted at 300mM concentration, and finally the protein was concentrated by ultrafiltration.

3. Gel migration Experiment (EMSA)

Primer P using pseudomonas aeruginosa genome as template _MDR2 -2F and primer P _MDR2 -2R, polymerase Chain Reaction (PCR), amplification of P _MDR2 The PCR amplification system of the promoter fragment is as follows:

the PCR procedure was: 3min at 95 ℃;30 cycles X (95 ℃ 15s,58 ℃ 15s,72 ℃ 30 s); 72 ℃ for 5min;16 ℃ infinity. The primer sequences are shown below:

P _MDR2 -2F：5’-TCGTTTCGATTCATCGGTGC-3’(SEQ ID NO.12)；

P _MDR2 -2R：5’-CGCAAACCTCGCCTTTGTGAAC-3’(SEQ ID NO.13)。

EMSA experiment verifies MexT protein and P _MDR2 Interaction between promoters: to each of the small PCR tubes, 0ng, 50ng, 100ng, 150ng, 200ng, 300ng, 400ng, 500ng of protein and 1.5. Mu.L of binding reaction solution were added, respectively, and after 30 minutes of reaction, 40ng of PMD 2 promoter fragment was added to the system, followed by 30 minutes of reaction, and finally 3. Mu.L of loading buffer was added to each of the small PCR tubes. The reaction system is loaded into an EMSA non-deforming PAGE gel, 120V and 400mA are run for 60min, the gel migration experiment result is shown in figure 4, and P _MDR2 The promoter fragment was migrated in the band when 50ng of MexT protein was added, the larger the amount of protein, the more evident the upward migration of the nucleic acid fragment was, and when 400ng of protein was added, the protein and nucleic acid were all combined, indicating that MexT protein and P _MDR2 The promoter can be combined and has strong combining ability.

Example 3: RT-qPCR experiments prove that the transcription of self-luminous gene luxC is up-regulated

(1) The strain was activated overnight at 37℃and transferred to 10mL LB liquid medium containing 50mg/L kanamycin, and transferred to 6 bottles altogether, and cultured until OD ₆₀₀ ＝0.6。

(2) To the bacterial suspension, 20. Mu.L of 2,4-DNT (final concentration: 0.25. 0.25 mM) was added at a mother liquor concentration of 125mM, and 3 replicates were made, while 20. Mu.L of absolute ethanol was added to the other 3 bottles of bacterial suspension as a control.

(3) After stimulation with 2,4-DNT for 0.5h, 1h, 1.5h, respectively, centrifugation was performed at 8000rpm at 4℃for 3min, the supernatant was discarded, the pellet was resuspended in sterile water, the supernatant was discarded by centrifugation and washing was repeated once.

(4) RNA from the above 6 samples was extracted, and the extracted RNA was reverse transcribed into cDNA using a reverse transcription kit (Vazyme, cat. No. R312-02).

(5) The reverse transcribed cDNA was diluted to 200 ng/. Mu.L and the RT-qPCR internal reference gene gapdh with the following primer sequences:

gapdh-F：5’-TATGACTCCACTCACGGC-3’(SEQ ID NO.14)；

gapdh-R：5’-AACCACTTTCTTCGCACC-3’(SEQ ID NO.15)；

LuxC-F：5’-ACACCCGATCACACGTTCAA-3’(SEQ ID NO.16)；

LuxC-R：5’-AACCACATCAGCACAGCTCA-3’(SEQ ID NO.17)。

the PCR system is as follows: sample cDNA 2. Mu.L, upstream and downstream primers 0.4. Mu.L, 2x clamQ SYBR qPCR Master Mix 10. Mu. L, H, respectively ₂ O 7.2μL。

The RT-qPCR procedure was: 3min at 95 ℃;30 cycles X (95 ℃ C. 10s,60 ℃ C. 30s,72 ℃ C. 30 s).

As shown in FIG. 5, the transcription of the luxC gene was significantly up-regulated after stimulation with 2,4-DNT compared to the ethanol-added control, up-regulated 5.38-fold at 0.5h, 4.04-fold at 1h, and 3.10-fold at 1.5h, indicating that the MexT regulatory protein sensed 2,4-DNT and activated downstream P after stimulation _MDR2 A promoter, thereby enhancing the transcriptional expression of the downstream reporter luxC.

Example 4: the enzyme-labeled instrument detects the intensity of spontaneous light generated by regulation after the MexT protein senses the stimulation of 2,4-DNT

1. Strain activation and culture

The strain was transferred to LB liquid medium containing 50mg/L kanamycin, and cultured overnight at 37℃to give a bacterial liquid.

10mL of M9 liquid medium containing 50mg/L kanamycin was added with 330. Mu.L of 60% glucose, 10. Mu.L of 1M magnesium sulfate stock solution, 200. Mu.L of bacterial liquid, and shake cultured at 37℃until the culture became OD ₆₀₀ =0.2 or so.

