CN110873790A - Whole-cell biosensor for detecting heavy metal ions in water-soluble sample and construction and application thereof - Google Patents
Whole-cell biosensor for detecting heavy metal ions in water-soluble sample and construction and application thereof Download PDFInfo
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Abstract
The invention discloses a whole-cell biosensor for detecting heavy metal ions in a water-soluble sample and construction and application thereof.A working object of the biosensor is escherichia coli, which has no risk of causing diseases and is simple and easy to operate, an SRrz cracking gene is used as a report element, so that microbial thalli are cracked, the turbidity of bacterial liquid is obviously changed, a result can be obtained by detecting through a visible spectrophotometer or directly observing through naked eyes, the response is rapid, only 30-60 min is needed from the contact of a sample to the result, β -galactosidase released to the outside of a cell is detected by using X-gal, the operation is simple and convenient, no expensive instrument is needed in application, the detection cost is greatly reduced, the field detection is possible, the accurate quantification of the activity of the extracellular β -galactosidase by using a developing gel containing oNPG is realized, the detection limit is low, and the detection result is accurate.
Description
Technical Field
The invention belongs to the technical field of environmental biology, and particularly relates to a whole-cell biosensor for high-sensitivity detection of heavy metal ions (divalent mercury ions or lead ions) in a water-soluble sample, and construction and application thereof.
Background
With the development of industry, heavy metal pollution becomes more serious and becomes one of outstanding environmental problems. Heavy metal has high toxicity, is easy to enrich in organisms and difficult to degrade, and brings great threat to human health after entering a food chain, so that the detection technology of heavy metal ions is very important. The detection technology of heavy metal ions mainly comprises an atomic absorption spectrometry, an atomic fluorescence spectrometry, an inductively coupled plasma method, an ultraviolet-visible spectrophotometry, a high performance liquid chromatography, an electrochemical analysis method, a biological detection method and a chemical color development method. The atomic absorption spectrometry, the atomic fluorescence spectrometry, the inductively coupled plasma method, the ultraviolet-visible spectrophotometry and the high performance liquid chromatography have the advantages of high sensitivity, high specificity and the like, but the required instruments are expensive, complex to operate and inconvenient to carry, are mainly used for laboratory detection and cannot be used as a field detection technology. The electrochemical analysis method and the chemical color development method are simple to operate and easy to miniaturize, can be used as a field detection technology, but the sensitivity of the electrochemical analysis method and the chemical color development method cannot meet the requirement. Because of serious heavy metal pollution in China, simple and convenient operation and high sensitivity, the on-site real-time detection of the heavy metal is more and more emphasized.
Specific microbial sensor detection is based on the response of engineered strains to specific small molecule substances, whose function is mainly performed by two elements. 1. The induction element composed of regulatory protein and promoter regulated by the regulatory protein can cause gene expression change after the induction element reacts with specific small molecular substance, so as to cause a series of phenomena. 2. A reporter element consisting of a protein which is controlled by the sensor element and is easy to detect. In 1997, Mistelli et al isolated a native Green Fluorescent Protein (GFP) from Aequorea Victoria. Because the fluorescent probe has fluorescence, does not need an additional substrate and is stable, the fluorescent probe is widely applied to a microbial sensor as a reporting element. Roberto FF et al, 2002, produced a microbial sensor for detecting As with GFP As a reporter element; in 2006, Lian VH et al detected heavy metal ions in soil based on GFP reporter elements; in 2016, Lara Bereza-Malcolm produced a biosensor capable of specifically detecting lead using GFP as a reporter element.
The advantages of the microbial sensor detection are low price, simple operation, high specificity and good sensitivity, but the existing biological detection method has the problems. Firstly, the detection period is longer, and green fluorescent protein has multiple advantages, but its maturation time is longer, often needs 2 ~ 3 hours just can the complete maturation, is unfavorable for the short-term test. Furthermore, the quantification of GFP requires the use of expensive instruments, which are not easily portable and do not facilitate on-site real-time detection.
The escherichia coli can overcome the defects, constructs the escherichia coli which is sensitive and can rapidly detect the heavy metal ions in the water, and lays a foundation for developing a cheap and portable microorganism field detection technology.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention mainly aims to provide a whole-cell biosensor for detecting heavy metal ions in a water-soluble sample.
The invention also aims to provide a construction method of the whole-cell biosensor for detecting heavy metal ions in the water-soluble sample.
The invention also aims to provide the application of the whole-cell biosensor for detecting heavy metal ions in the water-soluble sample.
Still another object of the present invention is to provide a method for rapidly detecting heavy metal ions using the whole-cell biosensor. The method has the advantages of convenient operation, high sensitivity, short time consumption, low cost, good visibility and easy quantification.
The microorganisms with heavy metal tolerance can cause the expression of a series of genes related to the heavy metal tolerance when being stimulated by corresponding heavy metals. The expression of these genes is mainly a class of regulatory proteins represented by MerR family proteins, which can activate non-optimal sigma 70-dependent promoters, whose-35 and-10 regions differ by a number of bases greater than the optimal length of 17 ± 1bp that can be recognized by the sigma 70 factor. When MerR protein is specifically combined with mercury ions, the configuration of the promoter can be changed, so that the promoter can be correctly recognized by sigma 70 factor, and the transcription of downstream genes is started. The invention uses MerR protein and a promoter regulated by the MerR protein and the like to control the expression of a downstream reporter gene and realize the qualitative and quantitative detection of heavy metal ions in the environment.
