Divalent mercury hypersensitive whole-cell sensor based on pigment signal and application of divalent mercury hypersensitive whole-cell sensor in environmental sample detection

Abstract

The invention discloses a divalent mercury hypersensitive whole-cell sensor based on a pigment signal and application of the divalent mercury hypersensitive whole-cell sensor in environmental sample detection. The invention provides a recombinant bacterium which is obtained by introducing a DNA fragment A and a DNA fragment B into a recipient bacterium, the DNA fragment A contains a Pmer gene, a merR gene and a vioABCE gene cluster; the Pmer is a bivalent mercury ion responsive bidirectional promoter, the merR gene expression is started in one direction, and the vioABCE gene cluster expression is started in the other direction; the DNA fragment B contains a constitutive promoter and a merC gene which is started and expressed by the constitutive promoter. The divalent mercury hypersensitive sensor constructed by the invention further reduces the sensing colorimetric detection limit and the naked eye direct reading judgment limit, and can be used for monitoring the mercury exposure of low-level normal people.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a divalent mercury ultrasensitive whole-cell sensor based on pigment signals and its application in environmental sample detection. Background Technology

[0002] Inorganic mercury exists primarily in the form of divalent mercury in various environmental media and organisms. As a global pollutant, its environmental persistence, biotransformation potential, and chronic toxicity pose a long-term threat to ecology and health. Long-term, low-dose exposure to inorganic mercury can lead to kidney damage such as nephropathy, and in severe cases, it can cause immunosuppression or teratogenic effects. Therefore, close monitoring of mercury exposure in high-risk populations and selection of appropriate mercury exposure assessment indicators are particularly important.

[0003] Current standards recommend instrumental analysis methods such as atomic absorption spectrometry (AAS) and inductively coupled plasma mass spectrometry (ICP-MS) for environmental mercury detection. However, due to equipment, technical, and cost barriers associated with physicochemical testing, mercury detection is primarily limited to public health institutions at the municipal level and above. Developing low-threshold, high-throughput detection methods for environmental mercury levels (whole-cell sensors) could serve as a powerful supplement to the gold standard "instrumental methods," becoming a primary screening tool for routine mercury exposure monitoring and potentially becoming as widely adopted in primary healthcare institutions as urinalysis.

[0004] Whole-cell biosensors consist of a sensing module for recognizing specific metal ions and a reporting module for signal output. Previous studies have shown that mercury can be transported across cells via channel proteins of other metals, but overexpression of mercury-specific channel proteins can significantly improve its transport efficiency.

[0005] There are currently no reports on whole-cell biosensors that use deoxyviolacetic violet pigment as an output signal in conjunction with divalent mercury channel proteins for environmental monitoring. Summary of the Invention

[0006] The purpose of this invention is to provide a divalent mercury ultrasensitive whole-cell sensor based on pigment signals and its application in environmental sample detection.

[0007] In a first aspect, the present invention claims protection for a recombinant bacterium.

[0008] The recombinant bacteria claimed in this invention are obtained by introducing DNA fragment A and DNA fragment B into recipient bacteria;

[0009] The DNA fragment A contains the Pmer, merR genes and the vioABCE gene cluster; the Pmer is a divalent mercury ion-responsive bidirectional promoter that initiates the expression of the merR gene in one direction and the expression of the vioABCE gene cluster in the other direction; the vioABCE gene cluster encodes vioA protein, vioB protein, vioC protein and vioE protein;

[0010] The DNA fragment B contains a constitutive promoter B and the merC gene expressed by the constitutive promoter.

[0011] In some embodiments, the DNA fragment A, between the merR gene and the Pmer, also contains a constitutive promoter A for initiating the expression of the merR gene; this constitutive promoter initiates the expression of the merR gene (to express it at a fixed level). Further, the nucleotide sequence of the constitutive promoter A is positions 436-494 of SEQ ID NO:1.

[0012] In some implementations, the nucleotide sequence of the Pmer is as shown in positions 495-565 of SEQ ID NO:1.

[0013] In some implementations, the amino acid sequence encoded by the merR gene is shown in SEQ ID NO:3.

[0014] In some embodiments, the amino acid sequence of the vioA protein is shown in SEQ ID NO:4; the amino acid sequence of the vioB protein is shown in SEQ ID NO:5; the amino acid sequence of the vioC protein is shown in SEQ ID NO:6; and the amino acid sequence of the vioE protein is shown in SEQ ID NO:7.

[0015] In some implementations, the amino acid sequence encoded by the merC gene is shown in SEQ ID NO:8.

[0016] In some implementations, the constitutive promoter B is P. J23119 Promoter. Further, the P J23119 The nucleotide sequence of the promoter is as shown in positions 1-52 of SEQ ID NO:2.

[0017] In some implementations, the nucleotide sequence of the merR gene is the reverse complementary sequence of positions 1-435 of SEQ ID NO:1.

[0018] In some embodiments, the nucleotide sequence of the gene encoding the vioA protein is shown in positions 608-1865 of SEQ ID NO:1; the nucleotide sequence of the gene encoding the vioB protein is shown in positions 1882-4878 of SEQ ID NO:1; the nucleotide sequence of the gene encoding the vioC protein is shown in positions 4896-6218 of SEQ ID NO:1; and the nucleotide sequence of the gene encoding the vioE protein is shown in positions 6236-6811 of SEQ ID NO:1.

[0019] In some implementations, the nucleotide sequence of the vioABCE gene cluster is shown as positions 608-6811 of SEQ ID NO:1.

[0020] In some implementations, the nucleotide sequence of the merC gene is shown as positions 53-475 of SEQ ID NO:2.

