A lead ion bacterial whole-cell biosensor with dual-color output signal and application

Abstract

The application discloses a lead ion bacterial whole-cell biosensor with double-color output signals and application. The application 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; on the DNA fragment A, a pbr bidirectional promoter is contained, one side of the pbr bidirectional promoter is a PbrR protein coding gene, and the other side is an alcohol-soluble PV synthesis gene cluster vioABDE. The recombinant bacterium can be used for preparing the lead ion bacterial whole-cell biosensor with double-color output signals. Through colorimetry, the recombinant bacterium can be used for qualitative and quantitative detection of the biologically available form of lead ions in environmental samples.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a lead ion bacterial whole-cell biosensor with dual-color output signals and its applications. Background Technology

[0002] Some heavy metals (such as lead, cadmium, mercury, and chromium) are known to have no biological function and are toxic to organisms even at extremely low concentrations. Furthermore, these elements are not biodegradable in the natural environment and exhibit bioaccumulation. The rapid global economic development in recent years has led to the "redistribution of heavy metals," triggering a series of serious pollution incidents, making environmental protection an increasingly widespread global concern. Reasonable and accurate monitoring of environmental heavy metal toxicities is a prerequisite for pollution control. Whole-cell biosensors, by simulating environmental biological perception of bioavailable heavy metals, integrate the functions of traditional methods such as physicochemical and biological monitoring of environmental pollution. They are of great significance for predicting the ecotoxicity and environmental risks of toxic heavy metals and are expected to become a powerful supplement to mainstream instrumental detection methods.

[0003] Using natural pigment synthesis gene clusters as reporter gene modules, this approach breaks through the bottleneck of traditional biosensor signals relying too heavily on instrument readings. It is expected to achieve low-cost, fast-response, and less instrument-dependent heavy metal biosensing based on color changes, enabling the naked-eye identification of heavy metal exposure. However, there are few reports on related research.

[0004] Proviolacein is a lipid-soluble pigment produced by *Chroma violaceum*. In recent years, with the elucidation of the proviolacein biosynthetic pathway, its heterologous synthesis has become possible. Based on the recently elucidated branched biosynthetic pathway of proviolacein, its intermediate, proviolacein (PV), is a green pigment. Currently, there are no reports on lead ion whole-cell biosensors for microorganisms using PV derivatives as output signals. Summary of the Invention

[0005] The purpose of this invention is to provide a lead ion bacterial whole-cell biosensor with dual-color output signals and its application.

[0006] In a first aspect, the present invention claims protection for a recombinant bacterium.

[0007] The recombinant bacteria claimed in this invention are obtained by introducing recombinant vector A into recipient bacteria.

[0008] The recombinant vector A contains DNA fragment A.

[0009] The DNA fragment A contains a pbr bidirectional promoter, with one side of the pbr bidirectional promoter being the PbrR protein-coding gene and the other side being the Proviolacein (PV) synthesis gene cluster vioABDE.

[0010] The Proviolacein (PV) synthetic gene cluster vioABDE is assembled in a tetracistronic form, encoding VioA, VioB, VioD, and VioE proteins.

[0011] The nucleotide sequence of the pbr bidirectional promoter is positions 442-526 of SEQ ID No. 1.

[0012] The amino acid sequence of the PbrR protein is shown in SEQ ID No. 2.

[0013] The amino acid sequence of the VioA protein is shown in SEQ ID No. 3.

[0014] The amino acid sequence of the VioB protein is shown in SEQ ID No. 4.

[0015] The amino acid sequence of the VioD protein is shown in SEQ ID No. 5.

[0016] The amino acid sequence of the VioE protein is shown in SEQ ID No. 6.

[0017] The nucleotide sequence of the gene encoding the PbrR protein is the reverse complementary sequence of positions 7-441 of SEQ ID No. 1.

[0018] The nucleotide sequence of the gene encoding the VioA protein is positions 569-1825 of SEQ ID No. 1.

[0019] The nucleotide sequence of the gene encoding the VioB protein is positions 1843-4839 of SEQ ID No. 1.

[0020] The nucleotide sequence of the gene encoding the VioD protein is positions 4857-6008 of SEQ ID No. 1.

[0021] The nucleotide sequence of the gene encoding the VioE protein is positions 6026-6601 of SEQ ID No. 1.

