CN113416685B

CN113416685B - Biosensor with signal amplification effect and capable of visually detecting explosive molecules and preparation method and application of biosensor

Info

Publication number: CN113416685B
Application number: CN202110755117.0A
Authority: CN
Inventors: 杨建明; 李美洁; 吕书喆; 汤若昊; 王兆宝; 梁波
Original assignee: Qingdao Agricultural University
Current assignee: Qingdao Agricultural University
Priority date: 2021-07-05
Filing date: 2021-07-05
Publication date: 2022-11-04
Anticipated expiration: 2041-07-05
Also published as: CN113416685A

Abstract

The invention discloses a biosensor with a signal amplification effect and capable of visually detecting explosive molecules, and a preparation method and application thereof. The host of the biosensor is escherichia coli, comprises a yqjfC55 promoter shown as SEQ ID No.1, and can start the expression of T7RNA Polymerase, so that the expression of mevalonate pathway and lycopene synthesis pathway genes started by the T7 promoter is activated, and visible lycopene is synthesized. The biosensor can sense the concentration of explosive molecules with low concentration, couple the concentration of the explosive molecules with the yield of lycopene and realize the visual detection of the explosive molecules, and the detection is simple and convenient to operate and has high safety.

Description

Biosensor with signal amplification effect for visually detecting explosive molecules and preparation method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering and molecular biology, and particularly relates to a biosensor with a signal amplification effect and capable of visually detecting explosive molecules, and a preparation method and application thereof.

Background

The residual explosives in the war zone (such as mines and the like) cause irreparable damage to life safety and ecosystems, so that the safe and effective detection of the mines is of great strategic significance. The biosensor can sense a specific compound and then generate a change which can be detected, thereby achieving the purpose of detecting the specific compound. The detection of residual mines by biosensors is an effective means.

The active ingredient of explosives such as land mine is TNT, which can be decomposed into various compounds such as 1, 3-dinitrobenzene (1, 3-DNB) and 2, 4-dinitrotoluene (2, 4-DNT). The Israel scientist Shimshon Belkin constructed a biosensing system for detecting 2,4-DNT using the GFP gene as a reporter element for the sensing element of the explosive molecule 2,4-DNT, namely the yqjFC55 promoter. The biological sensing system takes GFP as a reporting element, and when explosives are detected in the field, an instrument is required to be used for ultraviolet excitation with a specific wavelength and collection and analysis of a green fluorescent signal. However, the field condition is complex, and various non-GFP substances can emit green fluorescence under the excitation of ultraviolet light, so that an interference signal is generated; and the generated green fluorescence needs to be collected within a certain distance range, otherwise, the fluorescence is scattered, the farther the distance is, the weaker the signal is, and for explosive molecules, the long-distance detection is a basic requirement, and the detection at the closer distance can increase the danger of detection personnel.

Thus, there is a need for a biosensor that can detect explosive molecules quickly, easily, and safely.

Disclosure of Invention

In order to realize the visual detection of the explosive molecules, the invention provides a preparation method of a biosensor with a signal amplification effect and used for visually detecting the explosive molecules, the biosensor prepared by the preparation method and the application of the biosensor in the visual detection of the explosive molecules.

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

the invention provides a preparation method of a biosensor with a signal amplification effect and capable of visually detecting explosive molecules, which comprises the following steps:

(1) The IspA gene segment is amplified, purified and recovered, and pACYC-MvaE-MVaS plasmid utilizationBglII andNdei, after double digestion, purification and recovery, carrying out seamless connection on a plasmid digestion fragment and an IspA gene fragment, converting a connection product into an escherichia coli competent cell, and screening positive clones on an LB solid plate containing antibiotics to obtain a recombinant plasmid pACYC-mvaE-mvaS-IspA;