[0021] In some implementations, the nucleotide sequence of the DNA fragment A is as shown in SEQ ID NO:1.

[0022] In some implementations, the nucleotide sequence of the DNA fragment B is as shown in SEQ ID NO:2.

[0023] In some embodiments, DNA fragment A and DNA fragment B are introduced into the recipient bacteria in the form of a recombinant vector. Specifically, in some embodiments, the recombinant vector is a recombinant plasmid obtained by inserting DNA fragment A between the BglII and SacI restriction sites of the pET-21a(+) plasmid and DNA fragment B between the SacI and HindIII restriction sites.

[0024] In some embodiments, the recipient bacterium is *Escherichia coli*. Specifically, in some embodiments, the *Escherichia coli* is *Escherichia coli* TOP10, *Escherichia coli* Rosetta (DE3), or *Escherichia coli* BL21 (DE3).

[0025] Secondly, the present invention claims protection for the use of the recombinant bacteria described in the first aspect above in the application of, or in the preparation of, whole-cell biosensors for the detection of divalent mercury.

[0026] Thirdly, this invention claims protection for any of the following products or applications:

[0027] P1. A set of DNA fragments, consisting of DNA fragment A and DNA fragment B as described in the first aspect above;

[0028] P2. A DNA fragment formed by linking DNA fragment A and DNA fragment B as described in the first aspect above;

[0029] P3, the recombinant vector mentioned in the first aspect above;

[0030] The application of the complete set of DNA fragments described in P4 and P1, or the DNA fragment described in P2, or the recombinant vector described in P3 in the preparation of the recombinant bacteria described in the first aspect above;

[0031] The application of the complete set of DNA fragments described in P5 and P1, or the DNA fragment described in P2, or the recombinant vector described in P3, or the recombinant bacteria described in the first aspect above, in the preparation of a kit for detecting divalent mercury;

[0032] P6. A reagent kit for detecting divalent mercury, which is any one of the following (a1)-(a4):

[0033] (a1) Contains the recombinant bacteria described in the first aspect above;

[0034] (a2) Contains the recombinant vector and recipient bacteria described in P3;

[0035] (a3) Contains the complete set of DNA fragments, expression vector and recipient bacteria described in P1;

[0036] (a4) Contains the DNA fragment, expression vector and recipient bacteria described in P2;

[0037] Application of the kits described in P7 and P6 in the detection of divalent mercury.

[0038] In some embodiments, the recipient bacterium is *Escherichia coli*. Specifically, in some embodiments, the *Escherichia coli* is *Escherichia coli* TOP10, *Escherichia coli* Rosetta (DE3), or *Escherichia coli* BL21 (DE3).

[0039] In some embodiments, the expression vector is a plasmid capable of expression in *E. coli*. In some embodiments, the expression vector is the pET-21a(+) plasmid.

[0040] Fourthly, the present invention claims a method for detecting divalent mercury.

[0041] The method for detecting divalent mercury claimed in this invention may include the following steps: co-incubating the recombinant bacteria described in the first aspect above with the sample to be tested.

[0042] In some implementation cases, the content of divalent mercury in the sample to be tested is greater than or equal to 0.011 nM. In some implementation cases, the content of divalent mercury in the sample to be tested is 0.092-46.875 nM.

[0043] In some implementations, the NaCl concentration in the sample to be tested is 0-3.5 g / L. In other implementations, the NaCl concentration in the sample to be tested is 0.88-1.75 g / L.

[0044] In some embodiments, the co-incubation temperature is 30-40°C. In some embodiments, the temperature is 32-37°C. In some embodiments, the temperature is 37°C. In some embodiments, the co-incubation is performed at the above-mentioned temperature with shaking at 250 rpm for 4 hours.

[0045] In the method, the co-incubation is carried out in a liquid phase system. In some embodiments, the liquid phase system is LB liquid medium, or a mixture of LB liquid medium with purified water, drinking water, surface water, or seawater. That is, the test sample can be LB liquid medium containing divalent mercury, or purified water, drinking water, surface water, or seawater.

[0046] In the method, the sample to be tested may contain other metal ions besides divalent mercury, such as Pb(II), Cd(II), Zn(II), Mg(II), Cu(II), Mn(II) and / or Ca(II).

[0047] In the method described above, after co-incubation, the presence of divalent mercury in the sample to be tested can be determined based on the system color. Further, after co-incubation, if the system color is purple, the sample to be tested contains divalent mercury; otherwise, the sample to be tested does not contain divalent mercury.

[0048] The method may further include the following steps: after completing the co-incubation, taking a sample, then extracting with n-butanol and collecting the organic phase, and detecting the OD of the organic phase. 570 Value. Further, if the OD of the organic phase... 570 The value was significantly higher than that of the control group's OD. 570 If the value is positive, the sample contains divalent mercury; otherwise, the sample does not contain divalent mercury. The control group is a liquid that does not contain divalent mercury (such as LB liquid culture medium) used to replace the sample.

[0049] Regarding the aforementioned aspects, the detection of divalent mercury can be either qualitative or quantitative.

[0050] Regarding the aforementioned relevant aspects, the divalent mercury may be mercuric chloride, etc.

[0051] This invention innovatively incorporates the inorganic mercury transporter MerC and regulates its expression level by optimizing the constitutive promoter. Based on this, a bivalent mercury ultrasensitive sensor is constructed, which further reduces the detection limit of colorimetric sensing and the judgment limit of direct visual reading, and can be used for monitoring mercury exposure in low-level normal populations. Attached Figure Description

[0052] Figure 1 This diagram illustrates the molecular mechanism by which recombinant bacteria respond to divalent mercury sensing. A represents the molecular mechanism of recombinant bacteria responding to divalent mercury sensing; B is a detailed schematic diagram of the divalent mercury exposure protocol for recombinant bacteria in detecting environmental water samples.

