阅读说明：本技术 一种具有信号放大效应的可视化检测爆炸物分子的生物传感器及其制备方法和应用 (Biosensor with signal amplification effect and capable of visually detecting explosive molecules and preparation method and application of biosensor ) 是由 杨建明 李美洁 吕书喆 汤若昊 王兆宝 梁波 于 2021-07-05 设计创作，主要内容包括：本发明公开了一种具有信号放大效应的可视化检测爆炸物分子的生物传感器及其制备方法和应用。所述生物感应器的宿主为大肠杆菌,包含如SEQ ID NO.1所示的yqjfC55启动子,能够启动T7 RNA Polymerase的表达,从而激活由T7启动子所启动的甲羟戊酸途径和番茄红素合成途径基因的表达,从而合成肉眼可见的番茄红素。本发明的生物传感器能够感应低浓度的爆炸物分子浓度,将爆炸物分子浓度与番茄红素产量进行偶联,实现爆炸物分子的可视化检测,这种检测操作简单、方便、安全性高。(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 a T7 promoter is activated, and lycopene can be synthesized to be visible to the naked eye. 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 high in safety.)

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 clones to obtain recombinant plasmid pACYC-mvaE-mvaS-IspA-yqjfC55-T7 RNAP;

(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.

2. The method according to claim 1, wherein the promoter yqjfC55 is derived from Escherichia coliEscherichia coliThe nucleotide sequence is shown in SEQ ID NO. 1.

3. The method according to claim 1, wherein 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.

4. The method according to claim 1, wherein the promoter yqjfC55 is capable of promoting transcription of T7RNA Polymerase gene.

5. The method according to claim 1, wherein the recombinant plasmid pACYC-mvaE-mvaS-IspA-yqjfC55-T7RNAP is capable of exogenously expressing acetyl-coaEnzyme A acyltransferase/hydroxymethylglutaryl-coenzyme A reductase GenemvaE3-hydroxy-3-methylglutaryl coenzyme A synthase genemvaSAnd farnesyl/geranyl pyrophosphate synthasesIspA。

6. The method according to claim 1, wherein the expression vector downstream of the mevalonate pathway is capable of exogenously expressing mevalonate kinase geneERG12Phosphomevalonate kinase geneERG8Mevalonate Pyrophosphate decarboxylase GeneERG19And isopentenyl pyrophosphate isomerase geneIDII。

7. The method according to claim 1, wherein the lycopene synthesis gene plasmid is capable of expressing geranylgeranyl pyrophosphate synthase gene required for lycopene synthesiscrtEPhytoene synthase genecrtBAnd phytoene desaturase genecrtI。

8. A biosensor produced by the production method according to any one of claims 1 to 7.

9. Use of the biosensor of claim 8 for the visual detection of explosive molecules.

10. The use of claim 9, 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 the 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.

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 (such as landmines and the like) in the war zone cause irreparable damage to life safety and ecosystems, so that the safe and effective detection of the landmines 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 by using GFP gene as a reporter element for the sensing element of explosive molecule 2,4-DNT, namely yqjFC55 promoter. The biological sensing system takes GFP as a reporting element, and when explosives are detected in the field, an instrument is used for ultraviolet excitation with a specific wavelength and collection and analysis of a green fluorescence 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 used for visually detecting explosive molecules, which comprises the following steps:

(1) the IspA gene segment is amplified, purified and recovered, and the pACYC-MvaE-MvaS plasmid is utilizedBglII 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 promoter yqjfC55, T7RNA Polymerase gene and pACYC-mvaE-mvaS-IspA carrier fragment, then carrying out seamless connection on the amplification products of the three, transforming escherichia coli competent cells by the connection products, and screening positive clones on an LB solid plate containing antibiotics to obtain a recombinant plasmid pACYC-mvaE-mvaS-IspA-yqjfC55-T7 RNAP;

(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 openerThe active cell 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 was able to initiate 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 is as follows: after the biological sensor is cultured and activated, adding a sample to be tested, continuing culturing, and observing the color change of the 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.

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 ) The promoter is a strong promoter and is completely and exclusively controlled by T7RNA polymerase, the speed of synthesizing mRNA by the high-activity T7RNA polymerase is 5 times faster than that of the Escherichia coli RNA polymerase, when the two exist at the same time, the transcription of the host gene is not competitive with the T7 expression system, almost all cell resources can be used for expressing 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 T7RNA polymerase gene by yqjFC55 promoter and to control the expression of 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 the yqjfC55 promoter of the explosive molecule (2, 4-DNT) to be combined with the T7RNAP gene coding T7RNA Polymerase, and the T7RNA Polymerase can be combined with the T7 promoter, so as to start the expression of genes of a mevalonate pathway and a lycopene synthesis pathway downstream of the T7 promoter and synthesize the tomatoAnd (3) red pigment. The detection of the explosive molecule (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, and 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 constructed vector pACYA-mvaE-mvaS-IspA and the plasmid map (C) of pACYA-mvaE-mvaS-IspA detected by agarose gel electrophoresis (A), IspA fragment (B), and colony PCR verification (C).

