CN115975898A - Lead ion bacteria whole-cell biosensor with two-color output signals and application - Google Patents
Lead ion bacteria whole-cell biosensor with two-color output signals and application Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses a lead ion bacteria whole-cell biosensor with a two-color output signal and application thereof. The invention provides a recombinant bacterium, which is obtained by introducing a recombinant vector A into a recipient bacterium; the recombinant vector A contains a DNA fragment A; the DNA fragment A contains a pbr bidirectional promoter, one side of the pbr bidirectional promoter is a PbrR protein coding gene, and the other side of the pbr bidirectional promoter is an alcohol-soluble PV synthetic gene cluster vioABDE. The recombinant bacteria can be used for preparing a lead ion bacteria whole-cell biosensor with a two-color output signal. Through a colorimetric method, the recombinant strain can be used for qualitative and quantitative detection of lead ions in a bioavailable form in an environmental sample.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a lead ion bacteria whole cell biosensor with two-color output signals and application thereof.
Background
Some heavy metal elements (such as lead, cadmium, mercury, chromium, etc.) are known to have no biological function and to be toxic to organisms at very low concentrations. In addition, such elements are not biodegradable in the natural environment and are bioaccumulating. The rapid development of global economy in recent years has led to "heavy metal redistribution", thereby causing a serious pollution event, and environmental protection is becoming a global concern. The premise for carrying out pollution prevention and control is to reasonably and accurately monitor the heavy metal poison in the environment. The whole-cell biosensor senses the heavy metal in a bioavailable form by simulating environment biology, integrates the functions of traditional means such as environmental pollution physicochemical monitoring and biological monitoring, has great significance for predicting the ecological toxicity and environmental risk of toxic heavy metal, and is expected to become powerful supplement of the detection means of mainstream instruments.
The natural pigment synthetic gene cluster is used as a reporter gene module, the bottleneck that the traditional biological sensing signal excessively depends on reading of an instrument is broken through, heavy metal biological sensing based on color change, low cost, quick response and instrument dependence reduction is expected to be realized, exposure of heavy metals is recognized by naked eyes, and related researches are rarely reported.
Violacein is a fat-soluble pigment produced by chromobacterium violacearum. In recent years, heterologous synthesis of violacein has become possible with elucidation of the synthetic pathway of violacein. Based on the recently elucidated violacein branched biosynthetic pathway, the intermediate Proviolacein (PV) is a green pigment. At present, no relevant report of a lead ion microbial whole cell biosensor taking a PV derivative as an output signal exists.
Disclosure of Invention
The invention aims to provide a lead ion bacteria whole-cell biosensor with a two-color output signal and application thereof.
In a first aspect, the invention claims a recombinant bacterium.
The recombinant bacterium claimed by the invention is obtained by introducing the recombinant vector A into a recipient bacterium.
The recombinant vector A contains a DNA fragment A.
The DNA fragment A contains a pbr bidirectional promoter, one side of which is a PbrR protein coding gene, and the other side is Proviolacein (PV) synthetic gene cluster vioABDE.
The Proviolacein (PV) synthetic gene cluster, vioABDE, is assembled in the form of a tetracistron, encoding a VioA protein, a VioB protein, a VioD protein, and a VioE protein.
The nucleotide sequence of the pbr bidirectional promoter is 442 th to 526 th positions of SEQ ID No.1.
The amino acid sequence of the PbrR protein is shown as SEQ ID No. 2.
The amino acid sequence of the VioA protein is shown as SEQ ID No. 3.
The amino acid sequence of the VioB protein is shown in SEQ ID No. 4.
The amino acid sequence of the VioD protein is shown in SEQ ID No. 5.
The amino acid sequence of the VioE protein is shown as SEQ ID No. 6.
The nucleotide sequence of the coding gene of the PbrR protein is a reverse complementary sequence of 7 th to 441 th of SEQ ID No.1.
The nucleotide sequence of the VioA protein coding gene is 569-1825 th of SEQ ID No.1.
The nucleotide sequence of the coding gene of the VioB protein is 1843-4839 th of SEQ ID No.1.
The nucleotide sequence of the VioD protein coding gene is 4857-6008 th site of SEQ ID No.1.
The nucleotide sequence of the VioE protein coding gene is 6026-6601 th site of SEQ ID No.1.
Further, the nucleotide sequence of the alcohol-soluble PV synthetic gene cluster vioABDE is 569-6601 th site of SEQ ID No.1.