[0053] Figure 2 The following are time-dose curves showing the response of a series of recombinant bacteria to different concentrations of divalent mercury. A represents the band error plot of the dose-response curves of the series of recombinant bacteria to different concentrations of divalent mercury; B represents the regression analysis of the relationship between the deoxyviolacein signal of the series of recombinant bacteria and the concentration of divalent mercury, with the x-axis displaying the divalent mercury concentration on a log2 scale; C represents a representative extract of the pigment signal produced by the series of recombinant bacteria after exposure to divalent mercury.

[0054] Figure 3 The specific response capability of the recombinant strain TOP10 / pCon-DV-C is shown. A represents the pigment accumulation of the recombinant strain in response to different metal ions; B represents the bacterial concentration corresponding to different metal ions; C represents a representative extract of the pigments produced by the recombinant strain in response to different metal ions; and D represents the DV absorbance per unit cell concentration produced by the recombinant strain in response to different metal ions.

[0055] Figure 4 This section shows the interference resistance of the recombinant bacteria TOP10 / pCon-DV-C. A represents the absorbance values ​​of the pigments produced after co-culturing the recombinant bacteria with different metal ions; B represents the bacterial concentration after co-culturing the recombinant bacteria with different metal ions; and C represents a representative photograph of the recombinant bacterial extract.

[0056] Figure 5 This section presents the dose-response relationship of recombinant bacteria TOP10 / pCon-DV-C to divalent mercury at different temperatures. A is a waterfall plot of the dose-response curves of the recombinant bacteria in response to different concentrations of divalent mercury at different temperatures; B is a regression analysis of the relationship between the deoxyviolacein signal and the divalent mercury concentration at different temperatures, with the x-axis displaying the divalent mercury concentration on a log2 scale; C is a waterfall plot of the absorbance of the pigment signal produced by the recombinant bacteria after exposure to divalent mercury and the corresponding cell concentration at different temperatures; D is a representative extract of the pigment signal produced by the recombinant bacteria after exposure to divalent mercury.

[0057] Figure 6This section presents the dose-response relationship of recombinant bacteria to divalent mercury in different host cells. A is a bar graph showing the dose-effect of recombinant bacteria in different host cells responding to different concentrations of divalent mercury against the corresponding bacterial cell concentrations; B is a regression analysis of the relationship between deoxyviolacein signal and divalent mercury concentration in recombinant bacteria under different hosts, with the x-axis displaying divalent mercury concentration on a log2 scale; C is a representative extract graph of the pigment response of recombinant bacteria in different host cells to different concentrations of divalent mercury.

[0058] Figure 7 This section presents the tolerance of the recombinant bacterial TOP10 / pCon-DV-C to detection systems at different salt concentrations. Specifically, A is a bar chart showing the dose-response relationship of the recombinant bacteria to different NaCl concentrations and different divalent mercury concentrations; B is a regression analysis of the relationship between the deoxyviolacein signal and divalent mercury concentration in the recombinant bacteria at different NaCl concentrations, with the x-axis displaying the divalent mercury concentration on a log2 scale; C is a bar chart showing the dose-response relationship of the recombinant bacteria to different NaCl concentrations and different divalent mercury concentrations corresponding to the bacterial cell concentration; and D is a representative extract graph of the pigment signal produced by the recombinant bacteria after exposure to divalent mercury at different NaCl concentrations.

[0059] Figure 8 This study demonstrates the application of recombinant bacterial strain TOP10 / pCon-DV-C in the detection of divalent mercury in environmental water samples. A shows a bar chart of pigment signals produced by the recombinant bacteria in response to a series of divalent mercury concentrations in systems exposed to different environmental water compositions; B shows a regression analysis of the relationship between the deoxyviolacein signal and divalent mercury concentration in systems with different environmental water compositions, with the x-axis displaying divalent mercury concentration on a log2 scale; C shows a bar chart of bacterial cell concentrations corresponding to a series of divalent mercury concentrations in systems exposed to different environmental water compositions; and D shows a representative extract graph of the pigment signals produced by the recombinant bacteria in response to a series of divalent mercury concentrations in systems exposed to different environmental water compositions. Detailed Implementation

[0060] 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.

[0061] 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.

[0062] All quantitative data in the following implementation are the mean of at least three replicates.

[0063] Example 1: Construction of the Divalent Mercury Ultrasensitive Whole-Cell Sensor Based on Pigment Signals according to the present invention

[0064] I. Construction of recombinant plasmids pCon-DV, pCon-DV-C, and pCon-DV-TP

[0065] (1) Construction of recombinant plasmid pCon-DV

[0066] The recombinant plasmid pCon-DV was obtained by replacing the small fragment between the BglII and SacI restriction enzyme recognition sequences in the pET-21a(+) plasmid with DNA molecule A. The recombinant plasmid has been verified by sequencing. DNA molecule A is a synthetically produced double-stranded DNA molecule, as shown in SEQ ID NO:1. In SEQ ID NO:1, positions 1-485 are the reverse complementary sequence of the merR gene (encoding the MerR protein, as shown in SEQ ID NO:3), positions 436-494 are the constitutive promoter (which enables the merR gene to be expressed at a fixed level), positions 495-565 are the Pmer (a divalent mercury ion-responsive bidirectional promoter), positions 608-1865 are the vioA gene (encoding the vioA protein, as shown in SEQ ID NO:4), positions 1882-4878 are the vioB gene (encoding the vioB protein, as shown in SEQ ID NO:5), positions 4896-6218 are the vioC gene (encoding the vioC protein, as shown in SEQ ID NO:6), and positions 6236-6811 are the vioE gene (encoding the vioE protein, as shown in SEQ ID NO:7). The MerR protein and Pmer constitute a divalent mercury-responsive module. vioA, vioB, vioE, and vioC proteins are pigment reporter modules. L-tryptophan is converted into deoxyviolacein (DV) under the sequential action of vioA, vioB, vioE, and vioC proteins.