(2) Respectively amplifying yqjfC55 promoter, T7RNA Polymerase gene and pACYC-mvaE-mvaS-IspA carrier fragment, then seamlessly connecting the amplification products of the three genes, converting the connection product into escherichia coli competent cells, and screening positive clones on an LB solid plate containing antibiotics to obtain a recombinant plasmid pACYC-mvaE-mvaS-IspA-yqjfC55-T7RNAP;

(3) The recombinant plasmid pACYC-mvaE-mvaS-IspA-yqjfC55-T7RNAP, a mevalonic acid pathway downstream expression vector and a lycopene synthesis gene plasmid are co-transformed into an escherichia coli competent cell, and a positive clone is screened on an LB solid plate containing antibiotics to obtain an engineering strain XLYC3, namely the biosensor.

Further, the promoter yqjfC55 is derived from Escherichia coliEscherichia coliThe nucleotide sequence is shown in SEQ ID NO. 1.

Further, the T7RNA Polymerase gene is derived from Escherichia coliEscherichia coli。

Further, the T7RNA Polymerase gene has one of the following nucleotide sequences:

(1) A nucleotide sequence shown as SEQ ID NO. 6;

(2) A nucleotide sequence which has more than 90 percent of homology with the nucleotide sequence shown in SEQ ID NO.6 and can code T7RNA Polymerase.

Further, the promoter yqjfC55 is capable of promoting transcription of T7RNA Polymerase gene.

Further, the recombinant plasmid pACYC-mvaE-mvaS-IspA-yqjfC55-T7RNAP can exogenously express acetyl coenzyme A acyltransferase/hydroxymethyl glutaryl coenzyme A reductase genemvaE3-hydroxy-3-methylglutaryl coenzyme A synthase genemvaSAnd farnesyl/geranyl pyrophosphate synthasesIspA。

Furthermore, the downstream expression vector of the mevalonate pathway is ptrc-low, and can exogenously express mevalonate kinase geneERG12Phosphomevalonate kinase geneERG8Mevalonate Pyrophosphate decarboxylase GeneERG19And isopentenyl pyrophosphate isomerase geneIDII。

Furthermore, the lycopene synthesis gene plasmid is pET-lyc and can express the geranylgeranyl pyrophosphate synthase gene required by lycopenecrtEPhytoene synthase genecrtBAnd phytoene desaturase genecrtI。

Further, the host is Escherichia coliEscherichia coli Bl21。

The invention also provides the biosensor prepared by the preparation method.

The invention also provides the application of the biosensor in visual detection of explosive molecules.

Further, the use method of the biosensor comprises the following steps: after the biological sensor is cultured and activated, adding a sample to be tested, continuing culturing, and observing the color change of a culture solution; if the culture solution is not red, it is indicated that no explosive molecules exist in the sample to be detected, and if the culture solution is red, it is indicated that the explosive molecules exist in the sample to be detected, and the more obvious the red of the culture solution is, the higher the concentration of the explosive molecules in the sample to be detected is.

Further, the explosive molecule is DNT.

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

1. there are three genes in the lycopene operon,crtE、crtB、crtIencoding geranylgeranyl pyrophosphate synthase (CrtE), phytoene synthase (CrtB), phytoene desaturase (CrtI), respectively. The invention utilizes yqjFC55 induction element promoter and uses the promoter under the actioncrtE、crtB、crtIThe lycopene is synthesized by gene. T7 promoter (P) _T7 ) Is a strong promoter and is completely and specifically controlled by T7RNA polyThe synthase, and the high-activity T7RNA polymerase synthesizes mRNA 5 times faster than the Escherichia coli RNA polymerase, when the two exist at the same time, the transcription of the host self gene does not compete with the T7 expression system, almost all cell resources can be used for expressing the target protein, and the target protein can usually account for more than 50% of the total cell protein only a few hours after the induction expression. The invention uses the system to control the expression of the T7RNA polymerase gene by the yqjFC55 promoter and control the expression of the lycopene operoncrtE、crtB、crtIThe gene is expressed under the control of a strong promoter, T7. Under the induction action of explosive molecule 2,4-DNT, T7RNA polymerase is expressed and combined with a T7 promoter to start the expression of three downstream genes, thereby achieving the effect of signal amplification. Therefore, the visual biosensor constructed by the invention can induce yqjfC55 promoter of explosive molecule (2, 4-DNT) to combine with T7RNAP gene coding T7RNA Polymerase, and T7RNA Polymerase can combine with T7 promoter, thereby promoting the expression of genes of mevalonate pathway and lycopene synthesis pathway downstream of T7 promoter to synthesize lycopene. The detection of the explosive molecules (2, 4-DNT) can be achieved by visual observation, and the use is convenient and simple.