[0022] Furthermore, the nucleotide sequence of the alcohol-soluble PV synthesis gene cluster vioABDE is positions 569-6601 of SEQ ID No. 1.

[0023] Furthermore, the nucleotide sequence of the DNA fragment A is SEQ ID No. 1.

[0024] The recombinant vector A is obtained by replacing the DNA fragment A with a small fragment between the restriction sites BglII and SacI of the pET-21a(+) plasmid.

[0025] Furthermore, the recipient bacterium is Escherichia coli.

[0026] In a specific embodiment of the present invention, the Escherichia coli is Escherichia coli TOP10.

[0027] Secondly, this invention claims protection for a complete set of products.

[0028] The complete product claimed in this invention comprises:

[0029] (A1) The recombinant bacteria mentioned in the first aspect above; and

[0030] (A2) n-Butanol.

[0031] Furthermore, the complete set of products may also include the following:

[0032] (A3) Oxidizing agent.

[0033] In one embodiment of the present invention, the complete product is composed of (A1) and (A2).

[0034] In another embodiment of the present invention, the complete product is composed of (A1), (A2) and (A3).

[0035] In a specific embodiment of the present invention, the oxidant is hydrogen peroxide, specifically 30% hydrogen peroxide (mass fraction, with water as the matrix).

[0036] Thirdly, the present invention claims protection for the following applications:

[0037] (B1) The application of the recombinant bacteria described in the first aspect above or the complete set of products described in the second aspect above in the preparation of a lead ion bacterial whole-cell biosensor with a Proviolacein (PV) derivative as a two-color output signal.

[0038] (B2) The application of the recombinant bacteria described in the first aspect above or the complete set of products described in the second aspect above in the detection of lead ions.

[0039] Furthermore, the detection of lead ions involves qualitative and / or quantitative detection of lead ions in a liquid sample.

[0040] Fourthly, the present invention claims protection for any of the following methods:

[0041] Method 1: A method for detecting whether a liquid sample contains lead ions, which is method A, method B, method C, or method D:

[0042] Method A: Observation of bacterial culture under non-oxidative conditions may include the following steps: Place the recombinant bacteria described in the first aspect above into the liquid sample to be tested, and culture it at 37±2℃ (e.g., 37℃) with shaking for 5 hours. Then, add n-butanol to the culture system and vortex for 5 minutes (extraction). Let the extract stand at room temperature for more than 6 hours (e.g., 6-10 hours, specifically 6 hours). Observe the color change of the extract. If a brown color reaction is observed, the liquid sample to be tested contains or is a candidate for containing lead ions; otherwise, the liquid sample to be tested does not contain or is a candidate for not containing lead ions.

[0043] Method B: Bacterial suspension observation under oxidative conditions may include the following steps: Place the recombinant bacteria described in the first aspect above into the liquid sample to be tested, and culture it at 37±2℃ (e.g., 37℃) with shaking for 5 hours. Then, add n-butanol and an oxidant (e.g., hydrogen peroxide) to the culture system and vortex for 5 minutes (extraction). Let the extract stand at room temperature for more than 6 hours (e.g., 6-10 hours, specifically 6 hours). Observe the color change of the extract. If a green color reaction is observed, the liquid sample to be tested contains or is a candidate for containing lead ions; otherwise, the liquid sample to be tested does not contain or is a candidate for not containing lead ions.

[0044] Method C: A652 value detection method under non-oxidative conditions, which may include the following steps: placing the recombinant bacteria described in the first aspect above into the liquid sample to be tested, incubating at 37±2℃ (e.g., 37℃) with shaking for 5 hours, then adding n-butanol to the culture system, vortexing for 5 minutes (extraction), and then allowing the extract to stand at room temperature for more than 6 hours (e.g., 6-10 hours, specifically 6 hours), taking the supernatant organic phase to determine the A652 value, 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 tested is significantly greater than the A652 value of the control group, then the liquid sample to be tested contains or is a candidate for containing lead ions; otherwise, the liquid sample to be tested does not contain or is a candidate for not containing lead ions; wherein, the method for determining the A652 value of the control group differs 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 with a liquid sample that does not contain lead ions.