[0067] (2) Construction of recombinant plasmid pCon-DV-C

[0068] The small fragment between the SacI and HandIII restriction enzyme recognition sequences in recombinant plasmid pCon-DV was replaced with DNA molecule B to obtain recombinant plasmid pCon-DV-C. The recombinant plasmid has been sequenced and verified. DNA molecule B is a synthetically produced double-stranded DNA molecule, as shown in SEQ ID NO:2. In SEQ ID NO:2, positions 1-52 are the constitutive promoter P. J23119 The sequence, from positions 53 to 475, is the merC gene (encoding the MerC protein, as shown in SEQ ID NO:8). The MerC protein and P...J23119 It is a transmembrane transport module for divalent mercury.

[0069] (3) Construction of recombinant plasmid pCon-DV-TP

[0070] The small fragment between the SacI and HandIII restriction enzyme recognition sequences in recombinant plasmid pCon-DV was replaced with DNA molecule C to obtain recombinant plasmid pCon-DV-TP. The recombinant plasmid has been sequenced and verified. DNA molecule C is a synthetically produced double-stranded DNA molecule, as shown in SEQ ID NO:9. In SEQ ID NO:9, positions 1-52 are the constitutive promoter P. J23119 The sequence contains the merT gene (encoding the MerT protein) at positions 53-403, and the constitutive promoter P at positions 404-455. J23119 The sequence, from positions 456 to 731, is the merP gene (encoding the MerP protein). MerT protein, MerP protein, and P... J23119 It is also a transmembrane transport module for divalent mercury.

[0071] II. Construction of recombinant bacteria TOP10 / pCon-DV, TOP10 / pCon-DV-C, and TOP10 / pCon-DV-TP

[0072] The recombinant plasmids pCon-DV, pCon-DV-C, and pCon-DV-TP constructed in step one were transformed into Escherichia coli TOP10, and incubated upside down at 37°C for 12 h on LB agar plates containing 50 μg / mL ampicillin. Based on the different plasmids introduced, the resulting recombinant bacteria were named TOP10 / pCon-DV, TOP10 / pCon-DV-C, and TOP10 / pCon-DV-TP, respectively.

[0073] The recombinant bacteria described above constitute the whole-cell biosensor of this invention, used for the detection of divalent mercury. Single colonies were collected for further research.

[0074] Figure 1 This invention demonstrates the working principle of recombinant bacteria detecting divalent mercury. Constitutive promoter P J23119The merC or merTP gene is initiated to express and encode the corresponding protein (a divalent mercury transmembrane transporter). Pmer, along with another constitutive promoter, initiates the merR gene in one direction, encoding the MerR protein (a mercury ion-responsive transcriptional regulator), and expresses it at a fixed level. The MerR protein binds to and inhibits the promoter activity of Pmer in the other direction, and the vioABCE gene cluster is not expressed. When divalent mercury is present in the environment, mercury ions bind to the MerR protein, altering the binding conformation of the MerR protein and Pmer, thereby relieving the inhibition of Pmer by the MerR protein, and the downstream vioABCE gene cluster is expressed. Within a certain range, there is a dose-response relationship between the divalent mercury concentration and the vioABCE gene cluster expression level.

[0075] Example 2: Dose response of recombinant bacteria TOP10 / pCon-DV, TOP10 / pCon-DV-C, and TOP10 / pCon-DV-TP to divalent mercury.

[0076] 1. The recombinant bacteria TOP10 / pCon-DV, TOP10 / pCon-DV-C, and TOP10 / pCon-DV-TP constructed in Example 1 were inoculated into 2 mL LB liquid medium containing 50 μg / mL ampicillin and cultured overnight at 37°C and 250 rpm for 12-16 hours to activate the bacterial strains.

[0077] 2. Inoculate 1% (v / v) of overnight cultured TOP10 / pCon-DV, TOP10 / pCon-DV-C, and TOP10 / pCon-DV-TP bacterial suspension into 1 mL of fresh LB liquid medium (containing 50 μg / mL ampicillin). Add mercuric chloride to the medium using the 2-fold dilution method to final concentrations of 0, 0.011, 0.023, 0.046, 0.092, 0.183, 0.366, 0.732, 1.465, 2.930, 5.859, 11.719, 23.438, 46.875, 93.750, 187.5, 375, 750, 1500, and 3000 nM, respectively, and incubate at 37°C with shaking at 250 rpm for 4 hours.

[0078] 3. Take 100 μL of the sample (total 1 mL) obtained in step 2 into a 96-well plate and measure the bacterial concentration at 600 nm using a microplate reader.

[0079] 4. Transfer the remaining bacterial culture from step 3 (0.9 mL in total) to a 1.5 mL centrifuge tube, add 360 μL of n-butanol, vortex for 20 seconds to fully extract the pigment. After vortexing, centrifuge the tube at 12000 rpm for 5 minutes. Transfer 100 μL of the supernatant to a 96-well plate and measure the absorbance at 570 nm. The results are shown in Table 1. Figure 2 As shown.

[0080] Table 1. Absorbance values ​​of DV pigment produced by three groups of recombinant bacteria in response to different concentrations of mercuric chloride.