The lambda phage of E.coli is one of the most well studied and widely used phages at present. The genes causing cell lysis in bacteriophage include S, R and Rz, wherein the R gene encodes a water-soluble transglycosylase (transglycosylase) enzyme that causes hydrolysis of peptide bonds, breaking down peptidoglycan of cell wall. The product of the Rz gene may be an endopeptidase (endopeptidase) which cleaves the linkage between peptidoglycan and oligosaccharides and/or between peptidoglycan and the outer membrane of the cell wall. The products of both the R and Rz genes function to degrade the cell wall, while the S gene product functions to alter the permeability of the cytoplasmic membrane, forming a porous structure on the cytoplasmic membrane, so that the products of the R and Rz genes pass through the cytoplasmic membrane and act on the cell wall, disrupting the cell wall, releasing the intracellular material.
β -galactosidase (β -galactosidase) is widely present in various microorganisms and is a most mature reporter protein which catalyzes hydrolysis of β -galactoside bond in β -galactoside compounds, and catalyzes blue coloration of X-gal (5-Bromo-4-chloro-3-indol β -D-galactoside, 5-Bromo-4-chloro-3-indole- β -D-galactoside) hydrolysate, and is easy to observe and detect, and catalyzes hydrolysis of oNPG (o-Nitrophenyl- β -D-Galactopyranoside, o-Nitrophenyl β -D-Galactopyranoside) with yellow color, and reaction of β -galactosidase with oNPG is commonly used in activity test of β -galactosidase.
The invention uses recombinant colibacillus as heavy metal ion detecting bacteria, the strain connects the specific heavy metal ion response regulating protein and its corresponding promoter sequence with bacteriophage cracking protein SRRz gene, and then leads into the colibacillus after connecting with plasmid carrier, forms the colibacillus which can crack under heavy metal ion stimulation, namely the recombinant colibacillus used in the invention, when the recombinant colibacillus meets the corresponding heavy metal ion, the heavy metal ion enters into the bacteria body to combine with the regulating protein, thereby starts the expression of cracking gene SRRz, leads to the colibacillus cracking, and releases β -galactosidase.
The purpose of the invention is realized by the following technical scheme:
a whole cell biosensor for detecting heavy metal ions in a water-soluble sample is constructed by transforming a gene expression system induced by the heavy metal ions into escherichia coli;
the gene expression system induced by the heavy metal ions is sequentially connected with an escherichia coli terminator, a heavy metal ion response element, a phage lysis gene and an escherichia coli terminator from 5 'to 3';
the heavy metal ion response element can be any element responding to the heavy metal ion, and preferably contains a bidirectional promoter sequence; more preferably mercury (Hg) containing a bidirectional promoter sequence2+) Response protein (SEQID No.1) or lead (Pb) containing bidirectional promoter sequence2+) The response protein (SEQ ID No. 2).
The phage lysis gene can be any phage lysis gene, preferably the lysis gene SRrz of lambda phage, and has the nucleotide sequence of SEQ ID No.3 in the sequence table.
The Escherichia coli terminator can be any one of Escherichia coli terminators, and is preferably a terminator TrrnBThe nucleotide sequence is shown in SEQ ID No. 4.
The starting vector used by the heavy metal ion-induced gene expression system can be any escherichia coli vector, and preferably pSB1C3, pBluescript, pUC18, pUC19, pET series and other cloning expression vectors are selected. More preferably, pSB1C3 is used as a starting vector, and the constructed Escherichia coli cracking vectors are pSB1C3-MHg and pSB1C 3-MPb.
Coli BL21 is preferred.