Further, the nucleotide sequence of the DNA fragment A is SEQ ID No.1.
The recombinant vector A is obtained by replacing a small fragment between enzyme cutting sites BglII and SacI of pET-21a (+) plasmid with the DNA fragment A.
Further, the recipient bacterium is Escherichia coli.
In a specific embodiment of the invention, the E.coli is E.coli TOP10.
In a second aspect, the invention claims a kit.
The claimed kit of parts comprises:
(A1) A recombinant bacterium as hereinbefore described in the first aspect; and
(A2) And n-butanol.
Further, the kit may further comprise the following:
(A3) An oxidizing agent.
In one embodiment of the present invention, said kit consists of said (A1) and said (A2).
In another embodiment of the present invention, said kit consists of said (A1), said (A2) and said (A3).
In a specific embodiment of the invention, the oxidant is hydrogen peroxide, specifically 30% hydrogen peroxide (mass fraction, matrix is water).
In a third aspect, the invention claims the following application:
(B1) Use of a recombinant bacterium as hereinbefore described in the first aspect or a kit as hereinbefore described in the second aspect in the manufacture of a lead ion bacterial whole cell biosensor which provides a two-colour output signal in the form of a Proviolacein (PV) derivative.
(B2) Use of a recombinant bacterium as hereinbefore described in the first aspect or a kit as hereinbefore described in the second aspect for detecting lead ions.
Further, the lead ion detection is the qualitative and/or quantitative detection of lead ions on the liquid sample.
In a fourth aspect, the invention claims any of the following methods:
the method comprises the following steps: a method for detecting whether a liquid sample contains lead ions is method A or method B or method C or method D:
the method A comprises the following steps: the method for observing the bacterial liquid under the non-oxidation condition can comprise the following steps: placing the recombinant bacterium in the first aspect into the liquid sample to be tested, performing shake culture for 5h at 37 +/-2 ℃ (such as 37 ℃), adding n-butanol into the culture system, performing vortex shake for 5min (extraction), standing the extract at normal temperature for more than 6h (such as 6-10h, specifically such as 6 h), observing the color change of the extract, and if a brown color development reaction is observed, determining that the liquid sample to be tested contains or is candidate to contain lead ions; otherwise, the liquid sample to be tested does not contain or does not contain candidate lead ions.
The method B comprises the following steps: the method for observing the bacterial liquid under the oxidation condition can comprise the following steps: placing the recombinant bacterium in the first aspect into the liquid sample to be detected, performing shake culture for 5h at 37 +/-2 ℃ (such as 37 ℃), adding n-butanol and an oxidant (such as hydrogen peroxide) into a culture system, performing vortex shake for 5min (extraction), standing the extract at normal temperature for more than 6h (such as 6-10h, specifically such as 6 h), observing the color change of the extract, and if a green color reaction is observed, determining that the liquid sample to be detected contains or is candidate to contain lead ions; otherwise, the liquid sample to be detected does not contain or is candidate to contain lead ions.
The method C comprises the following steps: the A652 value detection method under non-oxidation conditions can comprise the following steps: placing the recombinant bacterium in the first aspect into the liquid sample to be tested, shake-culturing for 5h at 37 +/-2 ℃ (such as 37 ℃), then adding n-butanol into the culture system, vortex-shaking for 5min (extraction), standing the extract at normal temperature for more than 6h (such as 6-10h, specifically such as 6 h), taking the supernatant organic phase, and determining the A652 value, which is referred to as the A652 value of the liquid sample group to be tested; if the A652 value of the liquid sample group to be detected is significantly larger than the A652 value of the control group, the liquid sample to be detected contains or is candidate to contain lead ions; otherwise, the liquid sample to be detected does not contain or candidate does not contain lead ions; and the method for measuring the A652 value of the control group is different from the method for measuring the A652 value of the liquid sample group to be measured only in that the liquid sample to be measured is replaced by a liquid sample without containing lead ions.
The method D comprises the following steps: the method for detecting the A652 value under the oxidation condition can comprise the following steps: placing the recombinant bacteria in the first aspect into the liquid sample to be tested, shake-culturing for 5h at 37 +/-2 ℃ (such as 37 ℃), then adding n-butanol and an oxidant (such as hydrogen peroxide) into the culture system, vortex-shaking for 5min (extracting), standing the extract at normal temperature for more than 6h (such as 6-10h, specifically such as 6 h), taking the supernatant organic phase, and determining the A652 value, which is referred to as the A652 value of the liquid sample group to be tested; if the A652 value of the liquid sample group to be detected is obviously greater than the A652 value of the control group, the liquid sample to be detected contains or is candidate to contain lead ions; otherwise, the liquid sample to be detected does not contain or candidate does not contain lead ions; and the method for determining the A652 value of the control group is different from the method for determining the A652 value of the liquid sample group to be tested only in that the liquid sample to be tested is replaced by a liquid sample without containing lead ions.
