CN108998400B

CN108998400B - Bioengineering bacterium for reducing bivalent mercury ions and preparation method and application thereof

Info

Publication number: CN108998400B
Application number: CN201810732716.9A
Authority: CN
Inventors: 王丹; 陈阳清; 韦娜; 金楠皓; 郑亚楠; 庞婷; 刘婷; 梁乐桂
Original assignee: Nanning Normal University
Current assignee: Shenzhen Zhihui Huasheng Technology Co ltd
Priority date: 2018-07-05
Filing date: 2018-07-05
Publication date: 2022-01-28
Anticipated expiration: 2038-07-05
Also published as: CN108998400A

Abstract

The invention discloses a bioengineering bacterium for reducing divalent mercury ions, which contains an expression vector, wherein the expression vector is merR op-ompA-merA-pSB1A2, and comprises recombinant genes merR op-ompA-merA and pSB1A2 plasmid frameworks. The invention also discloses a preparation method and application of the bioengineering bacteria for reducing the divalent mercury ions. The bioengineering bacterium for reducing the divalent mercury ions provided by the invention can be used for treating Hg (II) pollution in a water environment. The bioengineering bacteria are thrown into a water environment polluted by Hg (II), and Hg (II) is reduced to Hg (0), so that the water environment is treated. The bioengineering bacteria of the invention are used for treating Hg (II) pollution in water environment, and have the advantages of ecological environmental protection, good selectivity, high sensitivity, low cost and reusability.

Description

Bioengineering bacterium for reducing bivalent mercury ions and preparation method and application thereof

Technical Field

The invention relates to the technical field of water pollution treatment, in particular to bioengineering bacteria for reducing bivalent mercury ions and a preparation method and application thereof.

Background

With the wide use of mercury, a part of mercury enters the environment along with the discharge of waste gas, waste water and waste residues, which causes serious pollution to the production and living and the ecological system of human beings and even harms the life health of human beings.

The biotoxicity of mercury is mainly caused by inorganic mercury and organic mercury compounds. The harm of organic mercury compounds to human bodies is far greater than that of inorganic mercury, but at present, inorganic mercury ions in a water pollution system are the main existing forms of mercury pollution and can be converted into organic mercury under the action of microorganisms. Therefore, the realization of the treatment of inorganic mercury ions in a water pollution system is particularly important.

Research on removing Hg (II) in wastewater by precipitation-adsorption synthesis method is carried out on panderine, rowavia and the like, and the research uses the common domestic garbage, namely pomelo peel, of southern people as raw material and ZnCl₂Pretreating pericarpium Citri Grandis to denature it, and treating with Na₂And adsorbing Hg (II) in the wastewater treated by the S precipitation method. The experiment discovers that a flocculating agent FeSO is added when the pH value is 9 and the adding amount of sodium sulfide is 0.36g/mL₄Under the condition, the removal rate of mercury ions in the wastewater can reach more than 96%.

The mainstream process of flue gas desulfurization widely implemented in factories in China can generate wastewater containing heavy metal mercury ions, and GuoMing et al explore the treatment of Hg (II) in simulated wastewater and carbide slag desulfurization wastewater by using chelating agent Dithiocarbamate (DTCR). The influence of DTCR on the removal of mercury ions in wastewater is researched by two factors, namely the dosage of DTCR and the pH, and the DTCR has high removal rate of the mercury ions and has little influence on the removal rate of Hg (II).

Zhuyimin and others select waste yeast used in the brewing industry to adsorb mercury ions in wastewater, and explore the effect of biological adsorption on removal of mercury ions in an aqueous phase by factors such as adsorption time, solution pH value, beer yeast amount, initial mass concentration and temperature of Hg (II) and the like to obtain the optimal adsorption condition of beer yeast on Hg (II), wherein when the pH value is 3 and the adsorption time is 15min, Hg (II) solution with the mass concentration of 2.6g/L is adsorbed, and the removal rate can reach 96%.

