CN113388566A - MR-1 novel bead for detecting explosive molecules and preparation method and application thereof - Google Patents

Abstract

The invention discloses a novel MR-1 bead for detecting explosive molecules, and a preparation method and application thereof. The MR-1 newThe beads are made of self-luminous operatorsluxABCDEThe report element and the promoter Pdnt-2 sensing element of the operon are prepared by amplifying, recombining, transfecting competent cells, and stirring with sodium alginate-agar-polyacrylic acid solution and calcium chloride solution until complete gelation. The novel MR-1 bead can sense explosive molecules 2, 4-dinitrotoluene (2, 4-DNT) with different concentrations, generate fluorescence with different intensities, can simply know the concentration of the explosive molecules through the fluorescence intensity, and further realizes real-time fluorescence detection of the explosive molecules. The MR-1 novel bead construction method is simple, convenient to operate, convenient to store and high in safety, and can improve the activity maintenance time of engineering strains and the safety and efficiency of explosive molecule detection.

Description

MR-1 novel bead for 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 novel MR-1 bead for detecting explosive molecules, and a preparation method and application thereof.

Background

The method has important strategic significance for national defense safety and social stability of China in rapid, efficient and safe detection of explosives such as DL in the environment. The traditional method for detecting the mine at present can not realize off-position detection and has certain limitations in the aspects of safety, accuracy and the like. Among other things, the main limiting factor impeding global mine efforts is not the actual removal of mines buried in the ground, but the determination of their exact location. Most of the existing landmine detection methods require detection personnel to enter a mine area for detection, and great danger exists. Thus, as a possible alternative, biosensors have been proposed which are able to sense explosive molecules and generate optical signals which are remotely analyzed by means of a signal receiving system, and finally the location of the explosive is determined.

The active ingredient of explosives, such as land mines, is TNT, which can be decomposed into a variety of compounds, such as 1, 3-dinitrobenzene (1, 3-DNB) and 2, 4-dinitrotoluene (2, 4-DNT), which, due to their high volatility, are often used as the characteristic chemicals for detecting the presence of explosives. Shimshon Belkin et al reported an Escherichia coli sensor strain that converts green fluorescent eggsFusion of white GFP Gene to E.coliPdnt-2In the gene promoter, the promoter can be induced by the effective component of the explosive, so that green fluorescence is generated. In addition, the strain carrying the promoter is fixed in the beading, but the beading needs a long time for sensing explosive molecules to generate fluorescent signals, and the high activity of the sensor strain in the beading cannot be guaranteed in the time, and then when eGFP is used as a reporter gene for detection, the strain emits fluorescence and needs irradiation of an excitation light source, which undoubtedly increases difficulty for outdoor real-time detection of explosives.

Thus, there remains a need for a method or sensor that can simply perform real-time detection of explosive molecules without excitation.

Disclosure of Invention

The invention provides a novel MR-1 bead for detecting explosive molecules, a preparation method and application thereof, and the novel MR-1 bead can realize real-time fluorescence detection of explosives and simply detect the explosive molecules with different concentrations.

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 novel MR-1 bead for detecting explosive molecules, which comprises the following steps:

(1) amplified self-luminescent operonluxABCDEThe gene fragment is verified, purified and recovered, and then used together with the plasmid pACYCDuet-1Not IAndKpn Idouble digestion is carried out, and then T4 ligase is used for digestingluxABCDEConnecting the operon fragment and the plasmid enzyme digestion fragment in a mass ratio of 5:2, converting a connecting product into an escherichia coli competent cell, and screening positive clone on an LB solid plate containing chloramphenicol to obtain a recombinant plasmid p-lux;

(2) amplifying promoter Pdnt-2 gene fragment, verifying, purifying and recovering, and simultaneously utilizing with recombinant plasmid p-luxNot IPerforming single enzyme digestion, connecting the Pdnt-2 gene fragment with the plasmid enzyme digestion fragment in a mass ratio of 1:25, converting a connecting product into an escherichia coli competent cell, and screening positive clones on an LB solid plate containing chloramphenicol to obtain a recombinant plasmid p-Pdnt-2-lux;

(3) transforming the recombinant plasmid p-Pdnt-2-lux into an escherichia coli competent cell, and screening positive clones on an LB solid plate containing chloramphenicol to obtain an engineering strain MR-1;

(4) the correctly sequenced engineered strain MR-1 was inoculated into 10 mL LB medium supplemented with 10 μ L kanamycin, cultured overnight at 37 ℃ with shaking at 200 rpm, and the bacterial culture was transferred to a new 10 mL LB medium at a volume ratio of 2% and grown under the same conditions to an optical density of 600 nm (OD)₆₀₀) 0.8, bacterial culture was obtained;