2. According to the visual biosensor constructed by the invention, the yqjfC55 promoter is used for starting the expression of T7RNA Polymerase, but the yqjfC55 promoter is not used for directly starting the expression of genes of a mevalonate pathway and a lycopene synthesis pathway, so that the strategy has a signal amplification effect, can improve the detection sensitivity of the biosensor, and has a wide application prospect.

Drawings

FIG. 1 is a plasmid map (D) of the vector pACYA-mvaE-mvaS-IspA constructed by detecting pACYC vector (A), ispA fragment (B), colony PCR verification (C) and agarose gel electrophoresis.

FIG. 2 is a plasmid map (E) of agarose gel electrophoresis detection of C55 fragment (A), T7 fragment (B), ispA vector (C), colony PCR verification (D) and constructed vector pACYA-mvaE-mvaS-IspA-yqjfC55-T7 RNAP.

FIG. 3 shows the induction results of visual detection of explosive molecules applied to XLYC3 recombinant strains in Erlenmeyer flasks; a is the result of visual observation, and B is the corresponding gray value.

Detailed Description

The present invention will be further described with reference to the following examples, but the present invention is not limited to these examples.

The examples do not show the specific techniques or conditions, and the techniques described in the literature in the field or the product specifications are followed. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available by purchase.

Example 1: gene acquisition and vector construction

1. Obtaining of genes

From Escherichia coli (A), (B)Escherichia coli) The nucleotide sequence of the promoter yqjfC55 in (1) is shown in SEQ ID No.1, and the promoter is chemically synthesized to pUC-57 vector by jinwei zhi corporation to obtain pUC-yqjfC55 vector.

2. Construction of pACYC-mvaE-mvaS-IspA expression vector

Using restriction enzyme 1Nde1030E (TaKaRa, cat No. 1621) and restriction enzyme 2Bgl II (TaKaRa, cat 1606) double restriction enzyme pACYC-mvaE-mvaS-GPPS ₂ The plasmid has an enzyme cutting system as follows:

plasmid or PCR product	3 μg
		10 ×Q.Cut Buffer	10 μL
Restriction enzyme
	1	5 μL
Restriction enzyme
	2	5 μL
Ultrapure water			Make up to 100 mu L

The enzyme digestion system is placed at 37 ℃ for incubation for 1h, and gel recovery and purification are carried out (FIG. 1A).

By Escherichia coliE. coli Taking a Bl21 (DE 3) genome as a template, carrying out Polymerase Chain Reaction (PCR) by using a primer IspA-F and a primer IspA-R, and amplifying an IspA gene fragment by using a PCR amplification system as follows:

the PCR procedure was: 95 ℃ for 3 min;30 cycles x (95 ℃ C15 s,55 ℃ C15 s,72 ℃ C1 min); 5 min at 72 ℃; and (3) 16 ℃ C ∞.

The primer sequences are shown below:

IspA-F：

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

IspA-R：

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

the PCR product was purified by gel recovery using a gel recovery purification kit (Vazyme, cat. No. DC 301-01) (FIG. 1B).