[0045] Method D: A652 value detection method under oxidative conditions, which may include the following steps: placing the recombinant bacteria described in the first aspect above into the liquid sample to be tested, incubating at 37±2℃ (e.g., 37℃) with shaking for 5 hours, then adding n-butanol and an oxidant (e.g., hydrogen peroxide) to the culture system, vortexing for 5 minutes (extraction), and then allowing the extract to stand at room temperature for at least 6 hours (e.g., 6-10 hours, specifically 6 hours), taking the supernatant organic phase to determine the A652 value, 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 tested is significantly greater than the A652 value of the control group, then the liquid sample to be tested contains or is a candidate for containing lead ions; otherwise, the liquid sample to be tested does not contain or is a candidate for not containing lead ions; wherein, the method for determining the A652 value of the control group differs 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 with a liquid sample that does not contain lead ions.

[0046] Method 2: A method for detecting lead ion content in liquid samples, which may be Method E or Method F:

[0047] Method E: A652 value detection method under non-oxidizing conditions, which may include the following steps:

[0048] (e1) Place the recombinant bacteria described in the first aspect above into a series of liquid samples with known lead ion concentrations, and culture them at 37±2℃ (e.g., 37℃) with shaking for 5h. Then add n-butanol to the culture system, vortex for 5min, and let the extract stand at room temperature for more than 6h (e.g., 6-10h, specifically 6h). Take the supernatant organic phase and determine the A652 value. Then plot a standard curve based on the lead ion concentration and the A652 value.

[0049] (e2) Replace the series of lead ion liquid samples with known concentrations in step (e1) with the liquid sample to be tested, repeat step (e1) to obtain the A652 value of the liquid sample to be tested, substitute it into the standard curve, and obtain the lead ion content in the liquid sample to be tested.

[0050] Method F: A652 value detection method under oxidizing conditions, which may include the following steps:

[0051] (f1) Place the recombinant bacteria described in the first aspect above into a series of liquid samples with known lead ion concentrations, and culture them at 37±2℃ (e.g., 37℃) with shaking for 5h. Then add n-butanol and oxidant (e.g., hydrogen peroxide) to the culture system, vortex for 5min (extraction), and let the extract stand at room temperature for more than 6h (e.g., 6-10h, specifically 6h). Take the supernatant organic phase and determine the A652 value. Then plot a standard curve based on the lead ion concentration and the A652 value.

[0052] (f2) Replace the series of lead ion liquid samples with known concentrations in step (f1) with the liquid sample to be tested, repeat step (f1) to obtain the A652 value of the liquid sample to be tested, substitute it into the standard curve, and obtain the lead ion content in the liquid sample to be tested.

[0053] Furthermore, the method (A652 value detection method under oxidizing conditions) is applicable when the test liquid sample contains lead ions of 0.183 nM or higher (e.g., 0.732 nM or higher). The method (A652 value detection method under non-oxidizing conditions) is applicable when the test liquid sample contains lead ions of 5.86 nM or higher.

[0054] In a specific embodiment of the present invention, the oxidant is 30% H2O2 (mass fraction, matrix is ​​water).

[0055] In each of the above non-oxidative treatment methods, the amount of n-butanol added is: 180-360 μL of n-butanol is added to every 900 μL of culture system containing the recombinant bacteria.

[0056] In each of the above oxidation treatment methods, the amount of n-butanol added is: 180-360 μL of n-butanol added to every 900 μL of culture system containing the recombinant bacteria; the amount of oxidant (such as 30% H2O2) added is: 50 μL of oxidant (such as 30% H2O2) added to every 900 μL of culture system containing the recombinant bacteria.

[0057] When the liquid sample to be tested is colorless or can be distinguished from its own color after a color reaction (visible to the naked eye), Method 1 preferably uses visual observation (i.e., Method A and Method B) to determine the result. When the liquid sample to be tested is difficult to distinguish from its own color after a color reaction (indistinguishable to the naked eye), Method 1 preferably uses the A652 value determination method (i.e., Method C and Method D) to determine the result.

[0058] In all of the above aspects, the liquid sample to be tested may be purified water, tap water, lake water, or soil extract solution suspected of containing lead ions.

[0059] In all of the above aspects, the lead ion is a divalent lead ion (Pb(II)).

[0060] In all 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, etc.

[0061] The recombinant bacteria of this invention can be used for the qualitative and quantitative detection of lead ions in bioavailable forms in environmental samples. Attached Figure Description

[0062] Figure 1 A diagram illustrating the molecular mechanism by which recombinant bacteria respond to lead ion sensing.