(5) weighing 1.1 g of sodium alginate solid, dissolving with deionized water to a constant volume of 50 mL, stirring the solution until all the sodium alginate is dissolved, preparing a sodium alginate solution with a concentration of 2.2% (w/v), adding 1.1% agar powder, and sterilizing the two solutions at high temperature and high pressure; preparing 10% polyacrylic acid solution and 0.1M calcium chloride solution; mixing the sodium alginate-agar mixed solution with the polyacrylic acid solution according to a certain proportion to ensure that the final concentration of the sodium alginate solution is 2 percent, the agar concentration is 1 percent and the final concentration of the polyacrylic acid solution is 0.9 percent, and stirring the mixture until the mixture is completely homogenized;

(6) centrifuging the bacterial culture at 8000 rpm, 4 ℃ for 4 min, discarding the supernatant, washing the precipitate twice with 5 mL of 1% (w/v) sodium chloride sterile saline, centrifuging at 5000 rpm for 4 min, weighing and resuspending the precipitate in 5 mL of sterile saline, adding a suitable volume of resuspended bacteria to a pre-prepared sodium alginate-agar-polyacrylic acid solution so that the concentration of bacteria in the solution is 0.2% (w/v), and gently stirring to ensure homogeneity;

(7) dripping the bacteria-sodium alginate-agar-polyacrylic acid solution into a gently stirred 0.1M calcium chloride solution by using a 1 mL syringe until complete gelation, filtering, washing with saline containing 0.5% (w/v) TWEEN 80, and filtering to obtain the MR-1 novel coagulated bead.

Further, the self-luminous operatorluxABCDEThe operon gene is derived from vibrio qinghaiensis, and the nucleotide sequence of the operon gene is shown in SEQ ID NO. 1.

Furthermore, the nucleotide sequence of the promoter Pdnt-2 gene is shown as SEQ ID NO. 2.

The invention also provides the novel MR-1 bead prepared by the preparation method of the novel MR-1 bead for detecting the explosive molecules.

Further, the novel MR-1 bead contains a self-luminous operatorluxABCDEAn operon gene and a promoter Pdnt-2 gene.

The invention also provides the novel MR-1 bead prepared by the preparation method of the novel MR-1 bead for detecting the explosive molecules.

Further, 10-20 novel MR-1 beads are put into a culture medium filled with a sample to be detected, and the fluorescence intensity is detected every 10-20 min by using a microplate reader, a plant living body fluorescence detector or a camera for 1-4 h.

Furthermore, the concentration of explosive molecules sensed by the MR-1 novel bead is 0.1 mg/L-5 mg/L.

Further, the explosive molecule is 2, 4-DNT.

Furthermore, the novel sensor strain bead can sense explosive molecules with different concentrations, so that self-luminescence with different intensities is generated, and the concentration of the explosive molecules and the self-luminescence value are coupled to achieve the purpose of monitoring the explosive molecules in real time.

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

1. the invention takes the reporter plasmid containing vibrio qinghai lux operon as the basic plasmid, the recombinant plasmid obtained is transferred into escherichia coli cells after being connected with a promoter capable of inducing 2,4-DNT, the biosensor capable of inducing explosive molecules is obtained, the biosensor is fixed in sodium alginate gel beads by utilizing a sodium alginate embedding method, the engineering strain gel beads can induce the explosive molecules with different concentrations, so that fluorescent signals with different degrees are generated, the purpose of detecting the explosive molecules can be achieved by detecting the intensity of the generated fluorescent light, the detection result is accurate and efficient, and the use is simple.

2. In the process of engineering strain immobilization, maintaining the preservation time of the engineering strain and the long-term detection activity are important. According to the invention, on the basis of the traditional preparation method, substances such as agar powder and the like are utilized, so that the water content in the coagulated beads can be kept for a long time, the preservation time of the immobilized coagulated beads of the engineering strain is prolonged, the activity of the sensor strain is maintained for a long time, the preservation time of the engineering strain is longer, and the engineering strain has long-time activity, so that the 2,4-DNT induction capability of the engineering strain is improved, and the engineering strain is more effectively applied to the practice.

3. The constructed engineering strain capable of inducing the molecules of the explosive to generate fluorescence is embedded in the improved sodium alginate bead to obtain the biological induction system capable of being detected in the field, so that the field detection of the explosive can be realized, the improved sodium alginate bead can maintain the long-time induction activity of the engineering strain, and the biological induction system has a great application prospect in the field detection of the explosive.