2. Preparation of 2,4-DNT solution

Preparing 20mg/mL mother liquor 2,4-DNT (100 mg 2,4-DNT dissolved in 5mL absolute ethanol);

the diluted 2,4-DNT solution was prepared in the following proportions:

100mg/L: 5. Mu.L of mother liquor+980. Mu. L M9 medium+15. Mu.L of absolute ethanol;

50mg/L: 2.5. Mu.L of mother liquor +980. Mu. L M9 Medium +17.5. Mu.L of absolute ethanol;

10mg/L: 0.5. Mu.L of mother liquor +980. Mu. L M9 Medium +19.5. Mu.L of absolute ethanol;

0mg/L: 980. Mu. L M9 Medium+20. Mu.L absolute ethanol.

The concentration of ethanol in each DNT solution was 2%, and the final concentration of ethanol after 10-fold dilution was 0.2%.

3. Sample addition

The 2,4-DNT mother liquor with concentration of 90 mu L of bacterial liquid plus 10 mu L of 0mg/L, 10mg/L, 50mg/L and 100mg/L is added into a 96 Kong Chunbai ELISA plate, so that the final concentration of 2,4-DNT is respectively 0mg/L, 1mg/L, 5mg/L and 10mg/L, 4 parallel concentrations are respectively carried out, and the sealing plate film (Biored, product number MSB 1001) is used for sealing.

4. Luminescence detection

The self-luminescence intensity of the strain is monitored in real time at a constant temperature of 30 ℃ by using an enzyme-labeled instrument (Biotek), and the detection is carried out once every 10min for 12 hours.

The results are shown in FIG. 6, with P alone after the addition of the 2,4-DNT inducer _MDR2 The engineering strain of promoter plasmid has no self-luminous signal generation, while the engineering strain with MexT protein gene has obvious self-luminous signal response to inducer 2,4-DNT, and P activated by MexT response with higher concentration of added 2,4-DNT _MDR2 The higher the activity of the promoter, the higher the self-luminous intensity thus produced, and the self-luminous signal value thereof is very significantly different even at the lowest 2,4-DNT concentration of 1mg/L from that at DNT concentration of 0. The invention is characterized in that the MexT regulatory protein capable of inducing 2,4-DNT stimulation signals is utilized to activate the downstream promoter, so that the characteristic of higher self-luminous intensity is generated, and a biosensor for inducing explosive molecules 2,4-DNT by utilizing the regulatory protein is established.

The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; while the invention has been described in detail with reference to the foregoing embodiments, it will be apparent to one skilled in the art that modifications may be made to the embodiments described, or equivalents may be substituted for elements thereof; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.

Sequence listing

<110> Qingdao university of agriculture

<120> a biosensor for responding to explosive molecules using transcription regulatory protein, and preparation method and application thereof

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gtgattacca cccatcatcc tgtacgtatc ggagaacaaa cgggcttact tgatcaaatg 3840