A construction method of a whole-cell biosensor for detecting heavy metal ions in a water-soluble sample specifically comprises the following steps: taking pSB1C3 as a starting vector:
(1) synthesizing response protein and bidirectional promoter sequences responding to mercury ions and lead ions respectively, wherein the sequences are shown as SEQ ID No.1 and SEQ ID No.2, and the sequences are EcoRI, NotI, XbaI, reverse complementary coding genes of the response protein, bidirectional promoters, SpeI, NotI and PstI in sequence from 5 'to 3';
(2) synthesizing a cleavage gene SRrz with the sequence shown in SEQ ID No.3, wherein the sequence is EcoRI, NotI, XbaI, SRrz cleavage gene, SpeI, NotI and PstI from 5 'to 3';
(3) synthesis of terminator TrrnBThe sequence is shown in SEQ ID No.4, and the sequence is EcoRI, NotI, XbaI and T in sequence from 5' to 3rrnBTerminator, SpeI, NotI and PstI;
(4) the protein containing mercury ion response protein and the bidirectional promoter sequence SEQ ID No.1 is subjected to double digestion by XbaI and PstI, and the protein contains a terminator TrrnBSequence SEQ ID No.4 was double digested with EcoRI and SpeI, vector pSB1C3 was double digested with EcoRI and PstI, and the three were cyclized by ligase to form pSB1C3-TrrnB-HgR, wherein TrrnBThe connection of HgR is carried out by means of SpeI and XbaI cleavage to generate the same tail sequence;
(5) the protein containing the lead ion response protein and the bidirectional promoter sequence SEQ ID No.2 is subjected to double digestion by XbaI and PstI, and the protein contains a terminator TrrnBSequence SEQ ID No.4EcoRI and SpeI are subjected to double digestion, the vector pSB1C3 is subjected to double digestion by EcoRI and PstI, and the three are cyclized under the action of ligase to form pSB1C3-TrrnB-PbR, wherein TrrnB-the ligation of PbR is performed by means of SpeI and XbaI cleavage to generate a homologous tail sequence;
(6) the sequence SEQ ID No.3 containing the SRrz cleavage gene is digested simultaneously with EcoRI and SpeI, containing the terminator TrrnBThe sequence SEQ ID No.4 is double-digested by XbaI and PstI, the vector pSB1C3 is double-digested by EcoRI and PstI, and the three are cyclized under the action of ligase to form pSB1C3-SRrz-TrrnBWherein SRrz-TrrnBThe connection of (a) is realized by means of SpeI and XbaI enzyme digestion to generate a homologous tail sequence for connection;
(7) plasmid pSB1C3-TrrnB-HgR plasmid pSB1C3-SRrz-T using SpeI and PstI double digestionrrnBXbaI and PstI are used for double enzyme digestion, and the target fragments are purified and then are connected to obtain the mercury ion response vector which is named as pSB1C 3-MHg.
(8) Plasmid pSB1C3-TrrnBThe plasmid pSB1C3-SRrz-T, obtained by double digestion of the PbR with SpeI and PstIrrnBXbaI and PstI are used for double enzyme digestion, and the target fragments are purified and then connected to obtain the lead ion response vector which is named as pSB1C 3-MPb.
(9) And (3) respectively transferring the vectors obtained in the steps (7) and (8) into E.coli BL21 competent cells to obtain recombinant escherichia coli for heavy metal detection, namely a whole-cell biosensor.
The whole-cell biosensor for detecting the heavy metal ions in the water-soluble sample is applied to the rapid detection of the heavy metal ions in the water-soluble sample.
The sensor cell can specifically complete sample detection in 30-45 min under the conditions of 15-40 ℃ and pH 4-9.
The heavy metal ion is preferably mercury (Hg)2+) Or lead (Pb)2+)。
The sensor cell can specifically detect 0-80 nM inorganic bivalent mercury ion pollution solution in 30-45 min at 15-40 ℃ and pH 4-9.
The sensor cell can specifically detect 0-2000 nM inorganic divalent lead ion polluted solution in 30-45 min at 15-40 ℃ and pH 4-9.
The content of heavy metal ions (inorganic bivalent mercury ions or lead ions) contained in the solution to be detected can be determined by detecting the cell density (spectrophotometry) of the biosensor or observing the turbidity of the bacterial liquid by naked eyes.
A method for rapidly detecting heavy metal ions by using the whole-cell biosensor comprises the following steps of incubating the whole-cell biosensor (recombinant escherichia coli) with the heavy metal ions, cracking escherichia coli cells and releasing β -galactosidase, wherein the result shows that the density of escherichia coli thalli is remarkably reduced, and the qualitative detection of whether a sample to be detected contains the heavy metal ions can be realized through quantitative detection by a spectrophotometer or visual observation.
The content of heavy metal ions (inorganic bivalent mercury ions or lead ions) contained in the solution to be detected can be quantified by detecting the enzyme activity of beta-galactosidase released outside the biosensor cell. The enzyme activity detection scheme is shown in one of the following schemes:
the first scheme is as follows: incubating lysate and a prefabricated gel block containing X-gal or oNPG, photographing the gel to convert the gel into a gray picture, and dividing the white color and the black color into a plurality of levels according to a logarithmic relation, wherein the range is from 0 to 255, the white color is 255, and the black color is 0; establishing a relation between the concentration of the heavy metal ions and the gray value of the gel, and drawing a standard curve of the developing gel; and carrying out semi-quantitative analysis on the concentration of the heavy metal ions in the sample to be detected, and calculating the ion concentration.
And in the second scheme, enzyme activity detection can be performed on β -galactosidase by using an enzyme-labeling instrument, and the ion concentration in the sample is accurately quantified through a standard curve.
The method specifically comprises the following steps:
the first scheme is as follows: semi-quantitative and semi-quantitative detection of heavy metal ions by chromogenic gel method
(A) Preparing an escherichia coli detection solution;
(B) adding 2% (m/v) agar powder into Z buffer, heating for dissolving, adding X-gal solution or oNPG solution to a final concentration of 1mg/mL, mixing, adding into 96-well plate, cooling at room temperature for solidification, and making into chromogenic gel;
(C) mixing the escherichia coli detection solution with heavy metal ion standard samples with different concentration gradients, and simultaneously taking the escherichia coli detection solution added with the same volume of pure solvent as a control; continuously culturing the samples for 20min at 35-37 ℃ and 220 rpm; adding the culture solution supernatant into a small hole containing the chromogenic gel, and incubating for 30min at 35-37 ℃; taking a picture of the gel, converting the picture into a grey-scale picture, and dividing the white color and the black color into a plurality of levels according to a logarithmic relation, wherein the range is from 0 to 255, the white color is 255, and the black color is 0; establishing a relation between the concentration of the heavy metal ions and the gray value of the gel, and drawing a standard curve of the developing gel;
(D) mixing a sample to be detected with the escherichia coli detection solution, and continuously culturing for 20min at 35-37 ℃ and 220 rpm; adding the culture solution supernatant into a small hole containing the chromogenic gel, and incubating for 30min at 35-37 ℃; taking a picture of the gel to convert the picture into a gray picture, and obtaining a gray value of the gel; and (C) calculating to obtain the concentration of the heavy metal ions in the sample to be detected according to the standard curve of the developing gel in the step (C).