Drawings

FIG. 1 is a plasmid map of the constructed vector p-luxQ 67.

FIG. 2 is a plasmid map of the constructed vector p-Pdnt-2-luxQ 67.

And FIG. 3 and FIG. 4 are the detection results of the constructed engineering strain MR-1 microplate reader.

FIG. 5 shows the result of the fluorescence detection of the improved engineering strain MR-1 synechocystis plant living body.

FIG. 6 shows the detection result of the constructed engineering strain MR-1 camera.

Detailed Description

The present invention will be further described with reference to the following specific 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

A compound derived from vibrio qinghai Q67 (Vibrio qinghaiensis Q67) ofluxABCDEThe nucleotide sequence of operon is shown as SEQ ID NO.1, and is chemically synthesized to p by Huada Gene companyUC-57 vector to obtain pUC-luxQ67 vector.

The nucleotide sequence of the Pdnt-2 promoter gene is shown as SEQ ID NO.2, and the gene is chemically synthesized on a pUC-57 vector by Huada Gene company to obtain a pUC-Pdnt-2 vector.

2. Construction of p-luxQ67 expression vector

And (3) carrying out Polymerase Chain Reaction (PCR) by using pUC-luxQ67 as a template and a primer luxQ67-F and a primer luxQ67-R, and amplifying a luxQ67 fragment, wherein a PCR amplification system is shown as follows:

the PCR procedure was: c, 3 min at 95 ℃; 30 cycles x (95 ℃ C15 s, 58 ℃ C15 s, 72 ℃ C4 min); c5 min at 72 ℃; and (3) 16 ℃ C ∞.

The primer sequences are shown below:

luxQ67-F：

5’-TCGACAAGCTTGCGGCCGCATGACAAAACACATACCATT-3’（SEQ ID NO.3）；

luxQ67-R：

5’-TACCAGACTCGAGGGTACCTTATACGTAGGCTAATCCATC-3’（SEQ ID NO.4）。

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

Using restriction enzyme 1Not I(TaKaRa, cat 1611) and restriction enzyme 2Kpn I(TaKaRa, cat # 1615) double digestion of pACYCDuet-1 plasmid, the digestion system is:

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

And (3) placing the enzyme digestion system at 37 ℃ for incubation for 1h, and performing gel recovery and purification.

The luxQ67 fragment was cloned on pACYCDuet-1 plasmid using a seamless clone, the system of which is shown below:

the ligation system was incubated at 50 ℃ for 30 min. Ligation product conversionE. coliDH5 alpha was competent, spread on LB solid plate containing 34 mg/L chloramphenicol, positive clones were PCR-screened, and the recombinant plasmid p-luxQ67 (FIG. 1) was extracted from the positive clones and identified by restriction enzyme digestion and sequencing.

3. Construction of p-Pdnt-2-luxQ67 expression vector

And (3) performing Polymerase Chain Reaction (PCR) by using pUC-Pdnt-2 as a template, a primer Pdnt-2-F and a primer Pdnt-2-R, and amplifying a Pdnt-2 fragment, wherein a PCR amplification system is as follows:

the PCR procedure was: c, 3 min at 95 ℃; 30 cycles x (95 ℃ 15 s, 55 ℃ 15 s, 72 ℃ 30 s); c5 min at 72 ℃; and (3) 16 ℃ C ∞.

The primer sequences are shown below:

Pdnt-2-R：

5’-ATGTGTTTTGTCATGCGGCCGCGACCTCACCCTCACCATTATTC-3’（SEQ ID NO.5）；

Pdnt-2-R：

5’-TCGACAAGCTTGCGGCCGCGCAAGCTCTTTTTTCAGTTG-3’ （SEQ ID NO.6）。

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

Using restriction endonucleasesNot I(TaKaRa, cat 1611) the p-luxQ67 plasmid was digested by the following enzymes:

plasmids	3 μg
		10 ×Q.Cut Buffer	10 μL
Restriction enzyme
		5 μL
Ultrapure water			Make up to 50 μ L

And (3) placing the enzyme digestion system at 37 ℃ for incubation for 1h, and performing gel recovery and purification.

The Pdnt-2 fragment was cloned on p-luxQ67 plasmid by seamless cloning, and the system is as follows:

the ligation system was incubated at 50 ℃ for 30 min. Ligation product conversionE. coliDH5 alpha competent cells, spread on LB solid plates containing 34 mg/L kanamycinAnd screening positive clones by PCR, extracting a recombinant plasmid p-Pdnt-2-luxQ67 (figure 2) from the positive clones, and identifying by restriction enzyme cutting and sequencing.