[0063] Figure 2 The images show the lead ion response time-pigment accumulation curves of the recombinant bacteria. A represents the time-pigment accumulation curve for the non-oxidative treatment group; B represents the time-pigment accumulation curve for the oxidative treatment group; C represents a photograph of the extract from the non-oxidative treatment group; and D represents a representative photograph of the extract from the oxidative treatment group.

[0064] Figure 3 The dose-response relationship of lead response in recombinant bacteria is shown. A represents the dose-pigment accumulation relationship in the non-oxidized group; B represents the dose-pigment accumulation relationship in the oxidized group; C represents the quantifiable dose-pigment accumulation curve in the non-oxidized group; D represents the quantifiable dose-pigment accumulation curve in the oxidized group; and E represents representative photographs of the extracts from the oxidized and non-oxidized groups.

[0065] Figure 4 The recombinant bacteria exhibit selectivity and resistance to interference. A represents the recombinant bacteria's response to non-oxidative treatment of pigment accumulation with different metal ions; B represents the recombinant bacteria's response to oxidative treatment of pigment accumulation with different metal ions; C represents the recombinant bacteria's response to non-oxidative treatment of pigment accumulation with a mixture of different metal ions; and D represents the recombinant bacteria's response to oxidative treatment of pigment accumulation with a mixture of different metal ions.

[0066] Figure 5 This study describes the detection of artificially contaminated lead in environmental samples using recombinant bacteria. A shows the dose-pigment accumulation relationship in the non-oxidized group; B shows the quantifiable dose-pigment accumulation curve in the non-oxidized group; C shows the dose-pigment accumulation relationship in the oxidized group; D shows the quantifiable dose-pigment accumulation curve in the oxidized group; E shows a representative photograph of the extract from the non-oxidized group; and F shows a representative photograph of the extract from the oxidized group. Detailed Implementation

[0067] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0068] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0069] Example 1: Construction and application of a lead ion bacterial whole-cell biosensor using an alcohol-soluble Proviolacein (PV) derivative as a two-color output signal.

[0070] I. Construction of recombinant plasmid pPb-vioABDE

[0071] The first step involved using pET-21a(+) as a vector to synthesize the PV gene cluster vioABDE (as shown in positions 569-6601 of SEQ ID No. 1). This PV gene cluster was then inserted into pET-21a(+) using NdeI / SacI. The recombinant plasmid that was verified to be correct by sequencing was named pET-vioABDE.

[0072] Structural description of pET-vioABDE: A recombinant plasmid in which the small fragment between NdeI and SacI in the pET-21a(+) vector is replaced with the DNA fragment shown at positions 569-6601 of SEQ ID No. 1.

[0073] The second step involves synthesizing the pbrR-pbr promoter gene module (as shown in positions 7-526 of SEQ ID No. 1), inserting it into pET-vioABDE using BglII / XbaI, and naming the recombinant plasmid that has been verified by sequencing as pPb-vioABDE.

[0074] Structural description of pPb-vioABDE: A recombinant plasmid in which the small fragment between BglII and XbaI in the pET-21a(+) vector is replaced with the DNA fragment shown in SEQ ID No. 1.

[0075] Positions 7-526 of SEQ ID No. 1 are the pbrR-pbr bidirectional promoter sequence, representing a Pb(II) ion sensing element. Specifically, positions 442-526 of SEQ ID No. 1 are the nucleotide sequence of the pbr bidirectional promoter, and positions 7-441 of SEQ ID No. 1 are the reverse complementary sequence of the pbrR gene, encoding the PbrR protein shown in SEQ ID No. 2.

[0076] Positions 569-6601 of SEQ ID No. 1 are vioABDE, assembled in a tetracistronic form, encoding four proteins. Positions 569-1825 of SEQ ID No. 1 are the vioA gene, encoding the VioA protein shown in SEQ ID No. 3. Positions 1843-4839 of SEQ ID No. 1 are the vioB gene, encoding the VioB protein shown in SEQ ID No. 4. Positions 4857-6008 of SEQ ID No. 1 are the vioD gene, encoding the VioD protein shown in SEQ ID No. 5. Positions 6026-6601 of SEQ ID No. 1 are the vioE gene, encoding the VioE protein shown in SEQ ID No. 6.