tcttacggtc gtttcgtttt aggcttaagt gactgtgtca atgacttcga aatggatttc 3900

tttaagagaa aacgtagctc tcaacagcaa caattcgaag catgttacga aattttaaat 3960

gaagcgctga cgacaaacta ttgtcaggca gatgatgact tctttaactt cccacgtatt 4020

tctgttaacc cgcattgtat tagcgaagta aaacaatata ttttagcttc aagcatgggc 4080

gtggttgaat gggcagcaag aaaaggattg ccactcactt accgctggag tgacagccta 4140

gcagaaaaag aaaaatacta tcagcgttat ctcgctgttg ctaaagagaa taatattgat 4200

gtatcaaata ttgaccacca attcccactg ctcgttaata tcaatgaaaa tcgtcgtatt 4260

gctcgagatg aagtaaggga gtatatacaa agttatgtga gtgaagccta ccctactgac 4320

cccaacattg agctaagagt agaagagctt attgagcagc atgctgtcgg caaagtggat 4380

gagtactacg actcaacaat gcacgcagta aaagttacag gttcaaaaaa tttattactc 4440

tcttttgaat caatgaaaaa taaagacgat gttaccaagc ttataaatat gtttaatcaa 4500

aaaatcaaag ataaccttat taaataattt aattacggat agatattttc gatatatcta 4560

agtcttacta ccatttatat aaactattta tacagataac gtttcatttg attaagtcag 4620

taaataattg ccattaatta atggcagtgc agatccttac actgccattt ataaattaaa 4680

taagggttaa catgtcaaca ttattaaata tagatgcaac tgaaattaag gtgagtacag 4740

aaatagatga tattattttt acatcatcac cgctaacgtt actatttgaa gatcaagaaa 4800

aaatacagaa agaacttatt ttggagtctt tccattatca ttacaatcat aataaagatt 4860

ataagtacta ttgtaatata caaggcgtag atgagaatat acagtccatt gacgatattc 4920

ctgtttttcc tacttcaatg ttcaagtact caagattaca tactgctgat gaatcaaata 4980

ttgaaaattg gtttactagt agtggtacaa agggagtcaa aagtcatata gctcgagatc 5040

ggcagagtat tgaacgcttg ctaggttctg ttaattacgg catgaaatac ttgggtgaat 5100

ttcacgagca tcaattagaa ctagtgaata tggggccaga tcgtttcagt gcgtcaaatg 5160

tttggtttaa atatgtaatg agcttagttc aattacttta cccaacaaca tttaccgttg 5220

aaaacgatga aatcgatttt gaacaaacca tcttagcgtt aaaagcaatt cagcgtaaag 5280

gaaaaggaat ttgtttaatt ggccctccgt attttattta tttgttatgc cactacatga 5340

aagagcataa tatcgaattt aatgctggtg cacatatgtt tatcattaca ggtgggggat 5400

ggaaaaccaa acaaaaagaa gcgctaaacc gacaagattt caatcaacta ttgatggaga 5460

cttttagcct tttccatgaa agtcaaattc gagatatctt taaccaagta gagctaaaca 5520

cttgtttctt tgaagacagc ctacagcgta aacatgtacc accgtgggta tatgctcgtg 5580

cgcttgatcc tgtcacttta acgcccgtag aagatggcca agagggcttg atgagttata 5640

tggatgcctc atctaccagc tacccgacat ttattgttac cgacgatatt ggtattgttc 5700

gccatctaaa agaaccagat ccattccaag gaacaacggt tgaaattgtt cgtcgtttaa 5760

atacgcgaga acaaaaagga tgttcactct caatggccac gagcctgaaa taaaagcagg 5820

gcttaatcat gatttttaat tgcaaggtta aaaaagtcga agcatctgac agccatattt 5880

acaaagtgtt tattaagcct gacaaatgct ttgattttaa agcgggtcaa tatgtaattg 5940

tgtatctcaa tggaaaaaat ttgccgtttt ctattgctaa ctgcccaact tgtaatgagc 6000

tccttgaatt acatgtagga ggttcggtaa aagaatccgc cattgaagct atttcgcact 6060

ttattaatgc atttatttat caaaaagaat ttacaatcga tgcaccacac ggtgatgcat 6120

ggctgagaga tgaaagccaa tcacctttac tacttatagc aggagggaca ggtttatcat 6180

atatcaatag cattttaagt tgttgtatta gtaaacagtt atctcagcct atctatcttt 6240

attggggagt aaataactgt aatttactct atgctgatca acaactaaaa acactcgccg 6300

cacaatacag aaatataaat tatattcctg tggtagagaa tttaaatact gactggcagg 6360

gaaaaattgg taatgttatt gacgcggtta ttgaagattt ttcagattta tctgactttg 6420

atatctatgt ctgcgggcca tttggtatga gccggactgc gaaagatatt ctgatctcac 6480

agaaaaaggc gaatatagga aaaatgtatt ctgatgcatt tagctatacg taa 6533

<210> 2

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

atgattaaga agatcccaat g 21

<210> 3

<211> 41

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

taccagactc gagggtacct tacgtatagc taaatgcatc a 41

<210> 4

<211> 220

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

tcgtttcgat tcatcggtgc gcttcgcctg aaggggggca ggggtgaatc ttaaggccaa 60

aggctgttac cctgcacggc tttacgggag ccaattgcag ggaatgtcag gtgcccaatc 120

gatgacaggc atgtcgacta ttaatgacca ctggtggtct aacagcaaaa gcccggatag 180

agtcagtggt aattagaggt tcacaaaggc gaggtttgcg 220

<210> 5

<211> 41

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

aggtcgacaa gcttgcggcc gctcgtttcg attcatcggt g 41

<210> 6

<211> 44

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

cattgggatc ttcttaatca tcgcaaacct cgcctttgtg aacc 44

<210> 7

<211> 1144

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

tcgcaaacct ctgcagtgca tcacggggtg aataacctca tgggttgtga ctgtatccgc 60

ccatgcctga caaaaccacc cgtcgttatt gataatggct atgcctgtca gtgatcctat 120

gcccctccgg cacctcgcca ggccccgccc cgtctcgcac gcaaggcttg acggcgagcc 180

cccgcggttg cagcctctag cccctggaaa cgaggaacgc catgaaccga aacgacctgc 240

gccgcgtcga tctgaacctg ctgatcgtgt tcgagaccct gatgcacgaa cgcagcgtga 300