Example 2: construction of biosensors

The p-Pdnt-2-LuxQ67 recombinant plasmid is transformedEscherichia coliBW25113 competent cells (purchased from Weidi Biotechnology, cat # DL 2050) were plated on LB solid plates containing 34 mg/L chloramphenicol, and positive clones were obtained by PCR screening, thereby obtaining the engineered strain MR-1 containing the vector p-Pcnt-2-luxQ 67.

Example 3: preparation of novel coagulated beads of detection engineering strain MR-1

1. Activation and culture of strains

The correctly sequenced engineered strain MR-1 was inoculated into 10 mL of LB medium supplemented with 10 μ L kanamycin, cultured overnight with shaking (200 rpm) at 37 ℃, and then the culture was transferred to a new 10 mL of LB medium at 2% and grown under the same conditions to an optical density of 600 nm (OD)₆₀₀) Is 0.8.

2. Preparation of fixed bacteria-related solution

(1) Preparation of sodium alginate solution with 2.2% (w/v) concentration: weighing 1.1 g of sodium alginate solid, dissolving with deionized water to a constant volume of 50 mL, stirring the solution until all the sodium alginate is dissolved, adding agar powder into the mixed sodium alginate according to 1.1%, and sterilizing with high pressure steam.

(2) 9% polyacrylic acid (PAA) solution (35% polyacrylic acid, molecular weight 250000): 9 mL of 35% (w/v) polyacrylic acid was adjusted to pH 7.0 with 10 mM NaOH, and ddH was added₂O was made to volume of 26 mL, then filter sterilized (0.22 μm biofilter) and stored at room temperature. A volume of PAA was added to the fully dissolved sodium alginate to a final concentration of 0.9%, 2% sodium alginate and 1% agar and stirred to homogeneity.

3. The bacterial culture was centrifuged at 8000 rpm, 4 ℃ for 4 min and the supernatant discarded. The pellet was resuspended in 5 mL of 1% w/v sodium chloride sterile saline and centrifuged again at 5000 rpm for 4 min. The supernatant was discarded, and the pellet was weighed and resuspended in 5 mL of sterile saline. An appropriate volume of resuspended bacteria was added to the previously prepared sodium alginate-agar-PAA solution to give a concentration of 0.2% (w/v) bacteria in the solution and gently stirred to ensure homogeneity.

4. The bacteria were immobilized in sodium alginate-agar-PAA coagulated beads by dropping the bacteria-sodium alginate-agar-PAA solution into a gently stirred calcium chloride (0.1M) solution with a 1 mL syringe (diameter 450 μ M).

5. Will react with Ca²⁺The beads formed after contact were stirred for an additional 30 min to ensure complete gelation. The beads were then filtered, washed with brine containing TWEEN 80 (0.5% w/v), filtered again and stored at 4 ℃.

Example 4: application of novel condensed bead detection of engineering strain MR-1 for detecting explosive molecules

1. Preparing 2,4-DNT solution

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

preparing a diluted 2,4-DNT solution according to the following proportion:

50 mg/L: 2.5. mu.L of the mother liquor + 980. mu. L M9 of the culture medium + 17.5. mu.L of absolute ethanol;

10 mg/L: 0.5. mu.L of the mother liquor + 980. mu. L M9 of the medium + 19.5. mu.L of absolute ethanol;

0 mg/L: 980 μ L M9 medium +20 μ L absolute ethanol.

Preparing 0.2 mg/mL 2,4-DNT dilution mother liquor (10 mu L of 20 mg/mL 2,4-DNT mother liquor and 990 mu L absolute ethyl alcohol);

preparing a diluted 2,4-DNT solution according to the following proportion:

1 mg/L: 5 μ L of diluted stock solution +980 μ L M9 medium +15 μ L of absolute ethanol;

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

2. Preparation of detection plate

Adding 0.15 g of agar powder into 9 mL of LB culture medium, heating until the agar powder is completely dissolved, adding 9 mu L of mother liquor with the concentration of 34 mg/L chloramphenicol and 1 mL of 2,4-DNT mother liquor with the concentrations of 0 mg/L, 1 mg/L, 10 mg/L and 50 mg/L into the culture medium when the temperature is reduced to 50-60 ℃, so that the final concentrations of 2,4-DNT are respectively 0 mg/L, 0.1 mg/L, 1 mg/L and 5 mg/L, adding the mixture into a 24-well plate, adding 1 mL of the mixture into each well, and setting 4 concentrations in parallel.

3. Fluorescence detection

Detection by a microplate reader: and (3) putting 10 beads into each hole, detecting the luminous intensity of all the beads in each hole once every 10 min for 20 h.