The second method comprises the following steps: a method for detecting the content of lead ions in a liquid sample can be method E or method F:
the method E comprises the following steps: the A652 value detection method under non-oxidation conditions can comprise the following steps:
(e1) Placing the recombinant strain in the first aspect into a series of lead ion liquid samples with known concentration, shake-culturing for 5h at 37 +/-2 ℃ (such as 37 ℃), adding n-butanol into a culture system, vortex-shaking for 5min, standing the extract at normal temperature for more than 6h (such as 6-10h, specifically such as 6 h), taking the supernatant organic phase to determine an A652 value, and then drawing a standard curve according to the lead ion concentration and the A652 value;
(e2) And (e) replacing the series of lead ion liquid samples with known concentrations in the step (e 1) with the liquid sample to be detected, repeating the step (e 1) to obtain the A652 value of the liquid sample to be detected, and substituting the A652 value into the standard curve to obtain the lead ion content in the liquid sample to be detected.
Method F: the method for detecting the A652 value under the oxidation condition can comprise the following steps:
(f1) Placing the recombinant bacteria in the first aspect into a series of lead ion liquid samples with known concentration, performing shake culture for 5h at 37 +/-2 ℃ (such as 37 ℃), then adding n-butanol and an oxidant (such as hydrogen peroxide) into a culture system, performing vortex shake for 5min (extraction), standing the extract at normal temperature for more than 6h (such as 6-10h, specifically such as 6 h), taking a supernatant organic phase to determine an A652 value, and then drawing a standard curve according to the lead ion concentration and the A652 value;
(f2) And (f) replacing the series of lead ion liquid samples with known concentrations in the step (f 1) with the liquid sample to be detected, repeating the step (f 1), obtaining the A652 value of the liquid sample to be detected, and substituting the A652 value into the standard curve to obtain the lead ion content in the liquid sample to be detected.
Further, the method (A652 value under oxidizing condition) is suitable for the case where the liquid sample to be tested contains lead ions of 0.183nM or more (e.g., 0.732nM or more). The method (A652 value detection method under non-oxidizing condition) is suitable for the condition that the liquid sample to be detected contains lead ions of more than 5.86 nM.
In a specific embodiment of the invention, the oxidant is 30% H 2 O 2 (mass fraction, matrix is water).
In each of the above non-oxidative treatments, the amount of n-butanol added was: adding 180-360 mu L of n-butyl alcohol into each 900 mu L of culture system containing the recombinant bacteria.
In the above oxidation treatment methods, the amount of n-butanol added is: adding 180-360 mu L of n-butanol into every 900 mu L of culture system containing the recombinant bacteria; oxidizing agent (e.g. 30% H) 2 O 2 ) The addition amount of (A) is as follows: adding 50 μ L of oxidant (such as 30% H) into 900 μ L of culture system containing the recombinant bacteria 2 O 2 )。
When the liquid sample to be detected is colorless or can be distinguished from the color of the liquid sample to be detected (can be distinguished by naked eyes) after superimposed color development reaction, the first method preferably adopts a naked eye observation method (namely the method A and the method B) to carry out result judgment. When the liquid sample to be detected is difficult to distinguish from the self color (can not be distinguished by naked eyes) after the superimposed color reaction, the method I preferably adopts the A652 value determination method (namely the method C and the method D) to carry out result judgment.
In the above aspects, the liquid sample to be tested may be purified water, tap water, lake water, or a soil extract solution suspected of containing lead ions.
In each of the above aspects, the lead ion is a divalent lead ion (Pb (II)).
In each of the above aspects, the lead ions are preferably present in the form of soluble divalent lead salts, such as lead acetate, lead chloride, lead nitrate, and the like.
The recombinant strain can be used for qualitative and quantitative detection of lead ions in a bioavailable form in an environmental sample.
Drawings
FIG. 1 is a mechanical diagram of recombinant bacteria response lead ion sensing molecules.