Scheme II: enzyme-linked immunosorbent assay for detecting heavy metal ions
(A) Preparing an escherichia coli detection solution;
(B) mixing an escherichia coli detection solution with heavy metal ion standard samples with different concentration gradients, and taking the escherichia coli detection solution added with a pure solvent with the same volume as the standard samples as a control, continuously culturing the samples for 20min at 35-37 ℃ and 220rpm, mixing a culture solution supernatant with a substrate X-gal solution or an oNPG solution, reacting for 30min at 35-37 ℃, determining β -galactosidase enzyme activity, establishing a relation between β -galactosidase enzyme activity and heavy metal ion concentration, and drawing a standard curve;
(C) mixing a sample to be detected with an escherichia coli detection solution, continuously culturing for 20min at 35-37 ℃ and 220rpm, mixing a culture solution supernatant with a substrate X-gal solution or an oNPG solution, reacting for 30min at 35-37 ℃, determining β -galactosidase enzyme activity, and calculating the concentration of heavy metal ions in the sample to be detected according to the standard curve in the step (B).
Preferably, the Z buffer described in step (one) (B): calculated as 50mL, the solution was buffered in 50mL of 1 XPBSTo the solution was added 0.12g MgSO4And 45. mu.L of β -mercaptoethanol.
Preferably, the standard curve of the developing gel described in step (a) (C) is as follows:
Hg2+，Y＝212.5-2.099X-0.02141X2，R2＝0.9922(0<X≤40nM)
Pb2+，Y＝244.7-0.13X-2.509*10-5X2，R2＝0.9850(0<X≤2000nM)
wherein X represents heavy metal ion concentration (nM), Y represents gel grayscale value, R2Is the correlation coefficient of the fitted curve.
Preferably, the standard curve described in step (two) (B) is as follows:
Hg2+，Y＝6.761*X-133.0，R2＝0.997(0<X≤80)
Pb2+，Y＝0.2208*X-25.68，R2＝0.982(0<X≤2000)
wherein X represents heavy metal ion concentration (nM), Y represents extracellular enzyme activity (U/mL), R2Is the correlation coefficient of the fitted curve.
The preparation method of the escherichia coli detection solution comprises the following steps:
(a) culturing the recombinant escherichia coli by using an LB solid culture medium to recover and activate the recombinant escherichia coli;
(b) picking a single colony to be inoculated into an LB liquid culture medium for shake culture overnight;
(c) mixing the raw materials in a ratio of 1: inoculating the strain into a fresh LB liquid culture medium in a volume ratio of 50-100, and culturing until the strain liquid OD600And (3) adding IPTG (isopropyl-beta-D-thiogalactoside) and culturing for 30min to obtain an escherichia coli detection solution, wherein the concentration of the IPTG is 0.4-0.8.
Preferably, the culturing in step (a) is carried out at 37 ℃ for 14 h.
Preferably, the shaking culture overnight in the step (b) is carried out at 37 ℃ and 220rpm for 12-16 h.
Preferably, the culturing condition in the step (c) is culturing at 35-37 ℃ and 220 rpm.
Preferably, the bacterial liquid OD6000.4 to 0.5.
Preferably, the concentration of IPTG is 0.1 mM.
Compared with the prior art, the invention has the following advantages and effects:
(1) the operation object is escherichia coli, no pathogenic risk exists, and the operation is simple and easy.
(2) By using the SRrz lytic gene as a reporter element, the response is quick, and only 30-60 min is needed from the contact of a sample to the acquisition of a result.
(3) The SRrz lytic gene is used as a report element, so that microbial thalli are lysed, the turbidity of microbial liquid is obviously changed, and the result can be obtained by detecting through a common visible spectrophotometer or directly observing through naked eyes.
(4) The β -galactosidase released outside the cell is detected by using the X-gal, the operation is simple, expensive instruments are not required to be used in the application, the detection cost is greatly reduced, and the field detection is possible.
(5) The enzymatic activity of the extracellular β -galactosidase is accurately quantified by using the chromogenic gel containing oNPG, the detection limit is low, and the detection result is accurate.
(6) The method can provide technical support for daily heavy metal pollution detection and sudden heavy metal pollution detection.
Drawings
FIG. 1 is a schematic diagram of the construction of a heavy metal-responsive vector.
FIG. 2 shows the change of the turbidity of the bacterial liquid when different detection bacteria are exposed to different heavy metal ion water samples for 0.5 hour.
FIG. 3 shows the extracellular β -galactosidase enzyme activity after each test bacterium was contacted with different heavy metal ions.