The bead detection results are shown in fig. 3 and 4, and the trend of the bead luminescence intensity under different concentrations is that the bead luminescence intensity gradually increases along with time, reaches the highest peak in about 4 hours, and gradually decreases along with time after 4 hours. Furthermore, the higher the concentration of 2,4-DNT, the higher the luminescence intensity of the beads, and the lower the concentration of 2,4-DNT to 0.1 mg/L, the more strongly responsive the novel MR-1 beads were.

And (3) detecting by using an improved coagulated-bead plant living body fluorescence detector: the improved novel beads are placed in an environment at-80 ℃ for 1 day, 7 days, 14 days, 30 days and 60 days to induce the fluorescence intensity of 2,4-DNT, and the concentration of the 2,4-DNT selected for detection is 5 mg/L.

The detection results of the improved novel beads are shown in fig. 5, the fluorescence intensity of the beads after being placed for 1 day, 7 days, 14 days and 30 days has no obvious difference, which indicates that the prepared novel engineering strain MR-1 beads still can keep higher detection activity after being placed for 30 days, the fluorescence intensity of the novel engineering strain beads after being placed for 60 days is slightly lower than that of the beads placed for a short time, but the detection activity still exists to a certain degree for 2, 4-DNT.

The photographing result is shown in FIG. 6, the higher the concentration of 2,4-DNT, the shorter the response time of the beads, especially at the concentration of 5 mg/L of 2,4-DNT, the fluorescence emitted from the beads can be monitored within 1 h. When the concentration of 2,4-DNT is as low as 0.1 mg/L, the fluorescence emitted by the beads can be monitored by the instrument at 4 h.

The detection results show that the novel engineered strain MR-1 bead prepared by the invention can generate stronger fluorescence induction on 2,4-DNT with the concentration as low as 0.1 mg/L, and the higher the concentration of the 2,4-DNT is, the higher the fluorescence intensity of the bead is, and the shorter the response time is.

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

<110> Qingdao agricultural university

<120> novel MR-1 bead for detecting explosive molecules, and preparation method and application thereof

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 6490

<212> DNA

<213> Vibrio qinghaiensis (Vibrio qingaiensis)