[0081]

[0082]

[0083] The above results indicate that recombinant bacteria TOP10 / pCon-DV, TOP10 / pCon-DV-C, and TOP10 / pCon-DV-TP can all respond to divalent mercury, and a dose-response relationship exists for all of them. With increasing divalent mercury concentration, pigment production initially increases and then decreases. The highest point of response for recombinant bacteria TOP10 / pCon-DV is approximately 375 nM, and a good linear relationship exists within the concentration range of 23.438–375 nM (R0). 2 =0.982)( Figure 2 (B) The highest value of the recombinant strain TOP10 / pCon-DV-C was approximately 46.875 nM, and a good linear relationship was observed within the concentration range of 0.092–46.875 nM (R0). 2 =0.994)( Figure 2 (B) The highest value of the recombinant strain TOP10 / pCon-DV-TP was approximately 23.438 nM, and a good linear relationship was observed in the concentration range of 0.366-23.438 nM (R0). 2 =0.965)( Figure 2 (B) Compared with the 0 concentration, the three groups of recombinant bacteria showed statistically significant differences starting from 0.011 nM (two-tailed t-test, P < 0.05), and the calculated limits of detection were all 0.011 nM. Based on comprehensive analysis, the recombinant bacteria TOP10 / pCon-DV-C was selected as the whole-cell biosensor for further experimental research.

[0084] Example 3: Specific response of recombinant bacteria TOP10 / pCon-DV-C to metal ions

[0085] 1. Inoculate a single colony of recombinant bacteria TOP10 / pCon-DV-C into 2 mL of LB liquid medium containing 50 μg / mL ampicillin, and incubate overnight at 37°C and 250 rpm in a shaking incubator.

[0086] 2. TOP10 / pCon-DV-C cultured overnight was added at a rate of 1% (v / v) to 1 mL LB liquid medium containing 50 μg / mL ampicillin. Stock solutions of Hg(II) (mercuric chloride), Pb(II) (lead nitrate), Cd(II) (cadmium chloride), Zn(II) (zinc sulfate heptahydrate), Mg(II) (magnesium chloride hexahydrate), Cu(II) (copper sulfate pentahydrate), Mn(II) (manganese chloride), and Ca(II) (calcium chloride) were added to the medium using a two-fold dilution method to achieve final concentrations of 0, 12.5, 25, 50 nM and 1, 2, 4 μM, respectively. The mixture was then incubated at 37°C and 250 rpm for 4 hours. The group with a final concentration of 0 served as the blank control, without the addition of any metal ions.

[0087] 3. Take 100 μL of the sample (total 1 mL) obtained in step 2 into a 96-well plate and measure the bacterial concentration at 600 nm using a microplate reader.

[0088] 4. Transfer the remaining bacterial culture from step 3 (0.9 mL in total) to a 1.5 mL centrifuge tube, add 360 μL of n-butanol, vortex for 20 seconds to fully extract the pigment. After vortexing, centrifuge the tube at 12000 rpm for 5 minutes. Transfer 100 μL of the supernatant to a 96-well plate and measure the absorbance at 570 nm. The results are shown in Tables 2 and 3. Figure 3 As shown.

[0089] Table 2. DV pigment absorbance values ​​of recombinant strain TOP10 / pCon-DV-C in response to metal ions.

[0090]

[0091]

[0092] Table 3. Detection values ​​of bacterial concentrations responding to metal ions by recombinant strain TOP10 / pCon-DV-C

[0093]

[0094]

[0095] The above results indicate that the recombinant bacterium TOP10 / pCon-DV-C exhibits a good specific response to Hg(II), and no response to other divalent metals was observed. Furthermore, only in Hg(II) was a clear color gradient change of pigments observed to the naked eye.

[0096] Example 4: Determination of the anti-interference effect of recombinant bacteria TOP10 / pCon-DV-C on metal ions

[0097] 1. Inoculate a single colony of recombinant bacteria TOP10 / pCon-DV-C into 2 mL of LB liquid medium containing 50 μg / mL ampicillin, and incubate overnight at 37°C and 250 rpm in a shaking incubator.

[0098] 2. TOP10 / pCon-DV-C cultured overnight was added at a ratio of 1% (v / v) to 1 mL LB liquid medium containing 50 μg / mL ampicillin. Stock solutions of Hg(II), Pb(II), Cd(II), Zn(II), Mg(II), Cu(II), Mn(II), and Ca(II) (using the same salts as in Example 3) were added to the medium and co-cultured to maintain a final Hg(II) concentration of 5 nM, while the concentrations of the other metals were 50 nM and 1 μM, respectively. The mixture was then incubated at 37°C and 250 rpm for 4 hours. A blank control without any added metal ions was also provided.

[0099] 3. Take 100 μL of the sample (total 1 mL) obtained in step 2 into a 96-well plate and measure the bacterial concentration at 600 nm using a microplate reader.

[0100] 4. Transfer the remaining bacterial culture from step 3 (0.9 mL in total) to a 1.5 mL centrifuge tube, add 360 μL of n-butanol, vortex for 20 seconds to fully extract the pigment. After vortexing, centrifuge the tube at 12000 rpm for 5 minutes. Transfer 100 μL of the supernatant to a 96-well plate and measure the absorbance at 570 nm. The results are shown in Table 4. Figure 4 As shown.

[0101] Table 4. DV pigment absorbance values ​​of recombinant strain TOP10 / pCon-DV-C in response to metal ions.

[0102]

[0103]

[0104] Note: "All" in the table refers to a mixture of all the metal ions that have appeared in the table.