[0077] When lead ions are present, the transcription and translation of four genes are activated, producing four pigment synthases that use tryptophan as a substrate to catalyze the production of proviolacein (PV). However, PV is unstable as an intermediate. After extraction with n-butanol, and followed by non-oxidative or oxidative standing, brown or green pigments can be produced, respectively. The molecular mechanism diagram is shown below. Figure 1 As shown.

[0078] II. Construction of recombinant bacteria TOP10 / pPb-vioABDE

[0079] The recombinant plasmid pPb-vioABDE constructed in step one was transformed into Escherichia coli TOP10 to obtain TOP10 / pPb-vioABDE.

[0080] III. Time-dose response to lead ions

[0081] 1. Inoculate the recombinant bacteria TOP10 / pPb-vioABDE constructed in step 2 into liquid LB medium containing 50 μg / mL ampicillin, and incubate overnight at 37°C and 250 rpm with shaking for 8-12 hours (only for bacterial activation).

[0082] 2. Inoculate 1% (v / v) of overnight bacterial culture into fresh LB medium (containing 50 μg / mL ampicillin), and supplement with Pb(II) (specifically lead acetate, but other soluble divalent lead salts are also acceptable) to final concentrations of 0, 0.15, and 1.5 μmol / L. Incubate at 37°C with shaking at 250 rpm, and take samples (2 × 1 mL) at 1-hour intervals, storing at 4°C. Sampling times are 0, 1, 2, 3, 4, 5, 6, and 7 hours.

[0083] 3. After collecting bacterial culture for 1-7 hours, take 100 μL from each group to test the bacterial concentration (OD600).

[0084] 4. Non-oxidative treatment group (brown): Add 180 μL of n-butanol to 900 μL of bacterial culture and vortex for 5 min.

[0085] 5. Oxidation treatment group (green): Add 180 μL n-butanol + 50 μL 30% H2O2 (mass fraction, substrate is water, the same below) to 900 μL bacterial culture, and vortex for 5 min.

[0086] Centrifuge at 6.3500 rpm for 5 min. After allowing the extracts from both the non-oxidized and oxidized groups to stand at room temperature for 6 h, 100 μL of the supernatant organic phase is aspirated and placed in a 96-well plate. The absorbance at 652 nm is measured. The results are shown in Tables 1 and 2. Figure 2 As shown.

[0087] Table 1. Detection values ​​of non-oxidized pigment A652 at different time points

[0088]

[0089] Table 2. Detection values ​​of pigment A652 in oxidized treatment groups at different time points

[0090]

[0091] The above results indicate that the recombinant bacteria exhibit a dose-response and time-response relationship in response to lead(II). Specifically, pigment production increases with prolonged induction time, and color development is visible to the naked eye after 5 hours, although the increase is not significant. Therefore, 5 hours of incubation was chosen as the detection time.

[0092] IV. Response to different concentrations of lead ions

[0093] 1. Inoculate the recombinant bacteria TOP10 / pPb-vioABDE constructed in step 2 into liquid LB medium containing 50 μg / mL ampicillin, and incubate overnight at 37°C and 250 rpm with shaking for 8-12 hours (only for bacterial activation).

[0094] 2. Inoculate 1% (v / v) of overnight bacterial culture into fresh LB medium (containing 50 μg / mL ampicillin), and add Pb(II) (specifically lead acetate, but other soluble divalent lead salts can also be used) by 2-fold dilution 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, 750, 1500, 3000, 6000, or 12000 nmol / L. Incubate at 37°C with shaking at 250 rpm for 5 h.

[0095] 3. Collect bacterial suspensions (2×1mL) from each group, and take 100μL to detect the bacterial suspension concentration (OD600).

[0096] 4. Non-oxidizing group: Add 360 μL of n-butanol to 900 μL of bacterial culture and vortex for 5 min; Oxidizing group: Add 360 μL of n-butanol + 50 μL of 30% H2O2 to 900 μL of bacterial culture and vortex thoroughly for 5 min.

[0097] Centrifuge at 5.3500 rpm for 5 min. After allowing the extracts from both conditions to stand at room temperature for 6 h, aspirate 100 μL of the supernatant organic phase and place it in a 96-well plate. Measure the absorbance at 652 nm. The results are shown in Table 3. Figure 3 As shown.