FIG. 2 is a response time-pigment accumulation curve of lead ions of recombinant bacteria. A is a non-oxidation treatment group time-pigment accumulation curve; b is an oxidation treatment group time-pigment accumulation curve; c is a photo of the non-oxidation treatment group extraction liquid; d is a representative photograph of the oxidation-treated group extract.
FIG. 3 shows the relationship between the response dose and effect of lead in recombinant bacteria. A is the non-oxidative treatment group dose-pigment accumulation relationship; b is the relationship between the dosage of the oxidation treatment group and the pigment accumulation; c is a quantifiable dose-pigment accumulation curve of the non-oxidative treatment group; d is a quantifiable dose-pigment accumulation curve of the oxidation treatment group; e is a photograph representing the extracts of the oxidation-treated group and the non-oxidation-treated group.
FIG. 4 shows the response selectivity and anti-interference ability of recombinant bacteria. A is pigment accumulation non-oxidation treatment of the recombinant bacteria responding to different metal ions; b is pigment accumulation oxidation treatment of the recombinant bacteria responding to different metal ions; c is pigment accumulation non-oxidation treatment of the recombinant bacteria responding to different metal ion mixtures; d is pigment accumulation oxidation treatment of the recombinant bacteria responding to different metal ion mixtures.
FIG. 5 is an environmental sample for detecting artificially contaminated lead by recombinant bacteria. A is the non-oxidative treatment group dose-pigment accumulation relationship; b is a quantifiable dose-pigment accumulation curve of the non-oxidation treatment group; c is the oxidation treatment group dose-pigment accumulation relation; d is a quantifiable dose-pigment accumulation curve of the oxidation treatment group; e is a representative photograph of the non-oxidation treatment group extract; f is a representative photograph of the oxidation treatment group extract.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 construction and application of lead ion bacteria whole cell biosensor using alcohol-soluble Proviolacein (PV) derivative as two-color output signal
1. Construction of recombinant plasmid pPb-vioABDE
Firstly, using pET-21a (+) as a vector, synthesizing a PV synthetic gene cluster vioABDE (shown as 569-6601 of SEQ ID No. 1) by using a whole gene, inserting NdeI/SacI into the pET-21a (+) and naming the recombinant plasmid with correct sequencing verification as pET-vioABDE.
Structural description of pET-vioABDE: a recombinant plasmid in which a small fragment between NdeI/SacI of pET-21a (+) vector is replaced with a DNA fragment shown at positions 569-6601 of SEQ ID No.1.
Secondly, the pbrR-pbr promoter gene module (shown as the 7 th-526 th sites of SEQ ID No. 1) is synthesized by the whole gene, bglII/XbaI is inserted into pET-vioABDE, and the recombinant plasmid which is verified to be correct by sequencing is named as pPb-vioABDE.
Structural description of pPb-vioABDE: the small fragment between BglII/XbaI of pET-21a (+) vector was replaced with a recombinant plasmid of the DNA fragment shown in SEQ ID No.1.
The 7 th to 526 th positions of SEQ ID No.1 are pbrR-pbr bidirectional promoter sequences, and are divalent lead ion Pb (II) sensing elements. Wherein, the 442 th to 526 th sites of SEQ ID No.1 are nucleotide sequences of pbr bidirectional promoters, the 7 th to 441 th sites of SEQ ID No.1 are reverse complementary sequences of pbrR genes, and the sequences encode PbrR proteins shown in SEQ ID No. 2.
Positions 569-6601 of SEQ ID No.1 are vioABDE, assembled in the form of a tetracistron, encoding 4 proteins. The 569-1825 th site of SEQ ID No.1 is a VioA gene which encodes VioA protein shown in SEQ ID No. 3. The 1843-4839 site of SEQ ID No.1 is a vioB gene and codes the VioB protein shown in SEQ ID No. 4. The 4857-6008 th position of SEQ ID No.1 is the vioD gene, coding the VioD protein shown in SEQ ID No. 5. The 6026 th to 6601 th sites of the SEQ ID No.1 are vioE genes which encode the VioE protein shown in the SEQ ID No. 6.
When lead ions appear, transcription and translation of 4 genes are activated, produced 4 pigment synthetases catalyze and generate Proviolacein (PV) by taking tryptophan as a substrate, but the PV serving as an intermediate is unstable, and brown or green can be produced respectively by non-oxidation standing or oxidation standing after n-butanol extraction. The molecular mechanism diagram is shown in FIG. 1 below.