Three PCR products were ligated by seamless cloning using 2 × Clon Express Mix (Vazyme, cat # C115) as follows:

PCR fragment of vector	0.03 pmol
		PCR fragment having inserted therein each gene	0.06 pmol
2×Clon Express Mix	5 μL
		Ultra-pure water	Make up to 10 mu L

The system is incubated at 50 ℃ for 30 min. Product conversionE. coli DH 5. Alpha. Was competent, spread on LB solid plate containing 34 mg/L chloramphenicol, positive clones were PCR-screened (FIG. 1C), and the recombinant plasmid pACYC-mvaE-mvaS-IspA was extracted from the positive clones (FIG. 1D), and identified by restriction enzyme digestion and sequencing.

3. Construction of pACYC-mvaE-mvaS-IspA-yqjfC55-T7RNAP expression vector

And (3) carrying out PCR by taking pUC-yqjfC55 as a template and using the primer IspA-C55-F and the primer IspA-C55-R to amplify the vector fragment, wherein a PCR amplification system is shown as follows:

the PCR procedure was: 30 cycles × (98 ° C10 s,60 ° C15 s,68 ° C90 s); and (3) 16 ℃ C ∞.

The primer sequences are shown below:

IspA-C55-F：

5’-CCGGTAAACCAGCAATAGACACGGTTTTGGCGTATGGAG-3’ （SEQ ID NO.4）；

IspA-C55-R：

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

the PCR product was gel-recovered and purified using a gel recovery and purification kit (Vazyme, cat. No. DC 301-01) (FIG. 2A).

With Escherichia coliE. coli The Bl21 (DE 3) genome is used as a template, and primer IspA-T7RNAP-F and primer IspA-T7RNAP-R are used for carrying out PCR (Polymerase chain reaction) to amplify a T7RNA Polymerase fragment (SEQ ID NO. 6), wherein the PCR amplification system is as follows:

the PCR procedure was: 95 ℃ for 3 min;30 cycles x (95, 58, 72); 5 min at 72 ℃; and 16 ℃ is infinity.

The primer sequences are shown below:

IspA-T7RNAP-F：

5’-GGAAGGAGGTAAAGATCTATGAACACG-3’（SEQ ID NO.7）；

IspA-T7RNAP-R：

5’-TTACGCGAACGCGAAGTCCG-3’ （SEQ ID NO.8）。

the PCR product was gel-recovered and purified using a gel recovery and purification kit (Vazyme, cat. No. DC 301-01) (FIG. 2B).

Taking pACYC-mvaE-mvaS-IspA as a template, carrying out Polymerase Chain Reaction (PCR) by using a primer IspA carrier-F and a primer IspA carrier-R, and amplifying a carrier fragment, wherein the PCR amplification system is shown as follows:

the PCR procedure was: 30 cycles x (98 ℃ C10 s,60 ℃ C15 s,68 ℃ C1 min30 s); and (3) 16 ℃ C ∞.

The primer sequences are shown below:

IspA vector-F:

5’- CGGACTTCGCGTTCGCGTAAtaagcggctatttaacgaccc-3’ （SEQ ID NO.9）；

IspA vector-R: 5 'tgtcttggtttaccgg-3' (SEQ ID NO. 10).

The PCR product was gel-recovered and purified using a gel recovery and purification kit (Vazyme, cat. No. DC 301-01) (FIG. 2C).

Three PCR products were ligated by means of seamless cloning using 2 XClon Express Mix (Vazyme, cat # C115) as follows:

PCR fragment of vector	0.03 pmol
		PCR fragment into which each gene was inserted	0.06 pmol
2×Clon Express Mix	5 μL
		Ultrapure water	Make up to 10 mu L

The system was incubated at 50 ℃ for 30 min. Product conversionE. coli DH 5. Alpha. Was competent, spread on LB solid plate containing 34 mg/L chloramphenicol, positive clones were PCR-screened (FIG. 2D), and recombinant plasmid pACYC-mvaE-mvaS-IspA-yqjfC55-T7RNAP (FIG. 2E) was extracted from the positive clones and identified by restriction enzyme digestion and sequencing. Through detection, the recombinant plasmid pACYC-mvaE-mvaS-IspA-yqjfC55-T7RNAP can exogenously express acetyl coenzyme A acyltransferase/hydroxymethyl glutaryl coenzyme A reductase genemvaE3-hydroxy-3-methylglutaryl coenzyme A synthase genemvaSAnd farnesyl/geranyl pyrophosphate synthasesIspA。