Although the current methods for treating mercury-containing wastewater are various and comprise a precipitation method, a chemical method, an adsorption method and the like, some defects are still not negligible. For example, the precipitation method is easy to generate secondary pollution, is easy to cause water hardening, and cannot completely treat mercury-containing wastewater. Although the adsorption method has the advantages of repeated use, various varieties, small generated secondary pollution and no new pollutant brought in during sewage treatment, the adsorbent is easy to saturate, high in treatment cost, poor in specificity and low in sensitivity, and can adsorb metal ions beneficial to human bodies while removing mercury ions, so that the ion balance of the water environment is damaged.

The biological adsorption method provides a new way for treating heavy metal ion mercury pollution, and the method displays the protein capable of adsorbing heavy metal ion mercury on the surface of bacteria by a microbial cell surface display technology, so that high-sensitivity and high-selectivity adsorption of mercury ions is realized. However, bacteria used for biosorption lose their ability to further reduce mercury contamination when the adsorption capacity is saturated.

With the rapid development of molecular biology technology, it becomes possible to construct gene recombinant microorganisms which can efficiently identify and treat mercury pollution by using genetic engineering technology, and a powerful tool is provided for treating mercury pollution in water environment. Therefore, the development of mercury pollution treatment technology which is ecological, environment-friendly, good in selectivity, high in sensitivity, low in cost and capable of being recycled is urgently needed.

Disclosure of Invention

An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.

Still another object of the present invention is to provide a bioengineering bacterium for reducing divalent mercury ions.

The invention also aims to provide a preparation method of the bioengineering bacteria for reducing the divalent mercury ions.

The invention also aims to provide the application of the bioengineering bacteria for reducing the bivalent mercury ions, and the bioengineering bacteria has the advantages of ecological environmental protection, good selectivity, high sensitivity, low cost and reusability.

To achieve these objects and other advantages in accordance with the purpose of the invention, there is provided a bioengineered bacterium for reduction of divalent mercury ions, comprising an expression vector merR op-ompA-merA-pSB1a2 comprising recombinant genes merR op-ompA-merA and pSB1a2 plasmid backbone.

The preparation method of the bioengineering bacteria for reducing the divalent mercury ions comprises the following steps:

firstly, utilizing PCR amplification reaction to respectively realize amplification of merR op, ompA and merA gene segments, connecting the amplified merR op gene segment and the ompA gene segment by an overlap extension PCR technology, and realizing amplification by the PCR amplification reaction to obtain a target gene segment merR op-ompA;

secondly, carrying out double enzyme digestion on the pSB1A2 plasmid framework and the target gene fragment merR op-ompA by using restriction endonucleases XbaI and SpeI, simultaneously exposing the same cohesive ends of the target gene fragment merR op-ompA and the pSB1A2 plasmid framework, and connecting the target gene fragment merR op-ompA and the pSB1A2 plasmid framework by using T4DNA ligase to realize the construction of the merR op-ompA-pSB1A2 plasmid;

thirdly, performing single enzyme digestion on the merR op-ompA-pSB1A2 plasmid and the amplified merA gene fragment by using a restriction enzyme SpeI, and then connecting the merR op-ompA-pSB1A2 plasmid and the amplified merA gene fragment by using T4DNA ligase to complete the construction of an expression vector merR op-ompA-merA-pSB1A 2;

and step four, transforming the expression vector merR op-ompA-merA-pSB1A2 into competent cells by a chemical transformation method to obtain the bioengineering bacteria.

Preferably, the target gene fragment merR op-ompA in the step one is obtained by the following method: and respectively taking the totally synthesized merR op, ompA and merA gene sequences as templates, carrying out a first PCR amplification reaction, connecting the amplified complementary fragments of the ends of the merR op and ompA gene fragments by an overlap extension PCR technology to obtain a connecting fragment merR op-ompA, carrying out a second PCR amplification reaction to realize the amplification of the connecting fragment merR op-ompA, and obtaining the target gene fragment merR op-ompA.

Preferably, the competent cells in step four are e.coli DH5 α competent cells.

Preferably, the primers to be added to the merR OP comprise ImeR-OP XF and OP-ompA UP in the first PCR amplification reaction, the primers to be added to the ompA comprise OP-ompA DN and OmpA SR, and the primers to be added to the merA comprise MerA SF and MerA SR; primers added for the second PCR amplification reaction included IMerr-OP XF and OmpA SR.