2. Construction of recombinant bacterium TOP10/pPb-vioABDE
And (3) transforming the recombinant plasmid pPb-vioABDE constructed in the step one into Escherichia coli TOP10 to obtain TOP10/pPb-vioABDE.
3. Time dose response to lead ions
1. The recombinant strain TOP10/pPb-vioABDE constructed in the second step is inoculated into a liquid LB culture medium containing 50. Mu.g/mL ampicillin, and is subjected to shaking culture at 37 ℃ and 250rpm overnight for 8-12 hours (only the activation of the strain).
2. 1% (volume ratio) of the overnight culture broth was inoculated into fresh LB medium (containing 50. Mu.g/mL ampicilin), supplemented with Pb (II) (specifically, lead acetate, but other soluble divalent lead salts can be used) to a final concentration of 0,0.15, 1.5. Mu. Mol/L, and cultured at 37 ℃ under shaking at 250rpm, sampled at intervals of 1h (2X 1 mL), and stored at 4 ℃. The sampling time was 0,1, 2, 3, 4, 5, 6, 7 hours, respectively.
3. After 1-7h of the bacterial solution was taken, 100. Mu.L of each group was taken to measure the bacterial solution concentration (OD 600).
4. Non-oxidative treatment group (brown): adding 180 mu L of n-butyl alcohol into 900 mu L of the bacterial liquid, and vortexing and shaking for 5min.
5. Oxidation treatment group (green): adding 180. Mu.L of n-butanol and 50. Mu.L of 30% H to 900. Mu.L of the bacterial solution 2 O 2 (mass fraction, matrix is water, the same below), vortex and shake for 5min.
Centrifuging at 6.3500rpm for 5min, standing the extract of non-oxidation and oxidation groups at room temperature for 6 hr, sucking out 100 μ L of supernatant organic phase, placing in 96-well plate, and measuring the light absorption value at 652 nm. The results are shown in table 1, table 2 and fig. 2.
TABLE 1 detection values of non-oxidized group dye A652 at different time points
TABLE 2 detection values of oxidation-treated dye A652 at different time points
The results show that the recombinant bacteria respond to the lead (II) in a dose-response and time-response relationship. Namely, the pigment generation is increased along with the prolonging of the induction time, and the color development is visible to naked eyes after 5h and is not obviously increased. Therefore, 5h of culture is selected as the detection time.
4. Response to different concentrations of lead ion
1. The recombinant strain TOP10/pPb-vioABDE constructed in the second step is inoculated into a liquid LB culture medium containing 50. Mu.g/mL ampicillin, and is subjected to shaking culture at 37 ℃ and 250rpm overnight for 8-12 hours (only the activation of the strain).
2. 1% (volume ratio) of the overnight culture broth was inoculated into fresh LB medium (containing 50. Mu.g/mL ampicilin), pb (II) (specifically, lead acetate, and other soluble divalent lead salts were used) was added by 2-fold dilution to a final concentration of 0,0.0915,0.183,0.366,0.732,1.46,2.93,5.86,11.7,23.4,46.9,93.8,187.5,375,750,1500,3000, 6000 or 12000nmol/L, and the mixture was cultured at 37 ℃ and 250rpm with shaking for 5 hours.
3. Each group of bacterial suspension (2X 1 mL) was collected, and 100. Mu.L of the test bacterial suspension concentration (OD 600) was collected.
4. Non-oxidizing group: adding 360 μ L n-butanol into 900 μ L bacteria solution, and vortex shaking for 5min; and (3) oxidation group: adding 360 mu L n-butanol and 50 mu L30% H into 900 mu L bacterial liquid 2 O 2 Vortex well for 5 minutes.
Centrifuging at 5.3500rpm for 5min, standing the extractive solutions at room temperature for 6 hr, collecting 100 μ L supernatant organic phase, placing in 96-well plate, and measuring 652nm absorbance. The results are shown in table 3 and fig. 3.
Table 3, pigment response detection results of recombinant bacteria to lead ions of different concentrations
The result shows that the recombinant bacterium TOP10/pPb-vioABDE has an obvious dose effect relationship in response to lead (II), the pigment production is improved along with the increase of the lead exposure dose, and the pigment accumulation is stable after 1500 nM.
The limit of detection after oxidative treatment will be lower, i.e. 0.183nM (ttest, statistically different from 0nM ratio) non-oxidative and higher, i.e. 5.86nM (ttest, statistically different from 0nM ratio).