<400> 1

atgacaaaac acataccatt tataatcaat ggcaagattt cgactgtctc aaaattagaa 60

aagccagaat tgaatagcgt tatttttcga gagaagactc tcgacttaga tattttaact 120

gaccatatta ctgatgaaat catagaccat aaaaaaactc gtgaattaac actcaataat 180

attgttaatt tcctttatac cgttggtcag cgttggaaaa atgaagaata caccagacga 240

cgtagttaca ttcgtgactt aaagaattac ttgggttatt ccgaagaaat ggccaaatta 300

gaaaccaatt ggatcgccat gattctgtgc tctaaaagtg cattgtatga cattgtacaa 360

actgagctag gctcacgtca tatattggat gaatggatag cacaagatga atgctatgtt 420

aaggcactgc ccaaaggccg ttcactccat ttattagcgg gcaatgtgcc tctttctggc 480

gtgacctcta ttttgcgcgc cattctgacc aagaatcaga taatcgtaaa aatgtcgtcg 540

aatgacccgt tcacccctca tgcattagcg atgagtttta tagatgtcga cccaaatcat 600

cccattaccc aatccatctc cgttatttat tggcctcata ctcaatgcac tcaggtagcc 660

cagcgtttaa tgcaaaagat ggatgtggtc gtcgcttggg gcggttcaga agcaatacgt 720

tgggcagccg agcacacgcc ttcgcatgct gagctcatta agtttggccc gaaaaaaagc 780

ctcactatta tcaatgatcc tgaaaattta atcgaagcgg ccgaaggagc agctcacgac 840

atctgttttt acgaccaaca agcctgcttt tctactcaaa atctttttta tctcggctct 900

cgttttcctg aatttaaaca agcgttaaga gagcagttac agcgctatgc tcgaattttg 960

ccgaaatcac agtccagtat tgatgaacaa gccgacttct ccttaacact gcgtgaatgc 1020

caatttgcgg ggtttacctc agaaatgggc agtcaacagg attggatgat gattgaatct 1080

ccagctggca cagagctgaa tcatccgcta gggcgctgca tttatctgca tcaaatggcg 1140

tcatttgagg aaattttgcc ctttgtgata aaaggacaaa ctcaaactgt ttccctattt 1200

ccttggtcct gttcatttca atatcgagat caactcgccg ctcacggggc tgaacgtatc 1260

gtcgaatctg gaatgaataa tattttccgc gtcggtggtg cccatgatgt catgcgccca 1320

ttacagcgtt tagtacgttt tatttcccat gaacgcccaa gccattttac aaccaaagat 1380

gtcgccgtag atattgaaca aacacgctat ttagaagaag acaaattctt agtctttgtt 1440

ccttagttac ttaaaccaac gtcataaagg agaaatgaat gtctcaccga tcacattggg 1500

aagtggatga accctgcacc attgatcatg tgatttcctt atctggaaat cagcaaatcc 1560

atgtttggga aacacctcca atacttaaat ttaatcagat gccaacacgg cggaatagta 1620

ttttgatcgc ttctggtttt gctcgtcgga tggaccattt tgcaggtcta gcacgatatt 1680

tgtcagccaa tggatttcat gtctatcgct atgattcact tcaccacgtt ggattaagct 1740

caggcacaat tgatcaatac acgatgagca ttggcaagca gagtcttgaa atcgtgatgg 1800

cttggcttaa taaccgtgga gtaacagacg ttggggtgat tgccgcaagc ttatccgctc 1860

gaattgctta cgccaccgtt aatgaacttg acctatcctt cttgatctcc gcggttggag 1920

ttgtgaatct gcgcgataca ttagagaaag ccttcggctt tgattatctc agtctcccta 1980

ttcaagattt gccaaacgat ctggattttg aaggccataa attaggtgct gaagtctttg 2040

ttcgtgattg ttttgcgaat aactgggata gcttttcatc caccgtcgaa caaatgcgcc 2100

atctctcaat ccctttcatc gcgttcaccg cgaatgggga tgattgggtc aaacaagacg 2160

aagtcattga acttctttct caactccctg caggtcaaag caagctctat tccctgcttg 2220

gaagttccca tgatctcggt gagaacttag tagtacttcg caacttctat caatcagtca 2280

ctaaagccgc actcgcactc gattcaaata gtttcgatat tgatattccg tttgtggagc 2340

ccacatttga acaattgact atcacgaccg tcaatgagcg tcgaatgaaa aatcaaattg 2400

aaactgaaac tgaaacggcc atgcaggctt aagcccgctc atacacttac ctaccttaat 2460

aaaggatatt ttatgaaatt tggaaatttc ttactgacct accaaccacc tgggttatca 2520

cagacagaag tgatccaacg tttggttaat cttggacgtg cttcagaatc atgcggtttc 2580

gaagcagttt ggttactaga acatcatttc actgagtttg gtctactcgg taatccttat 2640

gtcgcagccg ctaatctcct tggtgcaacg aaacatcttc atgtaggtac agcggcgatc 2700

gttctaccga ccgctcaccc catccgacaa cttgaagatg tgaacttgct cgatcaactt 2760

tcaaaaggac gttttcgctt tggcatttgt cgtggactct atgacaaaga ttttcgtgtg 2820

tttggcactg acatgaacaa cagccgcgcg ttgatggatt gttggtacga cttaatcacg 2880

acaggcatga cacaaggcac agtatccgct gataacgaac atatccattt ccctgaagtg 2940

aaagtccaac ccgcacccta tcaccgtagt ggtgctcctg tttatgtggt agcagagtct 3000

gcttcaacaa ctgagtgggc agcaaaacgt ggtttaccca tgattctaag ttggattatc 3060

aacacccatg agaaaaaagc gcagcttgat ctatataacg agatcgcttt agagcatgga 3120

cacgatatcc agaatattga tcactgcctc tcctacatca cctctgtaga tcatgattca 3180

caacacgcaa aagatatttg tcgccaattt ctcgcccact ggtatgactc ctatgtcaat 3240

gctacccgca tttttgatga ttcagaccaa actaaaggct atgactttaa taaaggacaa 3300