Example 2: construction of XLYC3 recombinant strains

pET-lys plasmid (cited from CN 112877342A), pACYC-mvaE-mvaS-IspA-yqjfC55-T7RNAP, pTrc-low plasmid (cited from Yan)g J, xiaoan M, su S, et al, enhancing production of bio-isoprene used hybrid MVA pathway and isoprene synthase in E. Coli, PLoS one 2012, 7E. coliBL21 (DE 3) competent cells were plated on LB solid plates containing 34 mg/L chloramphenicol, 100 mg/L ampicillin and 30 mg/L kanamycin to obtain positive clones, whereby an engineered strain XLYC3 containing the vectors pET-lyc, pACYC-mvaE-mvaS-IspA-C55-T7RNAP plasmid and ptrc-low plasmid was obtained.

Example 3: application of visual detection of explosive molecules

And (3) detection of the conical flask: selecting a single colony of the engineering escherichia coli XLYC3 obtained in the example 2 into 10 mL of LB liquid culture medium containing 34 mg/L of chloramphenicol, 100 mg/L of ampicillin and 30 mg/L of kanamycin, and carrying out overnight culture and activation in a shaking table at 37 ℃; then transferred to 100 mL M9 medium for amplification culture until OD ₆₀₀ 0.6-0.8, adding 0.5 mM isoproyl-beta-D-thiogalactoside (IPTG) for induction, setting 2 groups as positive control, adding 5 mg/L or 10 mg/L DNT respectively, performing shake culture at 30 ℃, and taking pictures.

The results are shown in fig. 3, where the culture medium red color was more pronounced for both DNT groups compared to the IPTG-induced group, and the red color was more pronounced with increasing time; and the culture broth red color of the group with high DNT concentration was more obvious than that of the group with low DNT concentration. The XLYC3 recombinant strain constructed by the invention can be used as a biosensor for sensing explosive molecules, can sense DNT to generate visible lycopene, and the higher the DNT concentration is, the higher the lycopene yield is, the more obvious the red color is, and the result shows that the effect of detecting the explosive molecules by the biosensor for generating lycopene is very obvious.

The above examples are only intended to illustrate the technical solution of the present invention, and 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 modifications may be made to the embodiments described in the foregoing embodiments, or equivalents may be substituted for some of the features thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.

Sequence listing

<110> Qingdao university of agriculture

<120> biosensor with signal amplification effect for visually detecting explosive molecules and preparation method and application thereof

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gatctttgtc aattttcctt gctaacaatc aacattcacc acatttatga ttttctccat 180

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cg 242

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<213> Artificial Sequence (Artificial Sequence)

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<213> Artificial Sequence (Artificial Sequence)

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ctttaccaga ctcgagatct ttatttatta cgctggatga tgtagtcc 48

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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ccggtaaacc agcaatagac acggttttgg cgtatggag 39

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<213> Artificial Sequence (Artificial Sequence)

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gtgttcatag atctttacct ccttccgcca ctcaggctgc tgat 44

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<213> Artificial Sequence (Artificial Sequence)