5. Selective response to metal ions
1. The recombinant strain TOP10/pPb-vioABDE constructed in the second step is inoculated into a liquid LB culture medium containing 50. Mu.g/mL ampicillin, and is subjected to shaking culture at 37 ℃ and 250rpm overnight for 8-12 hours (only the activation of the strain).
2. 1% (volume ratio) of the overnight culture broth was inoculated into fresh LB medium (containing 50. Mu.g/mL ampicillin).
3. Individual Metal Selectivity examination- -to the culture system of step 2, individual metal ions, pb (II), cr (III), hg (II), zn (II), mg (II), ni (II), mn (II), ca (II), fe (II), cd (II) or Cu (II) were added to a final concentration of 1.5. Mu.M each. The cells were cultured at 37 ℃ and 250rpm with shaking for 5 hours.
4. Mixed metals (anti-interference) study-to the culture system of step 2 the following ion mix was added to a final concentration of 1.5 μ M (each ion in each group is mixed equimolar). Pb (II), pb (II) + Cr (III), pb (II) + Hg (II), pb (II) + Zn (II), pb (II) + Mg (II), pb (II) + Ni (II), pb (II) + Mn (II), pb (II) + Ca (II), pb (II) + Fe (II), pb (II) + Cd (II), pb (II) + Cu (II), the above-mentioned various ions involved include Pb (II), or the above-mentioned various ions involved do not include Pb (II). The cells were cultured at 37 ℃ and 250rpm with shaking for 5 hours.
5. The control group was cultured without addition of metal salt at 37 ℃ and 250rpm for 5 hours with shaking.
6. Each group of bacterial suspension (2X 1 mL) was collected, and 100. Mu.L of the suspension was measured (OD 600).
7. Non-oxidizing group: adding 360 μ L n-butanol into 900 μ L bacterial liquid, and vortex shaking for 5min; and (3) oxidation group: adding 360 mu L n-butanol and 50 mu L30% H into 900 mu L bacterial liquid 2 O 2 Vortex well for 5 minutes.
Centrifuging at 8.3500rpm for 5min, standing the extractive solutions at room temperature for 6 hr, collecting 100 μ L supernatant organic phase, placing in 96-well plate, and measuring 652nm absorbance. The results are shown in table 4, table 5 and fig. 4.
TABLE 4 Absorbance of recombinant bacteria to produce pigments in response to various metal ions
TABLE 5 Absorbance of recombinant bacteria in response to Mixed Metal produced pigments
The results show that the single metal selectivity investigation result shows that the recombinant bacterium TOP10/pPb-vioABDE shows a highly specific response to Pb (II), and only a lead exposure group shows obvious pigment generation. The result of the study of mixed metal (anti-interference capability) shows that only the mercury and cadmium are doped, the response of the recombinant bacterium TOP10/pPb-vioABDE to lead is reduced, and in addition, all the cultures containing Pb (II) have obvious pigment generation, and the anti-interference capability is better.
6. Dose response of biosensors to Pb (II) in environmental samples
1. The recombinant strain TOP10/pPb-vioABDE constructed in the second step is inoculated into a liquid LB culture medium containing 50. Mu.g/mL ampicillin, and is subjected to shaking culture at 37 ℃ and 250rpm overnight for 8-12 hours (only the activation of the strain).
2. Purified water, tap water, lake water and soil extract are used as substrates (the dosage is 90 percent of volume) to prepare a liquid LB culture medium.
3. 1% (by volume) of the overnight culture was inoculated into fresh LB medium (containing 50. Mu.g/mL ampicillin).
4. Pb (II) (specifically, lead acetate, and other soluble divalent lead salts can be used) was added by 2-fold dilution.
The detection range of the non-oxidized group was smaller than that of the oxidized group, and Pb (II) was added to final concentrations of 0,1.46,2.93,5.86,11.7,23.4,46.9,93.8,187.5,375,750nM, respectively.
The oxidizing group was added Pb (II) to final concentrations of 0,0.0915,0.183,0.366,0.732,1.46,2.93,5.86,11.7,23.4,46.9,93.8,187.5,375,750nM, respectively.
The cells were cultured at 37 ℃ for 5 hours with shaking at 250 rpm.
5. Each group of bacterial suspension (2X 1 mL) was collected, and 100. Mu.L of the test bacterial suspension concentration (OD 600) was collected.