Preferably, the reaction temperature in the double digestion in step two is 37 ℃ and the reaction time is 1 hour.

Preferably, the reaction temperature in the single enzyme digestion in step three is 37 ℃ and the reaction time is 1 h.

Preferably, the first PCR amplification reaction comprises 30 cycles, wherein one cycle comprises denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 10s and extension at 72 ℃ for 5s in sequence; the second PCR amplification reaction comprises 30 cycles, wherein one cycle comprises denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 10s and extension at 72 ℃ for 10s in sequence.

The application of bioengineering bacteria for reducing bivalent mercury ions is provided, and the bioengineering bacteria are used for treating Hg (II) pollution in water environment.

Preferably, the bioengineering bacteria are thrown into a water environment polluted by Hg (II), and Hg (II) is reduced to Hg (0), so that the water environment is treated.

The invention at least comprises the following beneficial effects:

the bioengineering bacterium for reducing the divalent mercury ions provided by the invention can be used for treating Hg (II) pollution in a water environment. The bioengineering bacteria are thrown into a water environment polluted by Hg (II), and Hg (II) is reduced to Hg (0), so that the water environment is treated.

The invention successfully constructs the plasmid merR op-ompA-merA-pSB1A2 by utilizing genetic engineering means such as PCR amplification, enzyme digestion identification, connection, transformation and the like, the bioengineering bacteria containing the plasmid can regulate the expression of mercury ion reductase MerA on the surface of a microbial cell through a transcription activation mechanism of metal regulatory protein MerR, and the MerA protein can convert toxic Hg (II) into metal Hg (0) with low toxicity and volatility, thereby degrading mercury ions in a water environment. The mercury ion reductase MerA shows certain reduction effect on low-concentration and high-concentration mercury ions, and the reduction rate tends to increase along with time. The mercury ion reductase MerA only reduces mercury ions in the water environment, does not reduce other metal ions, and shows selectivity to Hg (II).

The bioengineering bacteria of the invention are used for treating Hg (II) pollution in water environment, and have the advantages of ecological environmental protection, good selectivity, high sensitivity, low cost and reusability.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 is a plasmid map of the plasmid backbone of pSB1A2 according to the present invention;

FIG. 2 is a flow chart of the construction of the expression vector of the present invention;

FIG. 3 is the electrophoretogram of the merR op, ompA, merA, merR op-ompA gene fragment after amplification according to the present invention;

FIG. 4 is the electrophoresis diagram of merR op-ompA-pSB1A2 plasmid and enzyme digestion identification according to the present invention;

FIG. 5 is the electrophoresis diagram of merR op-ompA-merA-pSB1A2 expression vector and enzyme digestion identification according to the present invention;

FIG. 6 is a diagram showing the reduction effect of the bioengineering bacteria and the control bacteria on mercury ions;

FIG. 7 shows the reduction effect of bioengineering bacteria according to the present invention on mercury ions of different concentrations;

FIG. 8 is a graph comparing the reduction performance of the bioengineering bacteria of the present invention on 8 metal ions.

Detailed Description

The present invention is further described in detail below with reference to the drawings and examples so that those skilled in the art can practice the invention with reference to the description.

The invention provides a bioengineering bacterium for reducing divalent mercury ions, which contains an expression vector, wherein the expression vector is merR op-ompA-merA-pSB1A2, and comprises recombinant genes merR op-ompA-merA and pSB1A2 plasmid frameworks. The plasmid skeleton of pSB1A2, which is a conventional material that can be prepared by the prior art, is presented by the subject group of professor Zhaojingzhi of Nanjing university for constructing a recombinant plasmid, namely an expression vector, the plasmid map of the plasmid skeleton of pSB1A2 is shown in FIG. 1, rfp in the map of pSB1A2 is removed by subsequent enzyme digestion reaction, and the construction of the system is not influenced.