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atgaacacga ttaacatcgc taagaacgac ttctctgaca tcgaactggc tgctatcccg 60

ttcaacactc tggctgacca ttacggtgag cgtttagctc gcgaacagtt ggcccttgag 120

catgagtctt acgagatggg tgaagcacgc ttccgcaaga tgtttgagcg tcaacttaaa 180

gctggtgagg ttgcggataa cgctgccgcc aagcctctca tcactaccct actccctaag 240

atgattgcac gcatcaacga ctggtttgag gaagtgaaag ctaagcgcgg caagcgcccg 300

acagccttcc agttcctgca agaaatcaag ccggaagccg tagcgtacat caccattaag 360

accactctgg cttgcctaac cagtgctgac aatacaaccg ttcaggctgt agcaagcgca 420

atcggtcggg ccattgagga cgaggctcgc ttcggtcgta tccgtgacct tgaagctaag 480

cacttcaaga aaaacgttga ggaacaactc aacaagcgcg tagggcacgt ctacaagaaa 540

gcatttatgc aagttgtcga ggctgacatg ctctctaagg gtctactcgg tggcgaggcg 600

tggtcttcgt ggcataagga agactctatt catgtaggag tacgctgcat cgagatgctc 660

attgagtcaa ccggaatggt tagcttacac cgccaaaatg ctggcgtagt aggtcaagac 720

tctgagacta tcgaactcgc acctgaatac gctgaggcta tcgcaacccg tgcaggtgcg 780

ctggctggca tctctccgat gttccaacct tgcgtagttc ctcctaagcc gtggactggc 840

attactggtg gtggctattg ggctaacggt cgtcgtcctc tggcgctggt gcgtactcac 900

agtaagaaag cactgatgcg ctacgaagac gtttacatgc ctgaggtgta caaagcgatt 960

aacattgcgc aaaacaccgc atggaaaatc aacaagaaag tcctagcggt cgccaacgta 1020

atcaccaagt ggaagcattg tccggtcgag gacatccctg cgattgagcg tgaagaactc 1080

ccgatgaaac cggaagacat cgacatgaat cctgaggctc tcaccgcgtg gaaacgtgct 1140

gccgctgctg tgtaccgcaa ggacaaggct cgcaagtctc gccgtatcag ccttgagttc 1200

atgcttgagc aagccaataa gtttgctaac cataaggcca tctggttccc ttacaacatg 1260

gactggcgcg gtcgtgttta cgctgtgtca atgttcaacc cgcaaggtaa cgatatgacc 1320

aaaggactgc ttacgctggc gaaaggtaaa ccaatcggta aggaaggtta ctactggctg 1380

aaaatccacg gtgcaaactg tgcgggtgtc gataaggttc cgttccctga gcgcatcaag 1440

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tggtgggctg agcaagattc tccgttctgc ttccttgcgt tctgctttga gtacgctggg 1560

gtacagcacc acggcctgag ctataactgc tcccttccgc tggcgtttga cgggtcttgc 1620

tctggcatcc agcacttctc cgcgatgctc cgagatgagg taggtggtcg cgcggttaac 1680

ttgcttccta gtgaaaccgt tcaggacatc tacgggattg ttgctaagaa agtcaacgag 1740

attctacaag cagacgcaat caatgggacc gataacgaag tagttaccgt gaccgatgag 1800

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ctggcttacg gtgttactcg cagtgtgact aagagttcag tcatgacgct ggcttacggg 1920

tccaaagagt tcggcttccg tcaacaagtg ctggaagata ccattcagcc agctattgat 1980

tccggcaagg gtctgatgtt cactcagccg aatcaggctg ctggatacat ggctaagctg 2040

atttgggaat ctgtgagcgt gacggtggta gctgcggttg aagcaatgaa ctggcttaag 2100

tctgctgcta agctgctggc tgctgaggtc aaagataaga agactggaga gattcttcgc 2160

aagcgttgcg ctgtgcattg ggtaactcct gatggtttcc ctgtgtggca ggaatacaag 2220

aagcctattc agacgcgctt gaacctgatg ttcctcggtc agttccgctt acagcctacc 2280

attaacacca acaaagatag cgagattgat gcacacaaac aggagtctgg tatcgctcct 2340

aactttgtac acagccaaga cggtagccac cttcgtaaga ctgtagtgtg ggcacacgag 2400

aagtacggaa tcgaatcttt tgcactgatt cacgactcct tcggtaccat tccggctgac 2460

gctgcgaacc tgttcaaagc agtgcgcgaa actatggttg acacatatga gtcttgtgat 2520

gtactggctg atttctacga ccagttcgct gaccagttgc acgagtctca attggacaaa 2580

atgccagcac ttccggctaa aggtaacttg aacctccgtg acatcttaga gtcggacttc 2640

gcgttcgcgt aa 2652

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<213> Artificial Sequence (Artificial Sequence)