[0105] The above results indicate that the recombinant bacterium TOP10 / pCon-DV-C exhibits strong resistance to interference from divalent mercury. When the recombinant bacterium was co-cultured with 5nM mercury ions at concentrations of 50 nM and 1 μM of seven metals, no pigment production was observed visually in any of the Hg(II)-free groups or the blank control group. There were statistically significant differences between all Hg(II)-free groups and the Hg(II)-only groups (P < 0.05). Therefore, it can be concluded that the recombinant bacterium TOP10 / pCon-DV-C exhibits a highly specific response to divalent mercury and is unaffected by interference from other heavy metal ions.

[0106] Example 5: Dosage response of recombinant bacteria TOP10 / pCon-DV-C to divalent mercury at different temperatures.

[0107] 1. Inoculate the recombinant bacteria TOP10 / pCon-DV-C into 2 mL LB liquid medium containing 50 μg / mL ampicillin, and incubate overnight at 37℃ and 250 rpm with shaking for 12-16 hours to activate the bacterial strain.

[0108] 2. Inoculate 1% (v / v) of overnight TOP10 / pCon-DV-C bacterial suspension into 1 mL of fresh LB liquid medium (containing 50 μg / mL ampicillin), and add mercuric chloride to the following concentrations using the 2-fold dilution method: 0, 0.011, 0.023, 0.046, 0.092, 0.183, 0.366, 0.732, 1.465, 2.930, 5.859, 11.719, 23.438, 46.875, 93.750, 187.50, 375, 750, 1500, and 3000 nM, respectively. Incubate at 30, 32, 35, 37, and 40 °C with shaking at 250 rpm for 4 hours.

[0109] 3. Take 100 μL of the sample (total 1 mL) obtained in step 2 into a 96-well plate and measure the bacterial concentration at 600 nm using a microplate reader.

[0110] 4. Transfer the remaining bacterial culture from step 3 (0.9 mL in total) to a 1.5 mL centrifuge tube, add 360 μL of n-butanol, vortex for 20 seconds to fully extract the pigment. After vortexing, centrifuge the tube at 12000 rpm for 5 minutes. Transfer 100 μL of the supernatant to a 96-well plate and measure the absorbance at 570 nm. The results are shown in Table 5. Figure 5 As shown.

[0111] Table 5. Absorbance values ​​of DV pigment produced by recombinant bacteria TOP10 / pCon-DV-C in response to mercuric chloride at different temperatures.

[0112]

[0113]

[0114] The results showed that the recombinant bacteria TOP10 / pCon-DV-C responded to divalent mercury at 32-40℃, and a dose-response relationship was observed in all cases. Under culture conditions of 30℃, the recombinant bacteria exhibited a good linear relationship within the concentration range of 0.732-46.875 nM (R0). 2 =0.983)( Figure 5 (B) The recombinant bacteria cultured at 32℃ showed a good linear relationship in the concentration range of 0.366-46.875 nM (R0). 2 =0.997)( Figure 5 (B) The recombinant bacteria cultured at 35℃ showed a good linear relationship in the concentration range of 0.366-46.875 nM (R0). 2 =0.998)( Figure 5 (B) The recombinant bacteria cultured at 37℃ showed a good linear relationship in the concentration range of 0.092-46.875 nM (R0). 2 =0.995)( Figure 5 (B) The recombinant bacteria cultured at 40℃ showed a good linear relationship in the concentration range of 0.366-11.719 nM (R0). 2 =0.994)( Figure 5 (B). Therefore, the optimal incubation temperature for recombinant bacteria TOP10 / pCon-DV-C with divalent mercury is 37℃.

[0115] Example 6: Dose-response relationship of recombinant bacteria in different host cells to divalent mercury

[0116] 1. Inoculate the recombinant bacteria Rosetta(DE3) / pCon-DV-C, BL21(DE3) / pCon-DV-C, and TOP10 / pCon-DV-C into 2 mL LB broth containing 50 μg / mL ampicillin, and incubate overnight at 37°C and 250 rpm with shaking for 12-16 hours to activate the bacterial strains. The construction method of the recombinant bacteria Rosetta(DE3) / pCon-DV-C and BL21(DE3) / pCon-DV-C is the same as in Example 1, except that the recipient bacteria, Escherichia coli TOP10, is replaced with Escherichia coli Rosetta(DE3) or Escherichia coli BL21(DE3).

[0117] 2. Inoculate 1% (v / v) of the overnight culture of the three recombinant bacteria into 1 mL of fresh LB liquid medium (containing 50 μg / mL ampicillin), and add mercuric chloride to the following concentrations by 2-fold dilution: 0, 0.011, 0.023, 0.046, 0.092, 0.183, 0.366, 0.732, 1.465, 2.930, 5.859, 11.719, 23.438, 46.875, 93.750, 187.50, 375, 750, 1500, and 3000 nM, respectively. Incubate at 37°C and 250 rpm for 4 hours with shaking.

[0118] 3. Take 100 μL of the sample (total 1 mL) obtained in step 2 into a 96-well plate and measure the bacterial concentration at 600 nm using a microplate reader.

[0119] 4. Transfer the remaining bacterial culture from step 3 (0.9 mL in total) to a 1.5 mL centrifuge tube, add 360 μL of n-butanol, vortex for 20 seconds to fully extract the pigment. After vortexing, centrifuge the tube at 12000 rpm for 5 minutes. Transfer 100 μL of the supernatant to a 96-well plate and measure the absorbance at 570 nm. The results are shown in Table 6. Figure 6 As shown.

[0120] Table 6. Absorbance values ​​of DV pigment produced by recombinant bacteria in response to mercuric chloride from different host cells.