tggcgagatt ttgtcttaaa aggccatcgt gatactaatc gccggattga ttacagctac 3360

gagatcaatc cggtaggaac gccagaagaa tgcattcgaa ttatccagca agacattgat 3420

acaacaggca ttactaatat ctgctgtgga tttgaagcca acggttcaga acaagaaatt 3480

atcgcctcaa tgaagttgtt ccaagctgag gtgatgcctc gcctcaaaaa cccaaactaa 3540

caccaactat tttcagagga agaacctatg aaattcggat tatttttctt aaatttcctt 3600

cattcaggtc aatcgtgcac agaagttttc gatgccatga tcgatagcgt gaactatgca 3660

gaaaaaggac attttgatcg actatttatt tatgaaaacc attttaatga ccatggtatt 3720

gtgggcgccc ccctcacagc ggccagtttt ttattgggca tgactgagcg tatcaaggtc 3780

ggttcgctca atcatgttct gacaactcac catcccgtcc gaacggctga agaaacggga 3840

ctactcgatc aaatgagtca aggccgtttc attttaggct tcagtgattg tgaaaaccgc 3900

gacgagatga tcttttttaa ccgcccttta gattctcagc agccgatatt tgaagcctgt 3960

tatcagatca taaatgatgc tttaacctcc ggttattgcc atccagataa tgacttttac 4020

agtttcccaa aaatttctgt taacccacac agttataccc aaggaggccc acaacagtat 4080

gtttatgcct ctagtacaca agtcgtggga tgggcagcta aacgggcatt accgctgacc 4140

tttaaatggg atgatagcaa cacgactcgg cagcagtatg cgcgacatta tcgagagacc 4200

gctaaaaaat acggtgtcga tgttcaagca gtgcgacatc aactcgcgtt attgatcaat 4260

caaaatgatg atggtgaaat cgcgcgtata gaggctcgcc aatacctcac tcaatacgtt 4320

atagaacgct accctagcga tgaaattgaa atcgtcttag aacgaatcat caaagagagt 4380

gctattggca cctacgaaga aagcactcaa gctgctcgta tggctattga gatgtgtgga 4440

gcttccgatc tgctgatctc cgttgagtcg attaaagagc cagctcaccg tctgcatgta 4500

ttggatgtga ttaatagcaa catcgctaaa taccatcaat aaaaggcttt tctttttccc 4560

ttgggccgtc attacctcct tcggcggcgg ccctttttta aggaataaac aatggatacc 4620

acactgccct taattgacaa gcaagccgtt gataaatttg atattgaagc aagctcctac 4680

cttgatgact taatcttcat gagtgaaccg aattcatggt catacgaaga gcaggaaaaa 4740

attcgacatg atgtgatcat gaaagccttt cgttggcact accaaaacaa tactgactat 4800

cgtcgttatt gccaaactat cggtattggc cttgaaatag agcatttgga cgatatcccc 4860

gtctatccca cgtcgatatt taaaaccatg cgagtgacca gcgcaaaacc tcaagagatc 4920

gagcattggt ttacaagcag tgggactcag ggacaaaaaa gtcatattcc acgagatcgt 4980

ctgagcattg agcgtttgtt aggttctgta aactatggaa tgaaattagt gggcgcatgg 5040

tttgaccatg agatggagct tgttaattta ggccccgatc gatttaacgc tcacaacata 5100

tggtttaaat atgtgatgag tcttgttgaa ttactctacc caaccgcttt tacagcaaaa 5160

gatgatctcg tggattttga ccaaaccctc atgcacttat atcgcattca aagcatggga 5220

aaaactactt gcttaattgg tcccccctat tttgtttatc ttctatgcca gcacatgaaa 5280

gcagaaaaaa tccacttcag agcagggcat aagctctaca tcataacggg tggtggttgg 5340

aaaactcacg aaaactccgc cttaaataga gatcaattca atcaattatt gatggaaact 5400

tttggtctca atgatataag ccaaatacgc gatacgttca accaagttga actcaacact 5460

tgcttttttg aagatgatca gcaacgcaaa tgtgttccgc cttgggttta tgctcgggct 5520

ttaaatcccc gaactctcca acctctgcct gatggggaaa taggtttaat gagttacatg 5580

gatgcctctg ctacaagcta tcctgcattt ttaattactg atgatcttgg ttacattcat 5640

cagcacactg aaaagattgc ctacacgacg gtacaaattg tacgcagaat caatacacga 5700

gcccaaaaag gctgtgccct aaagatgtca caacattttt cattccctca aatcacacct 5760

caataagaat aggaataaga aaatgaaatt ttcatgtcac gttcatacag tgcagcacct 5820

agcaccgggg atctaccaaa tcattttggc tccacactac cccttagatt ttaaagccgg 5880

acaatttttg aagctcacat tggcaggtaa agatcgctac ttttctattg ctagttgtcc 5940

ttcacaacca ggatttatag aactacatat tggaacctca aagactgatg aaggcattct 6000

tagtactatt gctgccttac atgagtttaa agaagctgaa cttccactag aaattgaagg 6060

accacttggt aatgcttggc tacgtaaaga gagcaacaat cccatattat ttattgctgg 6120

aggaacggga atatcctaca taatgagctt acttagaaat gctcttcata atcaactcga 6180

tcagagcatt tatctttact ggggagtgaa agggattaac caattgtatc ttcacccaga 6240

attattaatg ctttcagatc aatatcctaa tctacactat gtgtgttcgc tggaggaatc 6300

ctgcgaccaa ataatgagca gggaaggatt agtggttgat gcaatattaa atgacttttc 6360

agatttgcaa gattttgaca tctacttatg tgggccaata aatatgatta aagaaggcaa 6420

aaagtatcta cttgaaaaat gcaatgcaac catgggaaat atgtatggtg atggattagc 6480

ctacgtataa 6490

<210> 2

<211> 120

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gcaagctctt ttttcagttg tctccgaatg gctgcgattc tagcgcgtcg atcggtatag 60