<400> 7

ggaaggaggt aaagatctat gaacacg 27

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<213> Artificial Sequence (Artificial Sequence)

<400> 8

ttacgcgaac gcgaagtccg 20

<210> 9

<211> 41

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<213> Artificial Sequence (Artificial Sequence)

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cggacttcgc gttcgcgtaa taagcggcta tttaacgacc c 41

<210> 10

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<213> Artificial Sequence (Artificial Sequence)

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tgtctattgc tggtttaccg g 21

Claims

1. A method for preparing a biosensor for visually detecting explosive molecules with signal amplification effect, comprising the following steps:

(1) Amplifying, purifying and recovering the IspA gene segment, carrying out double digestion, purifying and recovering on the pACYC-MvaE-MvaS plasmid, carrying out seamless connection on the plasmid digestion segment and the IspA gene segment, converting a connecting product into a competent cell, and screening positive clones to obtain a recombinant plasmid pACYC-mvaE-mvaS-IspA;

(2) Respectively amplifying promoter yqjfC55, T7RNA polymerase gene and pACYC-mvaE-mvaS-IspA carrier fragment, then carrying out seamless connection on the amplification products of the three, converting the connection product into competent cells, and screening positive clone to obtain recombinant plasmid pACYC-mvaE-mvaS-IspA-yqjfC55-T7RNAP;

the promoter yqjfC55 is derived from Escherichia coli, and the nucleotide sequence of the promoter yqjfC55 is shown in SEQ ID NO. 1; the nucleotide sequence of the T7RNA polymerase gene is shown as SEQ ID NO. 6; the promoter yqjfC55 can start the transcription of the T7RNA polymerase gene;

(3) Co-transforming the recombinant plasmid pACYC-mvaE-mvaS-IspA-yqjfC55-T7RNAP, a mevalonic acid pathway downstream expression vector and a lycopene synthesis gene plasmid into competent cells, and screening positive clones to obtain an engineering strain XLYC3, namely the biosensor;

the recombinant plasmid pACYC-mvaE-mvaS-IspA-yqjfC55-T7RNAP can exogenously express acetyl coenzyme A acyltransferase/hydroxymethyl glutaryl coenzyme A reductase gene mvaE, 3-hydroxy-3 methyl glutaryl coenzyme A synthase gene mvaS and farnesyl/geranyl pyrophosphate synthase IspA; the downstream expression vector of the mevalonate pathway can exogenously express a mevalonate kinase gene ERG12, a phosphomevalonate kinase gene ERG8, a mevalonate pyrophosphate decarboxylase gene ERG19 and an isopentenyl pyrophosphate isomerase gene IDII; the lycopene synthesis gene plasmid can express geranylgeranyl pyrophosphate synthetase gene crtE, phytoene synthetase gene crtB and phytoene desaturase gene crtI required by lycopene synthesis.

2. A biosensor manufactured by the method of claim 1.

3. Use of a biosensor as claimed in claim 2 for the visual detection of explosive molecules.

4. The use of claim 3, wherein the biosensor is used by: after the biological sensor is cultured and activated, adding a sample to be tested, continuing culturing, and observing the color change of a culture solution; if the culture solution does not show red color, the explosive molecules do not exist in the sample to be detected, if the culture solution shows red color, the explosive molecules exist in the sample to be detected, and the more obvious the red color of the culture solution is, the higher the concentration of the explosive molecules in the sample to be detected is.