6. Non-oxidizing group: adding 360 mu L of n-butyl alcohol into 900 mu L of bacterial liquid, and performing vortex oscillation5min; and (3) oxidation group: adding 360 mu L n-butanol and 50 mu L30% H into 900 mu L bacterial liquid 2 O 2 Vortex well for 5 minutes.
Centrifuging at 7.3500rpm for 5min, standing the extractive solutions at room temperature for 6 hr, collecting 100 μ L supernatant organic phase, placing in 96-well plate, and measuring 652nm absorbance. The results are shown in table 6, table 7 and fig. 5.
TABLE 6 non-oxidized group test results of lead response pigment production of different environmental samples
Note: the limit of detection of lead ions in the samples from the various environments after the non-oxidative treatment was 5.86nM (ttest, statistically different from 0 nM).
TABLE 7 detection results of oxidation group generated by lead response pigment of different environmental samples
Note: the detection limit of lead ions in different environmental samples after oxidation treatment was 0.732nM (ttest, statistically different from 0 nM).
The result shows that the recombinant strain can respond to lead (II) in different environmental samples, can draw a lead exposure dose-pigment response curve in a certain range, and has application potential for quantitatively detecting environmental lead pollution.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Claims (10)
1. The recombinant strain is obtained by introducing a recombinant vector A into a recipient strain;
the recombinant vector A contains a DNA fragment A;
the DNA fragment A contains a pbr bidirectional promoter, one side of the pbr bidirectional promoter is a PbrR protein coding gene, and the other side of the pbr bidirectional promoter is Proviolacein synthetic gene cluster vioABDE.
2. The recombinant bacterium according to claim 1, wherein: the Proviolacein synthetic gene cluster vioABDE is assembled in the form of a tetracistron, encoding a VioA protein, a VioB protein, a VioD protein and a VioE protein.
3. The recombinant bacterium according to claim 1 or 2, wherein: the nucleotide sequence of the pbr bidirectional promoter is 442 th to 526 th sites of SEQ ID No. 1; and/or
The amino acid sequence of the PbrR protein is shown as SEQ ID No. 2; and/or
The amino acid sequence of the VioA protein is shown as SEQ ID No. 3; and/or
The amino acid sequence of the VioB protein is shown as SEQ ID No. 4; and/or
The amino acid sequence of the VioD protein is shown as SEQ ID No. 5; and/or
The amino acid sequence of the VioE protein is shown as SEQ ID No. 6.
4. The recombinant bacterium according to any one of claims 1 to 3, wherein: the nucleotide sequence of the coding gene of the PbrR protein is a reverse complementary sequence of the 7 th to 441 th positions of SEQ ID No. 1; and/or
The nucleotide sequence of the VioA protein coding gene is 569-1825 th of SEQ ID No. 1; and/or
The nucleotide sequence of the coding gene of the VioB protein is 1843-4839 th of SEQ ID No. 1; and/or
The nucleotide sequence of the VioD protein coding gene is 4857-6008 th site of SEQ ID No. 1; and/or
The nucleotide sequence of the VioE protein coding gene is 6026-6601 th site of SEQ ID No. 1;
further, the nucleotide sequence of the alcohol-soluble PV synthetic gene cluster vioABDE is 569-6601 th of SEQ ID No. 1;
further, the nucleotide sequence of the DNA fragment A is SEQ ID No.1.
5. The recombinant bacterium according to any one of claims 1 to 4, wherein: the recombinant vector A is obtained by replacing a small fragment between enzyme cutting sites BglII and SacI of a pET-21a (+) plasmid with the DNA fragment A;
and/or
The recipient bacterium is escherichia coli;
further, the Escherichia coli is Escherichia coli TOP10.
6. A kit comprising:
(A1) The recombinant bacterium of any one of claims 1-5; and
(A2) N-butanol.
7. The kit of claim 6, wherein: the kit further comprises the following:
(A3) An oxidizing agent;
further, the oxidant is hydrogen peroxide.
8. The following applications are provided:
(B1) Use of the recombinant bacterium according to any one of claims 1 to 5 or the kit according to claim 6 or 7 for the preparation of a lead-ion bacterial whole-cell biosensor which uses a Proviolacein derivative as a two-color output signal.
(B2) Use of the recombinant bacterium of any one of claims 1 to 5 or the kit of claim 6 or 7 for detecting lead ions;
further, the detection of the lead ions is the qualitative and/or quantitative detection of the lead ions on the liquid sample.