The invention provides a preparation method of bioengineering bacteria for reducing divalent mercury ions, which comprises the following steps:

firstly, utilizing PCR amplification reaction to respectively realize amplification of merR op, ompA and merA gene segments, connecting the amplified merR op gene segment and ompA gene segment by an overlap extension PCR technology, and realizing in-vitro amplification by the PCR amplification reaction to obtain a target gene segment merR op-ompA;

the gene sequence of merR is shown as SEQ ID NO.1, the gene sequence of op is shown as SEQ ID NO.2, the gene sequence of ompA is shown as SEQ ID NO.3, the gene sequence of merA is shown as SEQ ID NO.4, and the gene sequence of recombinant gene merR op-ompA-merA is shown as SEQ ID NO. 5.

The target gene fragment merR op-ompA in the step I is obtained by the following method: and respectively taking the totally synthesized merR op, ompA and merA gene sequences as templates, carrying out a first PCR amplification reaction, connecting the amplified complementary fragments of the ends of the merR op and ompA gene fragments by an overlap extension PCR technology to obtain a connecting fragment merR op-ompA, carrying out a second PCR amplification reaction to realize the amplification of the connecting fragment merR op-ompA, and obtaining the target gene fragment merR op-ompA. Table 1 below shows the first PCR amplification reaction (100 ul). Table 2 below shows the second PCR amplification reaction (100 ul).

TABLE 1

TABLE 2

In the first PCR amplification reaction, primers needing to be added to the merR OP comprise ImeR-OP XF and OP-ompA UP, primers needing to be added to the ompA comprise OP-ompA DN and OmpA SR, and primers needing to be added to the merA comprise MerA SF and MerA SR; the primers added in the second PCR amplification reaction include IMerr-OP XF and OmpASR, and the sequences of the primers are shown in Table 3 below. DNA polymerase is also added in the first PCR amplification reaction and the second PCR amplification reaction.

TABLE 3

The first PCR amplification reaction comprises 30 cycles, wherein one cycle comprises denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 10s and extension at 72 ℃ for 5s in sequence; the second PCR amplification reaction comprises 30 cycles, wherein one cycle comprises denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 10s and extension at 72 ℃ for 10s in sequence. The following table 4 shows the reaction procedure of the first PCR amplification reaction. The reaction procedure for the second PCR amplification reaction is shown in Table 5 below.

TABLE 4

TABLE 5

The gene fragments merR op, ompA, merA and merR op-ompA are amplified by a PCR program and then identified by agarose gel electrophoresis, and the electrophoresis result shows that the

lanes

1, 2, 3 and 4 have obvious single bands near 500bp, 500bp-750bp, 1500 bp-2000bp and 1000bp, and the single bands completely conform to the sizes of the merR op (506bp), ompA (549bp) and merA (1686bp) and the merR op-ompA (1054bp) gene fragments, as shown in FIG. 3, which indicates that the PCR amplification result is basically correct. In FIG. 3, 1 represents a merR op gene amplification product; 2 represents ompA gene amplification product; 3 represents a merA gene amplification product; 4 represents the merR op-ompA gene amplification product.

Secondly, carrying out double enzyme digestion on the pSB1A2 plasmid framework and the target gene fragment merR op-ompA by using restriction endonucleases XbaI and SpeI, simultaneously exposing the same cohesive ends of the target gene fragment merR op-ompA and the pSB1A2 plasmid framework, and connecting the target gene fragment merR op-ompA and the pSB1A2 plasmid framework by using T4DNA ligase to realize the construction of the merR op-ompA-pSB1A2 plasmid; the reaction temperature for carrying out double digestion is 37 ℃ and the reaction time is 1 h.

The target gene fragments merR op-ompA and pSB1A2 plasmid frameworks connected by an overlap extension PCR technology are treated by restriction enzymes XbaI and SpeI, and the concentration of the enzyme digestion products is tested after the enzyme digestion products are purified by a 1% agarose gel electrophoresis experiment and a gel cutting recovery experiment after the reaction system is reacted for 1h at the constant temperature of 37 ℃. After the enzyme digestion fragments merR op-ompA and the pSB1A2 plasmid skeleton are recovered by cutting gel, the merR op-ompA fragment and the pSB1A2 are connected under the action of T4 ligase, and the connecting product is transformed into E.coli DH5 alpha competent cells by a chemical method. Selecting a single clone to perform PCR identification according to the method of the step one, performing plasmid extraction and enzyme digestion identification experiments on the single clone with correct PCR identification, and respectively representing the results of double enzyme digestion identification of merR op-ompA-pSB1A2 plasmid, XbaI and SpeI by 1 and 2 in the figure 4 as shown in figure 4. Meanwhile, sequencing by Shanghai Biochemical company proves that the merR op-ompA fragment is successfully inserted into the XbaI/SpeI enzyme cutting sites of the pSB1A2 plasmid framework, and no mutation occurs in the construction process.