FIG. 4 is a color development gel image of each test bacterium when it is contacted with heavy metal ions of different concentrations for 0.5 hour. Wherein 1, 2 and 3 represent repeated samples respectively.
FIG. 5 is a standard curve of heavy metal ion developing gel.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents and the like used are, unless otherwise specified, reagents and materials obtained from commercial sources.
Example 1 obtaining of heavy Metal ion-responsive recombinant bacteria
A heavy metal response vector was constructed using pSB1C3 as the starting vector (iGEM, http:// parts.IGem. org/Part: pSB1C 3). The construction scheme of the heavy metal response carrier is shown in figure 1. The specific construction method comprises the following steps:
1. response proteins responding to mercury ions and lead ions and bidirectional promoter sequences (SEQ ID No.1 and SEQ ID No.2) are synthesized respectively, and the sequences are EcoRI, NotI, XbaI, a response protein reverse complementary coding gene, a bidirectional promoter, SpeI, NotI and PstI in sequence from 5 'to 3'.
2. Synthesizing a cleavage gene SRRz, wherein the sequence of the cleavage gene SRRz is shown in SEQ ID No.3, and the sequence of the cleavage gene SRRz is EcoRI, NotI, XbaI, SRRz, SpeI, NotI and PstI from 5 'to 3'.
3. Synthesis of terminator TrrnBThe sequence is shown in SEQ ID No.4, and the sequence is EcoRI, NotI, XbaI and T in sequence from 5' to 3rrnBTerminator, SpeI, NotI and PstI.
4. The protein containing mercury ion response protein and the bidirectional promoter sequence SEQ ID No.1 is subjected to double digestion by XbaI and PstI, and the protein contains a terminator TrrnBSequence SEQ ID No.4 was double digested with EcoRI and SpeI, vector pSB1C3 was double digested with EcoRI and PstI, and the three were cyclized by ligase to form pSB1C3-TrrnB-HgR, wherein TrrnBThe ligation of-HgR was performed by means of SpeI and XbaI cleavage to generate the homologous tail sequences.
5. The protein containing the lead ion response protein and the bidirectional promoter sequence SEQ ID No.2 is subjected to double digestion by XbaI and PstI, and the protein contains a terminator TrrnBSequence SEQ ID No.4 was double digested with EcoRI and SpeI, vector pSB1C3 was double digested with EcoRI and PstI, and the three were cyclized by ligase to form pSB1C3-TrrnB-PbR, wherein TrrnBThe ligation of-PbR is by means of SpeI and XbaI cleavage to generate homo-tailThe sequences are ligated.
6. The sequence SEQ ID No.3 containing the SRrz cleavage gene is digested simultaneously with EcoRI and SpeI, containing the terminator TrrnBThe sequence SEQ ID No.4 is double-digested by XbaI and PstI, the vector pSB1C3 is double-digested by EcoRI and PstI, and the three are cyclized under the action of ligase to form pSB1C3-SRrz-TrrnBWherein SRrz-TrrnBThe ligation of (2) was performed by means of SpeI and XbaI cleavage to generate the same tail sequence.
7. Plasmid pSB1C3-TrrnB-HgR plasmid pSB1C3-SRrz-T using SpeI and PstI double digestionrrnBXbaI and PstI are used for double enzyme digestion, and the target fragments are purified and then are connected to obtain the mercury ion response vector which is named as pSB1C 3-MHg.
8. Plasmid pSB1C3-TrrnBThe plasmid pSB1C3-SRrz-T, obtained by double digestion of the PbR with SpeI and PstIrrnBXbaI and PstI are used for double enzyme digestion, and the target fragments are purified and then connected to obtain the lead ion response vector which is named as pSB1C 3-MPb.
9. And (3) respectively transferring the vectors obtained in the steps (7) and (8) into E.coli BL21 competent cells according to a conventional method to obtain recombinant escherichia coli for detecting heavy metals, namely a whole-cell biosensor.
Example 2 fast detection of heavy metal ion concentration in samples by cell lysis efficiency
1. Resuscitation and activation of recombinant escherichia coli
The recombinant Escherichia coli was streaked from a refrigerator at-80 ℃ onto an LB plate medium and cultured for 14 hours at 37 ℃. And picking single colonies, inoculating the single colonies into an LB culture medium, and culturing for 12-16 h at 37 ℃ and 220 rpm.
2. Preparation of Escherichia coli detection solution
Resuscitated activated bacterial fluid is treated in a proportion of 1: inoculating the mixture into a fresh LB culture medium in a volume ratio of 50%, and culturing the mixture to OD under the conditions of 35-37 ℃ and 220rpm600Adding 0.1mM IPTG into the conical flask when the concentration is 0.4-0.8, and continuously culturing for 0.5h to obtain an escherichia coli detection solution.
3. Contact with the test sample
The escherichia coli detection solution in the conical flask is divided into 5mL test tubes, 10 mu L of test sample is added into the test tubes, and the culture is continued for 20min at 35-37 ℃ and 220 rpm.
4. Sample detection
Measuring OD with spectrophotometer600。
And (3) centrifuging the bacterial liquid for 2min at the temperature of 4 ℃ for 17000g, taking the supernatant into a 96-well plate, and preparing an β -galactosidase enzyme activity detection system according to the table 1.