[0121]

[0122] The results showed that all recombinant bacteria under the three host cells responded to divalent mercury, and a dose-response relationship was observed in all cases. Among them, the recombinant strain Rosetta(DE3) / pCon-DV-C had the highest pigment reporter value of 1.7, and exhibited a good linear relationship within the concentration range of 0.092-46.875 nM (R0.05). 2 =0.998)( Figure 6 (B) The recombinant strain BL21(DE 3) / pCon-DV-C exhibited a good linear relationship in the concentration range of 0.092-11.719 nM (R0). 2 =0.989)( Figure 6 (B) The recombinant strain TOP10 / pCon-DV-C exhibited good linearity in the concentration range of 0.092-46.875 nM (R0). 2 =0.994)( Figure 6 (Middle B). Combined Figure 6 The trend of bacterial concentration change in A showed that the decrease in recombinant bacteria TOP10 / pCon-DV-C was relatively stable, therefore TOP10 was selected as the host cell in this invention.

[0123] Example 7: Tolerance of recombinant bacteria TOP10 / pCon-DV-C to detection systems with different salt concentrations.

[0124] 1. Add TOP10 / pCon-DV-C cultured overnight at a ratio of 1% (v / v) to 1 mL of a solution containing 50...

[0125] In a medium containing μg / mL ampicillin (10% of 10×LB liquid medium with different NaCl concentrations and 90% sterile water, where % represents volume percentage), mercuric chloride was added to the medium using a 2-fold dilution method to final concentrations of 0, 0.391, 0.781, 1.56, 3.125, 6.25, 12.5, 25, 50, and 100 nM, and the medium was incubated at 37°C with shaking at 250 rpm for 4 hours.

[0126] 2. Take 100 μL of the sample (total 1 mL) obtained in step 2 into a 96-well plate and measure the bacterial concentration at 600 nm using a microplate reader.

[0127] 3. Transfer the remaining bacterial culture from step 3 (0.9 mL in total) to a 1.5 mL centrifuge tube, add 360 μL of n-butanol, vortex for 20 seconds to fully extract the pigment. After vortexing, centrifuge the tube at 12000 rpm for 5 minutes. Transfer 100 μL of the supernatant to a 96-well plate and measure the absorbance at 570 nm. The results are shown in Table 7. Figure 7 As shown.

[0128] Table 7. Absorbance values ​​of DV pigment produced by recombinant bacteria TOP10 / pCon-DV-C in response to mercuric chloride at different NaCl concentrations.

[0129]

[0130] Note: The NaCl concentration (%) in the table represents the NaCl content in 10×LB liquid medium as 0, 0.88, 1, 1.75, and 3.5%, respectively. The specific meaning of % is g / 100mL.

[0131] The above results indicate that the recombinant strain TOP10 / pCon-DV-C responds to divalent mercury at NaCl concentrations ranging from 0% to 3.5%, and that the recombinant strain at 0% and 3.5% NaCl concentrations exhibits a good linear relationship (R0) within the concentration range of 0.391–6.25 nM. 2 =0.980, 0.988)( Figure 7 (B) The recombinant bacteria at concentrations of 0.88%, 1%, and 1.75% showed a good linear relationship in the concentration range of 0.391-25 nM (R0). 2 =0.986, 0.994, 0.992)( Figure 7(B) In summary, the recombinant bacteria showed good detection ability for divalent mercury when the NaCl concentration was maintained in the range of 0.88-1.75%.

[0132] Example 8: Application of recombinant bacteria TOP10 / pCon-DV-C in the detection of divalent mercury in environmental water samples

[0133] 1. Prepare the culture medium by combining environmental water sample, 10×LB liquid medium (containing 1% NaCl, i.e., containing 10 g / L NaCl), 10×LB liquid medium (without NaCl), sterile water, and proportions as shown in Table 8:

[0134] Table 8. Culture medium preparation ratio (%) for environmental water sample testing

[0135]

[0136]

[0137] 2. The recombinant bacteria TOP10 / pCon-DV-C, which had been cultured overnight, was added at a ratio of 1% (v / v) to 1 mL of medium containing 50 μg / mL ampicillin (10% 10×LB liquid medium and 90% sterile water). Mercuric chloride was added to the medium using the 2-fold dilution method to final concentrations of 0, 1.563, 3.125, 6.25, 12.5, 25, 50, and 100 nM, respectively. The medium was then incubated at 37°C with shaking at 250 rpm for 4 hours.

[0138] 3. Take 100 μL of the sample (total 1 mL) obtained in step 2 into a 96-well plate and measure the bacterial concentration at 600 nm using a microplate reader.

[0139] 4. Transfer the remaining bacterial culture from step 3 (0.9 mL in total) to a 1.5 mL centrifuge tube, add 360 μL of n-butanol, vortex for 20 seconds to fully extract the pigment. After vortexing, centrifuge the tube at 12000 rpm for 5 minutes. Transfer 100 μL of the supernatant to a 96-well plate and measure the absorbance at 570 nm. The results are shown in Table 9. Figure 8 As shown.

[0140] Table 9. DV pigment absorbance values ​​of recombinant bacteria TOP10 / pCon-DV-C at different addition ratios in environmental samples.

[0141]

[0142] The above results indicate that the recombinant bacteria TOP10 / pCon-DV-C exhibits a significant dose-response relationship to divalent mercury in environmental water samples, and demonstrates good linearity in the detection concentration range of 0-12.5 nM for purified water, drinking water, surface water, and seawater. Figure 8 (B) This suggests that the whole-cell biosensor of the present invention (i.e., recombinant bacteria TOP10 / pCon-DV-C) has the potential for application in the ultrasensitive quantitative detection of divalent mercury.