[0098] Table 3. Detection results of pigment response of recombinant bacteria to different concentrations of lead ions

[0099]

[0100]

[0101] The above results indicate that the recombinant bacteria TOP10 / pPb-vioABDE responds to lead(II) with a significant dose-response relationship. As the lead exposure dose increases, pigment production increases, and pigment accumulation stabilizes after 1500 nM.

[0102] The detection limit is lower after oxidation treatment, i.e., 0.183 nM (t test, statistically different from 0 nM). The detection limit is higher for non-oxidized products, i.e., 5.86 nM (t test, statistically different from 0 nM).

[0103] V. Selective response to metal ions

[0104] 1. Inoculate the recombinant bacteria TOP10 / pPb-vioABDE constructed in step 2 into liquid LB medium containing 50 μg / mL ampicillin, and incubate overnight at 37°C and 250 rpm with shaking for 8-12 hours (only for bacterial activation).

[0105] 2. Inoculate 1% (v / v) of overnight culture medium into fresh LB medium (containing 50 μg / mL ampicillin).

[0106] 3. Single Metal Selectivity Study – Add the following single metal ions to the culture system from step 2: Pb(II), Cr(III), Hg(II), Zn(II), Mg(II), Ni(II), Mn(II), Ca(II), Fe(II), Cd(II), or Cu(II), to a final concentration of 1.5 μM. Incubate at 37℃ and 250 rpm with shaking for 5 h.

[0107] 4. Investigation of Mixed Metals (Anti-interference Ability) --- The following ions were added to the culture system in step 2 to a final concentration of 1.5 μM (equal molar mixture of each ion in each group): 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). All the ions mentioned above include Pb(II), or all the ions mentioned above do not include Pb(II). The mixture was incubated at 37℃ and 250 rpm with shaking for 5 h.

[0108] 5. The control group was not given any metal salts and was cultured at 37℃ and 250 rpm for 5 hours with shaking.

[0109] 6. Collect bacterial suspensions (2×1mL) from each group, and take 100μL to detect the bacterial suspension concentration (OD600).

[0110] 7. Non-oxidizing group: Add 360 μL of n-butanol to 900 μL of bacterial culture and vortex for 5 min; Oxidizing group: Add 360 μL of n-butanol + 50 μL of 30% H2O2 to 900 μL of bacterial culture and vortex thoroughly for 5 min.

[0111] Centrifuge at 3500 rpm for 5 min. After allowing the extracts from both conditions to stand at room temperature for 6 h, aspirate 100 μL of the supernatant organic phase and place it in a 96-well plate. Measure the absorbance at 652 nm. The results are shown in Tables 4 and 5. Figure 4 As shown.

[0112] Table 4. Absorbance of pigments produced by recombinant bacteria in response to various metal ions

[0113]

[0114]

[0115] Table 5. Absorbance of pigments produced by recombinant bacteria in response to mixed metals

[0116]

[0117] The above results indicate that the single metal selectivity study showed that the recombinant strain TOP10 / pPb-vioABDE exhibited a highly specific response to Pb(II), with only the lead-exposed group showing significant pigment production. The mixed metal (interference resistance) study showed that only the incorporation of mercury and cadmium reduced the response of the recombinant strain TOP10 / pPb-vioABDE to lead. Furthermore, all cultures containing Pb(II) showed significant pigment production, demonstrating good interference resistance.

[0118] VI. Dose response of biosensors to Pb(II) in environmental samples

[0119] 1. Inoculate the recombinant bacteria TOP10 / pPb-vioABDE constructed in step 2 into liquid LB medium containing 50 μg / mL ampicillin, and incubate overnight at 37°C and 250 rpm with shaking for 8-12 hours (only for bacterial activation).

[0120] 2. Prepare liquid LB medium using purified water, tap water, lake water, and soil extract as substrates (90% by volume).

[0121] 3. Inoculate 1% (v / v) of overnight culture solution into fresh LB medium (containing 50 μg / mL ampicillin) of the above four types.

[0122] 4. Add Pb(II) (specifically lead acetate, but other soluble divalent lead salts can also be used) by dilution by 2 times.

[0123] The detection range of the non-oxidized group was relatively smaller than that of the oxidized group. The final concentrations of Pb(II) added were 0, 1.46, 2.93, 5.86, 11.7, 23.4, 46.9, 93.8, 187.5, 375, and 750 nM, respectively.