TABLE 1 enzyme activity assay systems
| Reagent | Volume of |
| Z buffer | 136μL |
| oNPG(4mg/mL) | 44μL |
| Lysate centrifugation supernatant | 0.2OD600 |
The preparation method of the Z buffer comprises the following steps: to 50mL of 1 XPBS buffer solution was added 0.12g MgSO4And 45. mu.L of β -mercaptoethanol.
β -galactosidase enzyme activity definition:
U＝1000×[(OD420-1.75×OD550)]/(T×V×OD600)
wherein, OD420、OD550And OD600For the absorbance of the sample at the corresponding wavelength, T is the reaction time after addition of the reaction substrate oNPG, and V is the volume of the sample after dilution (unit: mL).
The experimental results show that when the test bacteria are contacted with samples containing different concentrations of heavy metal ions, the test bacteria are subjected to thallus lysis to release the intracellular β -galactosidase, and the released enzyme quantity is related to the ion concentration, as shown in FIG. 2.
According to the detection graph, the detection standard curve of the corresponding heavy metal ions is shown in FIG. 3.
Hg2+，Y＝6.761*X-133.0，R2＝0.997(0<X≤80)
Pb2+，Y＝0.2208*X-25.68，R2＝0.982(0<X≤2000)
Wherein X represents heavy metal ion concentration (nM), Y represents extracellular enzyme activity (U/mL), R2Is the correlation coefficient of the fitted curve.
Example 3 semi-quantitative Rapid detection of heavy Metal ion gel in combination with chromogenic gel method
1. Preparation of a developing gel
1.1 Add 0.4g of agar powder into 20mL of Z buffer, heat to dissolve in microwave oven, and keep in 70 deg.C water bath for further use.
1.2 adding 40mg/mL of X-gal solution into the solution until the final concentration is 1mg/mL, uniformly mixing, sucking 200 mu L of the mixture, adding the mixture into a 96-well plate, cooling and solidifying at room temperature, and storing at 4 ℃ for later use. Can be stored for more than 3 months.
2. Drawing a standard curve of the developing gel
The test bacteria were activated, cultured and contacted with the samples to be tested as in example 2 steps 1-3. Add 10. mu.L of the bacterial solution to the wells containing the detection gel and incubate at 37 ℃ for 30 min. The gels exhibited different shades of blue at different metal ion concentrations (fig. 4).
The gel was photographed with a camera and converted to a grey scale photograph, dividing the relationship between white and black into several levels ranging from 0 to 255, with white being 255 and black being 0. Establishing a relation (figure 5) between the metal ion concentration and the gel gray value, and drawing a standard curve of the developing gel; the correlation coefficient of the fitting curve reaches R2＝0.9922(Hg2+) And R2＝0.9850(Pb2+)。
According to FIG. 5, the standard curve for the developed gel is as follows:
Hg2+，Y＝212.5-2.099X-0.02141X2，R2＝0.9922(0<X≤40nM)
Pb2+，Y＝244.7-0.13X-2.509*10-5X2，R2＝0.9850(0<X≤2000nM)
wherein X represents heavy metal ion concentration (nM), Y represents gel grayscale value, R2Is the correlation coefficient of the fitted curve.
3. Heavy metal ion sample detection
The test bacteria were activated, cultured and contacted with the sample to be tested as in example 2 steps 1-3. Add 10. mu.L of the bacterial solution to the wells containing the detection gel and incubate at 37 ℃ for 30 min. And scanning the gel for photographing, and calculating to obtain the corresponding concentration of the heavy metal ions in the sample according to the standard curve of the developing gel.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> university of southern China's science
Guangzhou Delong environmental testing technology Co., Ltd
<120> whole-cell biosensor for detecting heavy metal ions in water-soluble sample and construction and application thereof
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<222>(1)..(22)
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<220>
<222>(23)..(457)
<223> reverse complementary sequence of mercury ion response protein coding gene
<220>
<222>(458)..(528)
<223> bidirectional promoter sequence
<220>
<222>(529)..(549)
<223> the corresponding cleavage sites are SpeI, NotI and PstI in this order
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ccgctgcag 549
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<220>
<222>(1)..(22)
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<220>
<222>(23)..(457)
<223> reverse complementary sequence of lead ion responsive protein coding gene
<220>
<222>(458)..(542)
<223> bidirectional promoter sequence
<220>
<222>(543)..(563)
<223> the corresponding cleavage sites are SpeI, NotI and PstI in this order
<400>2
gaattcgcgg ccgcttctag agttagccgc tggtctggct gttggtcgcg ctttcgccgt 60
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tttcacgcag atcaaccagg tgacgtttca gctggagcag cgcgctaaca cgcatttcaa 180
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<400>3
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ctggcgtggt cggagggaac tgataacgga cgtcagaaaa ccagaaatca tggttatgac 660
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gcattgcagc agattaagga gcgtggcgct ttacctatga ttgatcgtgg tgatatccgt 900
caggcaatcg accgttgcag caatatctgg gcttcactgc cgggcgctgg ttatggtcag 960
ttcgagcata aggctgacag cctgattgca aaattcaaag aagcgggcgg aacggtcaga 1020
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tctgcctgtc atgggctgtt aatcattacc gtgataacgc cattacctac aaagcccagc 1140
gcgacaaaaa tgccagagaa ctgaagctgg cgaacgcggc aattactgac atgcagatgc 1200
gtcagcgtga tgttgctgcg ctcgatgcaa aatacacgaa ggagttagct gatgctaaag 1260
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cagtctgtca gtcagtgcgt gaagccacca ccgcctccgg cgtggataat gcagcctccc 1380
cccgactggc agacaccgct gaacgggatt atttcaccct cagagagagg ctgatcacta 1440
tgcaaaaaca actggaagga acccagaagtatattaatga gcagtgcaga tagagttgcc 1500
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<210>4
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<222>(1)..(22)
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<220>
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<400>4
gaattcgcgg ccgcttctag agaggcatca aataaaacga aaggctcagt cgaaagactg 60
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gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc 180
ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg acggatggcc tttttgcgtt 240
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Claims (10)
1. A whole-cell biosensor for detecting heavy metal ions in a water-soluble sample is characterized in that: the whole-cell biosensor is constructed by transforming a gene expression system induced by heavy metal ions into escherichia coli;
the gene expression system induced by the heavy metal ions is sequentially connected with an escherichia coli terminator, a heavy metal ion response element, a phage lysis gene and an escherichia coli terminator from 5 'to 3'.