9. Any one of the following methods:
the method comprises the following steps: a method for detecting whether a liquid sample contains lead ions is a method A or a method B or a method C or a method D:
the method A comprises the following steps: the method comprises the following steps: placing the recombinant strain of any one of claims 1 to 5 in the liquid sample to be tested, shake-culturing at 37 ± 2 ℃ for 5h, adding n-butanol into the culture system, extracting for 5min, standing the extract at normal temperature for more than 6h, observing the color change of the extract, and if a brown color reaction is observed, determining that the liquid sample to be tested contains or is selected to contain lead ions; otherwise, the liquid sample to be detected does not contain or candidate does not contain lead ions;
the method B comprises the following steps: the method comprises the following steps: placing the recombinant bacterium of any one of claims 1-5 in the liquid sample to be tested, shake-culturing for 5h at 37 ± 2 ℃, then adding n-butanol and an oxidant into the culture system, extracting for 5min, standing the extract at normal temperature for more than 6h, observing the color change of the extract, and if a green color reaction is observed, determining that the liquid sample to be tested contains or is a candidate for containing lead ions; otherwise, the liquid sample to be detected does not contain or candidate does not contain lead ions;
the method C comprises the following steps: the method comprises the following steps: placing the recombinant strain of any one of claims 1-5 in the liquid sample to be tested, shake-culturing at 37 ± 2 ℃ for 5h, adding n-butanol into the culture system, extracting for 5min, standing the extract at normal temperature for more than 6h, taking the supernatant organic phase to determine the A652 value, which is referred to as the A652 value of the liquid sample group to be tested; if the A652 value of the liquid sample group to be detected is obviously greater than the A652 value of the control group, the liquid sample to be detected contains or is candidate to contain lead ions; otherwise, the liquid sample to be detected does not contain or candidate does not contain lead ions; wherein the A652 value of the control group is determined by a method which is different from the A652 value of the liquid sample group to be detected only in that the liquid sample to be detected is replaced by a liquid sample without containing lead ions;
the method D comprises the following steps: the method comprises the following steps: placing the recombinant bacterium of any one of claims 1-5 in the liquid sample to be tested, shake-culturing for 5h at 37 ± 2 ℃, then adding n-butanol and an oxidant into the culture system, extracting for 5min, standing the extract for more than 6h at normal temperature, taking the supernatant organic phase to determine the A652 value, which is referred to as the A652 value of the liquid sample group to be tested; if the A652 value of the liquid sample group to be detected is significantly larger than the A652 value of the control group, the liquid sample to be detected contains or is candidate to contain lead ions; otherwise, the liquid sample to be detected does not contain or candidate does not contain lead ions; wherein the A652 value of the control group is determined by a method which is different from the A652 value of the liquid sample group to be detected only in that the liquid sample to be detected is replaced by a liquid sample without containing lead ions;
the second method comprises the following steps: a method for detecting the content of lead ions in a liquid sample is a method E or a method F:
the method E comprises the following steps: the method comprises the following steps:
(e1) Placing the recombinant strain of any one of claims 1-5 in a series of lead ion liquid samples with known concentration, shake-culturing for 5h at 37 +/-2 ℃, then adding n-butanol into the culture system for extraction for 5min, standing the extract at normal temperature for more than 6h, taking the organic phase of the supernatant to determine the A652 value, and then drawing a standard curve according to the lead ion concentration and the A652 value;
(e2) Replacing the series of lead ion liquid samples with known concentrations in the step (e 1) with the liquid sample to be detected, repeating the step (e 1) to obtain an A652 value of the liquid sample to be detected, and substituting the A652 value into the standard curve to obtain the lead ion content in the liquid sample to be detected;
method F: the method comprises the following steps:
(f1) Placing the recombinant strain of any one of claims 1-5 in a series of lead ion liquid samples with known concentration, shake-culturing for 5h at 37 +/-2 ℃, then adding n-butanol and an oxidant into the culture system, extracting for 5min, standing the extract for more than 6h at normal temperature, taking the supernatant organic phase to determine an A652 value, and then drawing a standard curve according to the lead ion concentration and the A652 value;
(f2) And (f) replacing the series of lead ion liquid samples with known concentrations in the step (f 1) with the liquid sample to be detected, repeating the step (f 1), obtaining the A652 value of the liquid sample to be detected, and substituting the A652 value into the standard curve to obtain the lead ion content in the liquid sample to be detected.
10. A recombinant bacterium or a kit or use or method according to any one of claims 1 to 9, wherein: the lead ions are divalent lead ions.
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