[0124] The oxidation group was treated with 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, and 750 nM.

[0125] Incubate at 37℃ and 250 rpm for 5 h with shaking.

[0126] 5. Collect bacterial suspensions (2×1mL) from each group, and take 100μL to detect the bacterial suspension concentration (OD600).

[0127] 6. Non-oxidizing group: Add 360 μL of n-butanol to 900 μL of bacterial culture and vortex for 5 min; Oxidizing group: Add 360 μL of n-butanol + 50 μL of 30% H2O2 to 900 μL of bacterial culture and vortex thoroughly for 5 min.

[0128] Centrifuge at 7.3500 rpm for 5 min. After allowing the extracts from both conditions to stand at room temperature for 6 h, aspirate 100 μL of the supernatant organic phase and place it in a 96-well plate. Measure the absorbance at 652 nm. The results are shown in Tables 6 and 7. Figure 5 As shown.

[0129] Table 6. Detection results of non-oxidized groups of lead-responsive pigments in different environmental samples.

[0130]

[0131]

[0132] Note: The detection limit for lead ions in different environmental samples after non-oxidation treatment was 5.86 nM (t test, which showed a statistically significant difference compared to 0 nM).

[0133] Table 7. Results of Oxidation of Lead-Responding Pigments in Different Environmental Samples

[0134]

[0135] Note: The detection limit of lead ions in different environmental samples after oxidation treatment was 0.732 nM (t test, which was statistically different from 0 nM).

[0136] The above results indicate that the recombinant bacteria can respond to lead(II) in different environmental samples and can plot lead exposure dose-pigment response curves within a certain range, showing potential for quantitative detection of environmental lead pollution.

[0137] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Recombinant bacteria are obtained by introducing recombinant vector A into recipient bacteria; The recombinant vector A contains DNA fragment A; The DNA fragment A contains a pbr bidirectional promoter, with the gene encoding the PbrR protein on one side and the proviolacein synthesis gene cluster on the other side. vioABDE ; The Proviolacein synthetic gene cluster vioABDE It is assembled in a tetracistronic form and encodes VioA, VioB, VioD and VioE proteins; The nucleotide sequence of the pbr bidirectional promoter is positions 442-526 of SEQ ID No. 1; The amino acid sequence of the PbrR protein is shown in SEQ ID No. 2; The amino acid sequence of the VioA protein is shown in 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 in SEQ ID No. 6; The nucleotide sequence of the gene encoding the PbrR protein is the reverse complementary sequence of positions 7-441 of SEQ ID No. 1; The nucleotide sequence of the gene encoding the VioA protein is positions 569-1825 of SEQ ID No. 1; The nucleotide sequence of the gene encoding the VioB protein is positions 1843-4839 of SEQ ID No. 1; The nucleotide sequence of the gene encoding the VioD protein is positions 4857-6008 of SEQ ID No. 1; The nucleotide sequence of the gene encoding the VioE protein is positions 6026-6601 of SEQ ID No. 1; The Proviolacein synthetic gene cluster vioABDE The nucleotide sequence is positions 569-6601 of SEQ ID No. 1; The nucleotide sequence of the DNA fragment A is SEQ ID No. 1; The recipient bacterium is Escherichia coli.

2. The recombinant bacteria according to claim 1, characterized in that: The recombinant vector A is formed by replacing the restriction enzyme sites of the pET-21a(+) plasmid with the DNA fragment A. Bgl II and Sac This was obtained after a short segment between I.

3. The recombinant bacteria according to claim 1 or 2, characterized in that: The Escherichia coli mentioned is the Escherichia coli TOP10.

4. Complete product set, including: (A1) The recombinant bacteria according to any one of claims 1-3; and (A2) n-Butanol.

5. The complete set of products according to claim 4, characterized in that: The complete set of products also includes the following: (A3) Oxidizing agent.

6. The complete set of products according to claim 5, characterized in that: The oxidant is hydrogen peroxide.

7. The application of the recombinant bacteria according to any one of claims 1-3 or the complete product according to any one of claims 4-6 in the preparation of a lead ion bacterial whole-cell biosensor with a Proviolacein derivative as a two-color output signal; The lead ions are divalent lead ions.