2. The whole-cell biosensor for detecting heavy metal ions in a water-soluble sample according to claim 1, wherein:
the heavy metal ion response element is a heavy metal ion response element containing a bidirectional promoter sequence.
3. The whole-cell biosensor for detecting heavy metal ions in a water-soluble sample according to claim 1 or 2, wherein:
the heavy metal ion response element is mercury response protein containing a bidirectional promoter sequence shown in SEQ ID No.1 or lead response protein containing a bidirectional promoter sequence shown in SEQ ID No. 2;
the bacteriophage lysis gene is a lysis gene SRrz of lambda bacteriophage and has a nucleotide sequence shown in SEQ ID No. 3;
the Escherichia coli terminator is a terminator TrrnBThe nucleotide sequence is shown in SEQ ID No. 4.
4. The whole-cell biosensor for detecting heavy metal ions in a water-soluble sample according to claim 1 or 2, wherein:
the starting vector used by the heavy metal ion-induced gene expression system is pSB1C3, pBluescript, pUC18, pUC19 or pET series;
coli BL 21.
5. A construction method of a whole-cell biosensor for detecting heavy metal ions in a water-soluble sample is characterized by comprising the following steps: taking pSB1C3 as a starting vector:
(1) synthesizing response protein and bidirectional promoter sequences responding to mercury ions and lead ions respectively, wherein the sequences are shown as SEQ ID No.1 and SEQ ID No.2, and the sequences are EcoRI, NotI, XbaI, reverse complementary coding genes of the response protein, bidirectional promoters, SpeI, NotI and PstI in sequence from 5 'to 3';
(2) synthesizing a cleavage gene SRrz with the sequence shown in SEQ ID No.3, wherein the sequence is EcoRI, NotI, XbaI, SRrz cleavage gene, SpeI, NotI and PstI from 5 'to 3';
(3) synthesis of terminator TrrnBThe sequence is shown in SEQ ID No.4, and the sequence is EcoRI, NotI, XbaI and T in sequence from 5' to 3rrnBTerminator, SpeI, NotI and PstI;
(4) the protein containing mercury ion response protein and the bidirectional promoter sequence SEQ ID No.1 is subjected to double digestion by XbaI and PstI, and the protein contains a terminator TrrnBSequence SEQ ID No.4 was double digested with EcoRI and SpeI, vector pSB1C3 was double digested with EcoRI and PstI, and the three were cyclized by ligase to form pSB1C3-TrrnB-HgR, wherein TrrnBThe connection of HgR is carried out by means of SpeI and XbaI cleavage to generate the same tail sequence;
(5) the protein containing the lead ion response protein and the bidirectional promoter sequence SEQ ID No.2 is subjected to double digestion by XbaI and PstI, and the protein contains a terminator TrrnBSequence SEQ ID No.4 was double digested with EcoRI and SpeI, vector pSB1C3 was double digested with EcoRI and PstI, and the three were cyclized by ligase to form pSB1C3-TrrnB-PbR, wherein TrrnBThe linkage of-PbR is by means of SpeI and XbaICarrying out enzyme digestion to generate a sequence with the same tail for connection;
(6) the sequence SEQ ID No.3 containing the SRrz cleavage gene is digested simultaneously with EcoRI and SpeI, containing the terminator TrrnBThe sequence SEQ ID No.4 is double-digested by XbaI and PstI, the vector pSB1C3 is double-digested by EcoRI and PstI, and the three are cyclized under the action of ligase to form pSB1C3-SRrz-TrrnBWherein SRrz-TrrnBThe connection of (a) is realized by means of SpeI and XbaI enzyme digestion to generate a homologous tail sequence for connection;
(7) plasmid pSB1C3-TrrnB-HgR plasmid pSB1C3-SRrz-T using SpeI and PstI double digestionrrnBXbaI and PstI double enzyme digestion is adopted, and the target fragments are purified and then connected to obtain a mercury ion response vector which is named as pSB1C 3-MHg;
(8) plasmid pSB1C3-TrrnBThe plasmid pSB1C3-SRrz-T, obtained by double digestion of the PbR with SpeI and PstIrrnBXbaI and PstI double enzyme digestion are adopted, and a target fragment is purified and then connected to obtain a lead ion response vector which is named as pSB1C 3-MPb;
(9) and (3) respectively transferring the vectors obtained in the steps (7) and (8) into E.coli BL21 competent cells to obtain recombinant escherichia coli for heavy metal detection, namely a whole-cell biosensor.