cccgcgccgc agagaaactg ttcctcggcc agccggccag ccggccatca gcgccgcgct 360

gtcgcgcctg cgcacgctgt tcgacgaccc gctgttcgtc cgtaccggac gcagcatgga 420

gcccaccgcg cgagcccagg aaatcttcgc ccacctgtcg ccggcgctgg attccatctc 480

caccgccatg agtcgcgcca gcgagttcga tccggcgacc agcaccgcgg tgttccgcat 540

cggcctttcc gacgacgtcg agttcggcct gttgccgccc ctgctccgcc gcctgcgcgc 600

ggaggcgccg gggttcgtcc tcgtcgtgcg ccgcgccaac tatctattga tgccgaacct 660

gctggcctcg ggggagatct cggtgggcgt cagctacacc gacgaactgc cggccaacgc 720

caagcgcaag accgtgcgcc gcagcaagcc gaagatcctc cgcgccgact ccgcgcccgg 780

ccagctgacc ctcgacgact attgcgcgcg accgcacgcg ctggtgtcct tcgccggcga 840

cctcagcggc ttcgtcgacg aggagctgga aaaattcggc cgcaagcgca aggtggtcct 900

ggcggtgccg cagttcaacg gcctcggcac cctcctggcc ggcaccgaca tcatcgccac 960

cgtgcccgac tacgccgccc aggcgctgat cgccgccggc ggcctacgcg ccgaggaccc 1020

accgttcgag acccgggcct tcgaactgtc gatggcttgg cgcggcgccc aggacaacga 1080

tccggccgaa cgctggctgc gctcgcggat cagcatgttc atcggcgatc cggacagtct 1140

ctga 1144

<210> 8

<211> 40

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

tcgacaagct tgcggccgct cgcaaacctc tgcagtgcat 40

<210> 9

<211> 41

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

atgaatcgaa acgagcggcc gctcagagac tgtccggatc g 41

<210> 10

<211> 38

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

tgccgcgcgg cagccatatg cctgtcagtg atcctatg 38

<210> 11

<211> 37

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

tcgagtgcgg ccgcaagctt tcagagactg tccggat 37

<210> 12

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

tcgtttcgat tcatcggtgc 20

<210> 13

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

cgcaaacctc gcctttgtga ac 22

<210> 14

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

tatgactcca ctcacggc 18

<210> 15

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

aaccactttc ttcgcacc 18

<210> 16

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

acacccgatc acacgttcaa 20

<210> 17

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

aaccacatca gcacagctca 20

Claims

1. A biosensor that responds to an explosive molecule with a transcriptional regulatory protein, comprising in order:

p derived from Pseudomonas aeruginosa _mexT The sequence of the MexT gene fragment is shown in SEQ ID NO. 7;

p derived from Pseudomonas aeruginosa _MDR2 The promoter gene fragment has a sequence shown as SEQ ID NO. 4;

self-luminescence from Protous binidusluxABCDEThe sequence of the operon is shown as SEQ ID NO. 1.

2. The method for manufacturing a biosensor according to claim 1, comprising the steps of:

(1) Amplified self-luminescenceluxABCDEOperon and P _MDR2 After the promoter gene fragment and purification recovery, the promoter gene fragment and the promoter gene fragment are connected to pACYCDuet-1 plasmid which is subjected to double enzyme digestion, competent cells are transformed by the product, positive clones are screened on LB solid plates containing antibiotics, and recombinant plasmid P-P is obtained _MDR2 -luxPleio；

(2) Amplification of P _mexT After purification and recovery of the fragment of the MexT gene, it is ligated to the singly digested P-P _MDR2 -luxPleioOn plasmid, the product is transformed into competent cells, positive clones are screened on LB solid plates containing antibiotics, and recombinant plasmid P-P is obtained _mexT -MexT-P _MDR2 -luxPleio；

(3) Recombinant plasmid P-P _mexT -MexT-P _MDR2 -luxPleioTransforming into competent cells to obtain engineering strain MEXT, culturing and activating in LB liquid medium containing antibiotics, and transferring to M9 medium for culturing to obtain the biosensor.

3. Use of the biosensor of claim 1 for the real-time detection of an explosive molecule, wherein the explosive molecule is 2,4-DNT.

4. Use of a biosensor according to claim 3 for the real-time detection of explosives molecules, characterized in that the method of use of the biosensor is: after the biosensor and the sample to be detected are uniformly mixed in a volume ratio of 9:1, a pure white ELISA plate is added and then is sealed, the spontaneous light intensity is monitored in real time by using an ELISA reader at a constant temperature of 30 ℃, and the spontaneous light intensity is monitored once every 20 min, so that 12h is monitored altogether.

5. Use of a biosensor according to claim 3 for the real-time detection of explosives molecules, characterized in that the concentration range of the explosives molecules detected by the biosensor is: 1 mg/L-10 mg/L.