8. The application of the recombinant bacteria according to any one of claims 1-3 or the complete product according to any one of claims 4-6 in the detection of lead ions; The lead ions are divalent lead ions.

9. The application according to claim 8, characterized in that: The detection of lead ions involves the qualitative and / or quantitative detection of lead ions in a liquid sample.

10. A method for detecting whether a liquid sample contains lead ions, which is method A, method B, method C, or method D: Method A: includes the following steps: placing the recombinant bacteria of any one of claims 1-3 into the liquid sample to be tested, incubating with shaking at 37±2℃ for 5 hours, then adding n-butanol to the culture system for extraction for 5 minutes, allowing the extract to stand at room temperature for more than 6 hours, and observing the color change of the extract. If a brown color reaction is observed, the liquid sample to be tested contains or is a candidate for containing lead ions; otherwise, the liquid sample to be tested does not contain or is a candidate for not containing lead ions. Method B includes the following steps: placing the recombinant bacteria of any one of claims 1-3 into the liquid sample to be tested, incubating with shaking at 37±2℃ for 5 hours, then adding n-butanol and an oxidant to the culture system for extraction for 5 minutes, allowing the extract to stand at room temperature for more than 6 hours, and observing the color change of the extract. If a green color reaction is observed, the liquid sample to be tested contains or is a candidate for containing lead ions; otherwise, the liquid sample to be tested does not contain or is a candidate for not containing lead ions. Method C: includes the following steps: placing the recombinant bacteria described in any one of claims 1-3 into the liquid sample to be tested, incubating with shaking at 37±2℃ for 5 hours, then adding n-butanol to the culture system for extraction for 5 minutes, allowing the extract to stand at room temperature for at least 6 hours, taking the supernatant organic phase to determine the A652 value, 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 tested is significantly greater than the A652 value of the control group, then the liquid sample to be tested contains or is a candidate for containing lead ions; otherwise, the liquid sample to be tested does not contain or is a candidate for not containing lead ions; wherein, The method for determining the A652 value of the control group differs from that for determining the A652 value of the test liquid sample group only in that the test liquid sample is replaced with a liquid sample that does not contain lead ions. Method D: includes the following steps: placing the recombinant bacteria of any one of claims 1-3 into the liquid sample to be tested, incubating with shaking at 37±2℃ for 5h, then adding n-butanol and an oxidant to the culture system for extraction for 5min, allowing the extract to stand at room temperature for more than 6h, taking the supernatant organic phase to determine the A652 value, 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 tested is significantly greater than the A652 value of the control group, then the liquid sample to be tested contains or is a candidate for containing lead ions; otherwise, the liquid sample to be tested does not contain or is a candidate for not containing lead ions; wherein, the method for determining the A652 value of the control group differs 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 with a liquid sample that does not contain lead ions; The lead ions are divalent lead ions.

11. A method for detecting lead ion content in a liquid sample, wherein method E or method F is provided: Method E: includes the following steps: (e1) Place the recombinant bacteria of any one of claims 1-3 in a series of liquid samples of known lead ion concentrations, and culture them at 37±2℃ with shaking for 5h. Then add n-butanol to the culture system for extraction for 5min. Let the extract stand at room temperature for more than 6h, take the supernatant organic phase and determine the A652 value. Then plot a standard curve based on the lead ion concentration and the A652 value. (e2) Replace the series of lead ion liquid samples with known concentrations in step (e1) with the liquid sample to be tested, repeat step (e1) to obtain the A652 value of the liquid sample to be tested, substitute it into the standard curve, and obtain the lead ion content in the liquid sample to be tested. Method F includes the following steps: (f1) Place the recombinant bacteria of any one of claims 1-3 in a series of liquid samples of known lead ion concentrations, and culture them at 37±2℃ with shaking for 5h. Then add n-butanol and oxidant to the culture system for extraction for 5min. Let the extract stand at room temperature for more than 6h, take the supernatant organic phase and determine the A652 value. Then plot a standard curve based on the lead ion concentration and the A652 value. (f2) Replace the series of lead ion liquid samples with known concentrations in step (f1) with the liquid sample to be tested, repeat step (f1) to obtain the A652 value of the liquid sample to be tested, substitute it into the standard curve, and obtain the lead ion content in the liquid sample to be tested; The lead ions are divalent lead ions.

Images