6. The use of the whole-cell biosensor for detecting heavy metal ions in a water-soluble sample according to any one of claims 1 to 4 for rapidly detecting heavy metal ions in a water-soluble sample.
7. Use according to claim 6, characterized in that:
the whole cell sensor cell can complete sample detection in 30-45 min under the conditions of 15-40 ℃ and pH 4-9.
8. A method for rapidly detecting heavy metal ions by using the whole-cell biosensor as claimed in any one of claims 1 to 4, comprising the steps of:
the first scheme is as follows: incubating lysate of a whole-cell biosensor incubated with heavy metal ions with a prefabricated gel block containing X-gal or oNPG, photographing the gel to convert the gel block into a gray picture, and dividing the white color and the black color into a plurality of levels according to a logarithmic relationship, wherein the range is from 0 to 255, the white color is 255, and the black color is 0; establishing a relation between the concentration of the heavy metal ions and the gray value of the gel, and drawing a standard curve of the developing gel; carrying out semi-quantitative analysis on the concentration of heavy metal ions in a sample to be detected, and calculating the ion concentration;
and secondly, carrying out enzyme activity detection on β -galactosidase by using an enzyme-labeling instrument, and accurately quantifying the ion concentration in the sample through a standard curve.
9. The method according to claim 8, characterized in that it comprises in particular the steps of:
the first scheme is as follows: semi-quantitative and semi-quantitative detection of heavy metal ions by chromogenic gel method
(A) Preparing an escherichia coli detection solution;
(B) adding 2% m/v agar powder into Z buffer, heating to dissolve, adding X-gal solution or oNPG solution to a final concentration of 1mg/mL, mixing, adding into a 96-well plate, and cooling at room temperature to solidify to obtain a chromogenic gel for later use;
(C) mixing the escherichia coli detection solution with heavy metal ion standard samples with different concentration gradients, and simultaneously taking the escherichia coli detection solution added with the same volume of pure solvent as a control; continuously culturing the samples for 20min at 35-37 ℃ and 220 rpm; adding the culture solution supernatant into a small hole containing the chromogenic gel, and incubating for 30min at 35-37 ℃; taking a picture of the gel, converting the picture into a grey-scale picture, and dividing the white color and the black color into a plurality of levels according to a logarithmic relation, wherein the range is from 0 to 255, the white color is 255, and the black color is 0; establishing a relation between the concentration of the heavy metal ions and the gray value of the gel, and drawing a standard curve of the developing gel;
(D) mixing a sample to be detected with the escherichia coli detection solution, and continuously culturing for 20min at 35-37 ℃ and 220 rpm; adding the culture solution supernatant into a small hole containing the chromogenic gel, and incubating for 30min at 35-37 ℃; taking a picture of the gel to convert the picture into a gray picture, and obtaining a gray value of the gel; calculating the concentration of the heavy metal ions in the sample to be detected according to the standard curve of the developing gel in the step (C);
scheme II: enzyme-linked immunosorbent assay for detecting heavy metal ions
(A) Preparing an escherichia coli detection solution;
(B) mixing an escherichia coli detection solution with heavy metal ion standard samples with different concentration gradients, and taking the escherichia coli detection solution added with a pure solvent with the same volume as the standard samples as a control, continuously culturing the samples for 20min at 35-37 ℃ and 220rpm, mixing a culture solution supernatant with a substrate X-gal solution or an oNPG solution, reacting for 30min at 35-37 ℃, determining β -galactosidase enzyme activity, establishing a relation between β -galactosidase enzyme activity and heavy metal ion concentration, and drawing a standard curve;
(C) mixing a sample to be detected with an escherichia coli detection solution, continuously culturing for 20min at 35-37 ℃ and 220rpm, mixing a culture solution supernatant with a substrate X-gal solution or an oNPG solution, reacting for 30min at 35-37 ℃, determining β -galactosidase enzyme activity, and calculating the concentration of heavy metal ions in the sample to be detected according to the standard curve in the step (B).
10. The method of claim 9, wherein:
the preparation method of the escherichia coli detection solution comprises the following steps:
(a) culturing the recombinant escherichia coli by using an LB solid culture medium to recover and activate the recombinant escherichia coli;
(b) picking a single colony to be inoculated into an LB liquid culture medium for shake culture overnight;
(c) mixing the raw materials in a ratio of 1: inoculating the strain into a fresh LB liquid culture medium in a volume ratio of 50-100, and culturing until the strain liquid OD600And (3) adding IPTG (isopropyl-beta-D-thiogalactoside) and culturing for 30min to obtain an escherichia coli detection solution, wherein the concentration of the IPTG is 0.4-0.8.
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