cgctatagat caagtctgat agttaaaatc accagcatga ataatggtga gggtgaggtc 120

<210> 3

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tcgacaagct tgcggccgca tgacaaaaca cataccatt 39

<210> 4

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

taccagactc gagggtacct tatacgtagg ctaatccatc 40

<210> 5

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgtgttttg tcatgcggcc gcgacctcac cctcaccatt attc 44

<210> 6

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

tcgacaagct tgcggccgcg caagctcttt tttcagttg 39

Claims (10)

1. A method for preparing novel MR-1 beads for the detection of explosive molecules, comprising the steps of:

(1) amplified self-luminescent operonluxABCDEPurifying and recovering the operon gene fragment, carrying out double enzyme digestion with a plasmid pACYCDuet-1, and carrying outluxABCDEConnecting the region fragment and the plasmid enzyme digestion fragment in a mass ratio of 5:2, converting the connecting product into competent cells, and screening positive clones to obtain a recombinant plasmid p-lux;

(2) amplifying a promoter Pdnt-2 gene fragment, purifying and recovering, performing single enzyme digestion on the gene fragment and a recombinant plasmid p-lux, connecting the plasmid enzyme digestion fragment and the Pdnt-2 gene fragment according to the mass ratio of 25:1, converting a connecting product into a competent cell, and screening positive clone to obtain the recombinant plasmid p-Pdnt-2-lux;

(3) transforming the recombinant plasmid p-Pdnt-2-lux into competent cells, and screening positive clones to obtain an engineering strain MR-1;

(4) culturing and activating the engineering strain MR-1 in an LB culture medium containing antibiotics to obtain a bacterial culture;

(5) and (2) centrifuging and resuspending the bacterial culture at low temperature, adding the bacterial culture into a sodium alginate-agar-polyacrylic acid solution, uniformly stirring, dripping the bacterial culture into a calcium chloride solution, continuously stirring until the bacterial culture is completely gelatinized, and filtering and cleaning to obtain the MR-1 novel coagulated beads.

2. The method for preparing MR-1 novel beads for detecting explosive molecules according to claim 1, wherein the bacterial concentration in the solution is 0.2-0.5% (w/v) after the addition of sodium alginate-agar-polyacrylic acid solution in step (5).

3. Method for preparing a novel MR-1 beading for detecting explosive molecules according to claim 1, characterized in that the self-luminescent operonluxABCDEThe operon gene is derived from vibrio qinghaiensis, and the nucleotide sequence of the operon gene is shown in SEQ ID NO. 1.

4. The method for preparing a novel MR-1 bead set for detecting explosive molecules as claimed in claim 1, wherein the nucleotide sequence of the promoter Pdnt-2 gene is shown in SEQ ID No. 2.

5. The method for preparing the MR-1 novel agglutinated bead for detecting the explosive molecules according to claim 1, wherein the engineering strain MR-1 with correct sequencing in the step (4) is inoculated into 10 mL of LB culture medium added with 10 mu L kanamycin, cultured overnight at 37 ℃ under 200 rpm shaking, and then the culture is transferred into new 10 mL of LB culture medium by 2% and grown under the same conditions until the optical density is 600 nm and 0.8, so as to obtain the bacterial culture.

6. The novel MR-1 beading prepared by the method for preparing novel MR-1 beading for detecting explosive molecules according to any one of claims 1 to 5.

7. Use of the novel MR-1 beading according to claim 6 for real-time detection of explosive molecules.

8. The use according to claim 7, wherein the MR-1 novel beads are put into a culture medium containing a sample to be detected in an amount of 10-20 beads, and the fluorescence intensity is detected every 10-20 min by using a microplate reader, a plant living body fluorescence detector or a camera for 1-4 h.

9. Use according to claim 7, wherein the concentration of explosive molecules sensed by the MR-1 novel beads is between 0.1 mg/L and 5 mg/L.

10. Use according to claim 7, characterized in that the explosive molecule is 2, 4-DNT.

Images