[0143] The present invention has been described in detail above. Those skilled in the art will recognize that 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. While specific embodiments have been provided, 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.

Claims

1. A recombinant bacterium, characterized in that: The recombinant bacteria were obtained by introducing DNA fragment A and DNA fragment B into the recipient bacteria; The DNA fragment A contains the Pmer, merR genes and the vioABCE gene cluster; the Pmer is a divalent mercury ion-responsive bidirectional promoter that initiates the expression of the merR gene in one direction and the expression of the vioABCE gene cluster in the other direction; the vioABCE gene cluster encodes vioA protein, vioB protein, vioC protein and vioE protein; The DNA fragment B contains a constitutive promoter B and the merC gene expressed by the constitutive promoter.

2. The recombinant bacteria according to claim 1, characterized in that: On the DNA fragment A, between the merR gene and the Pmer, there is also a constitutive promoter A for initiating the expression of the merR gene; Furthermore, the nucleotide sequence of the constitutive promoter A is shown as positions 436-494 of SEQ ID NO:1; and / or The nucleotide sequence of the Pmer is as shown in positions 495-565 of SEQ ID NO:1; and / or The amino acid sequence encoded by the merR gene is shown in SEQ ID NO:3; and / or The amino acid sequence of the vioA protein is shown in SEQ ID NO:4; and / or, the amino acid sequence of the vioB protein is shown in SEQ ID NO:5; and / or, the amino acid sequence of the vioC protein is shown in SEQ ID NO:6; and / or, the amino acid sequence of the vioE protein is shown in SEQ ID NO:

7. and / or The amino acid sequence encoded by the merC gene is shown in SEQ ID NO:8; and / or The nucleotide sequence of the constitutive promoter B is as shown in positions 1-52 of SEQ ID NO:

2.

3. The recombinant bacteria according to claim 1 or 2, characterized in that: The nucleotide sequence of the merR gene is the reverse complementary sequence of positions 1-435 of SEQ ID NO:1; and / or The nucleotide sequence of the gene encoding the vioA protein is shown in positions 608-1865 of SEQ ID NO:1; and / or, the nucleotide sequence of the gene encoding the vioB protein is shown in positions 1882-4878 of SEQ ID NO:1; and / or, the nucleotide sequence of the gene encoding the vioC protein is shown in positions 4896-6218 of SEQ ID NO:1; and / or, the nucleotide sequence of the gene encoding the vioE protein is shown in positions 6236-6811 of SEQ ID NO:

1. Furthermore, the nucleotide sequence of the vioABCE gene cluster is shown in positions 608-6811 of SEQ ID NO:1; and / or The nucleotide sequence of the merC gene is shown in positions 53-475 of SEQ ID NO:

2.

4. The recombinant bacteria according to any one of claims 1-3, characterized in that: The nucleotide sequence of the DNA fragment A is shown in SEQ ID NO:1; and / or The nucleotide sequence of the DNA fragment B is shown in SEQ ID NO:

2.

5. The recombinant bacteria according to any one of claims 1-4, characterized in that: The DNA fragment A and the DNA fragment B were introduced into the recipient bacteria in the form of a recombinant vector; Furthermore, the recombinant vector is a recombinant plasmid obtained by inserting the DNA fragment A between the restriction sites BglII and SacI of the pET-21a(+) plasmid and inserting the DNA fragment B between the restriction sites SacI and HindIII.

6. The recombinant bacteria according to any one of claims 1-5, characterized in that: The recipient bacterium is Escherichia coli; Furthermore, the Escherichia coli is Escherichia coli TOP10, Escherichia coli Rosetta (DE3), or Escherichia coli BL21 (DE3).

7. The use of the recombinant bacteria according to any one of claims 1-6 in the preparation of whole-cell biosensors for the detection of divalent mercury.

8. Any of the following products or applications: P1. A set of DNA fragments, comprising DNA fragment A and DNA fragment B as described in any one of claims 1-6; P2. A DNA fragment formed by linking DNA fragment A and DNA fragment B as described in any one of claims 1-6; P3, the recombinant vector as described in claim 5; The use of the complete set of DNA fragments described in P4 and P1, or the DNA fragment described in P2, or the recombinant vector described in P3 in the preparation of any of the recombinant bacteria described in claims 1-6; The use of the complete set of DNA fragments described in P5 and P1, or the DNA fragment described in P2, or the recombinant vector described in P3, or any of the recombinant bacteria described in claims 1-6, in the preparation of a kit for detecting divalent mercury; P6. A reagent kit for detecting divalent mercury, which is any one of the following (a1)-(a4): (a1) Contains any of the recombinant bacteria described in claims 1-6; (a2) Contains the recombinant vector and recipient bacteria described in P3; (a3) Contains the complete set of DNA fragments, expression vector and recipient bacteria described in P1; (a4) Contains the DNA fragment, expression vector and recipient bacteria described in P2; Application of the kits described in P7 and P6 in the detection of divalent mercury.

9. A method for detecting divalent mercury, comprising the following steps: co-incubating the recombinant bacteria as described in any one of claims 1-6 with the sample to be tested.

10. The method according to claim 9, characterized in that: The content of divalent mercury in the sample to be tested is greater than or equal to 0.011 nM; Furthermore, the content of divalent mercury in the sample to be tested is 0.092-46.875 nM; and / or The concentration of NaCl in the sample to be tested was 0-3.5 g / L; Furthermore, the NaCl concentration in the sample to be tested is 0.88-1.75 g / L; and / or In the method, the co-incubation temperature is 30-40℃; Furthermore, the temperature is 32-37°C; Furthermore, the temperature is 37°C.

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