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 in XLYC3 recombinant strain applied 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 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

From Escherichia coli (Escherichia coli) The nucleotide sequence of the promoter yqjfC55 is shown in SEQ ID NO.1, and the promoter is chemically synthesized into a pUC-57 vector by Jinwei Zhi corporation to obtain a pUC-yqjfC55 vector.

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

Using restriction endonucleasesEnzyme 1NdeІ (TaKaRa, cat 1621) and restriction enzyme 2Bgl II (TaKaRa, cat 1606) double restriction enzyme pACYC-mvaE-mvaS-GPPS₂The 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 was incubated at 37 ℃ for 1h for gel recovery and purification (FIG. 1A).

With Escherichia coliE. coli Bl21(DE3) genome is template, primer IspA-F and primer IspA-R, Polymerase Chain Reaction (PCR) is carried out, and IspA gene fragment is amplified, wherein the PCR amplification system is shown as follows:

the PCR procedure was: c, 3 min at 95 ℃; 30 cycles x (95 ℃ C15 s, 55 ℃ C15 s, 72 ℃ C1 min); c5 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 gel-recovered and purified using a gel recovery and purification kit (Vazyme, cat. No. DC 301-01) (FIG. 1B).

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 having inserted therein each gene	0.06 pmol
2×Clon Express Mix	5 μL
		Ultrapure water	Make up to 10 mu L

The system is incubated at 50 ℃ for 30 min. Product conversionE. coliDH 5. alpha. was competent, spread on LB solid plates containing 34 mg/L chloramphenicol, positive clones were PCR-screened (FIG. 1C), and the recombinant plasmid pACYC-mvaE-mvaS-IspA (FIG. 1D) was extracted from the positive clones 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 using pUC-yqjfC55 as a template, a primer IspA-C55-F and a primer IspA-C55-R, and amplifying a vector fragment, wherein a PCR amplification system is shown as follows:

the PCR procedure was: 30 cycles x (98 ℃ 10 s, 60 ℃ 15 s, 68 ℃ 90 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 Bl21(DE3) genome as template, primer IspA-T7RNAP-F and primer IspA-T7RNAP-R, and PCR was performed to amplify a T7RNA Polymerase fragment (SEQ ID NO. 6) in the following PCR amplification system:

the PCR procedure was: c, 3 min at 95 ℃; 30 cycles × (95 ° C15 s, 58 ° C15 s, 72 ° C20 s); c5 min at 72 ℃; and (3) 16 ℃ C ∞.

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).

Using 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 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'-tgtctattgctggtttaccgg-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 having inserted therein each gene	0.06 pmol
2×Clon Express Mix	5 μL
		Ultrapure water	Make up to 10 mu L

The system is incubated at 50 ℃ for 30 min. Product conversionE. coliDH 5. alpha. was competent, spread on LB solid plates containing 34 mg/L chloramphenicol, positive clones were PCR-screened (FIG. 2D), and the 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 Strain

The plasmid pET-lyc (cited in CN 112877342A), pACYC-mvaE-mvaS-IspA-yqjfC55-T7RNAP, pTrc-low plasmid (cited in Yang J, Xiaoan M, Su S, et al, Enhancing production of bio-isopropyl using hybrid MVA pathway and isopropyl synthase in E. coli, PLoS one 2012; 7: e 33509.) were co-transformedE. coliBL21(DE3) 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, thereby obtaining an engineered strain XLYC3 containing the vectors pET-lyc, pACYC-mvaE-mvaS-IspA-C55-T7RNAP plasmid and ptrc-low plasmid.

Example 3: application of visual detection of explosive molecules

And (3) detection of the conical flask: selecting a single colony of engineering escherichia coli XLYC3 obtained in example 2 to be placed in 10 mL LB liquid culture medium containing 34 mg/L chloramphenicol, 100 mg/L ampicillin and 30 mg/L kanamycin, and carrying out shaking table overnight culture and activation at 37 ℃; transferring to 100 mL M9 culture medium for amplification culture to OD₆₀₀0.6-0.8, 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 solution red color is more pronounced for both DNT groups compared to the IPTG-induced group, and the red color is 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 result shows that the effect of detecting the explosive molecules by the biosensor for generating lycopene is very obvious as the concentration of DNT is higher, the yield of lycopene is higher and red is more obvious.

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

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cg 242

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atcggtcggg ccattgagga cgaggctcgc ttcggtcgta tccgtgacct tgaagctaag 480

cacttcaaga aaaacgttga ggaacaactc aacaagcgcg tagggcacgt ctacaagaaa 540

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tctgctgcta agctgctggc tgctgaggtc aaagataaga agactggaga gattcttcgc 2160

aagcgttgcg ctgtgcattg ggtaactcct gatggtttcc ctgtgtggca ggaatacaag 2220

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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

<210> 7

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ggaaggaggt aaagatctat gaacacg 27

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ttacgcgaac gcgaagtccg 20

<210> 9

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

cggacttcgc gttcgcgtaa taagcggcta tttaacgacc c 41

<210> 10

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tgtctattgc tggtttaccg g 21