The double digestion reaction system of the target gene fragment merR op-ompA and pSB1A2 plasmid backbone is shown in Table 6 below. Then, a target gene fragment merR op-ompA is constructed to a pSB1A2 plasmid skeleton through a ligation reaction, so that the construction of a merR op-ompA-pSB1A2 vector is realized, a specific reaction system is shown in Table 7, a small amount of ligation products are taken for transformation after being kept at a constant temperature of 16 ℃ overnight, a single clone with correct PCR identification and enzyme digestion identification is selected for overnight culture in an LB (LB) culture medium and then sent to Shanghai bio-company for sequencing, and the vector merR-ompA-pSB1A2 with a correct sequencing result is subjected to subsequent experiments.

TABLE 6

TABLE 7

Thirdly, performing single enzyme digestion on the merR op-ompA-pSB1A2 plasmid and the amplified merA gene fragment by using a restriction enzyme SpeI, and then connecting the merR op-ompA-pSB1A2 plasmid and the amplified merA gene fragment by using T4DNA ligase to complete the construction of an expression vector merR op-ompA-merA-pSB1A2, wherein the overlapping PCR in the figure 2 is the overlapping extension PCR technology; the reaction temperature when single enzyme digestion is carried out in the third step is 37 ℃, and the reaction time is 1 h.

Treating the amplified merA gene fragment and merR op-ompA-pSB1A2 by using a restriction enzyme SpeI, reacting the reaction system at the constant temperature of 37 ℃ for 1h, purifying the digestion product by a 1% agarose gel electrophoresis experiment and a gel cutting recovery experiment, and then testing the concentration of the digestion product. As shown in FIG. 5, 1 and 2 in FIG. 5 represent the results of enzyme digestion identification of expression vectors merR op-ompA-merA-pSB1A2 and SpeI, respectively, and it is proved that after the merR op-ompA fragment is successfully ligated to plasmid pSB1A2, the merR op-ompA-pSB1A2 and the merA fragment are digested with SpeI alone, and then T4 ligase is used to successfully construct plasmid merR op-ompA-merA-pSB1A 2. And then transferred into a competent cell E.coli DH5 alpha, and sequenced to prevent the base deletion or mutation. Sequencing by Shanghai Bionical company proves that the merA fragment is successfully inserted between the pSB1A2 vector and the merR op-ompA fragment, and mutation does not occur in the construction process.

The single-enzyme reaction system of merR op-ompA-pSB1A2 and the amplified merA gene fragment is shown in Table 8 below. And then constructing the amplified merA gene fragment onto merR op-ompA-pSB1A2 through a ligation reaction, so as to realize the construction of an expression vector merR op-ompA-merA-pSB1A2, wherein a specific reaction system is shown in Table 9, then transforming, selecting a single clone with correct PCR identification and enzyme digestion identification, culturing the single clone in an LB culture medium overnight, sending the single clone to Shanghai bio-company for sequencing, and marking and preserving the expression vector merR op-ompA-merA-pSB1A2 with correct sequencing result for subsequent performance test experiments.

TABLE 8

TABLE 9

And step four, transforming the expression vector merR op-ompA-merA-pSB1A2 into competent cells by a chemical transformation method to obtain the bioengineering bacteria. The competent cells in step four were e.coli DH5 α competent cells as host bacteria. The specific transformation process is as follows: (1) thawing the subpackaged competent cells E.coli DH5 alpha on ice from a nitrogen tank; (2) adding 1ul expression vector merR op-ompA-merA-pSB1A2, flicking the tube wall to mix, and standing on ice for 30 min; (3) carrying out water bath heat shock for 90s at 42 ℃ to avoid shock; (4) quickly putting back on ice for 5 min; (5) adding 500ul of sterilized LB (without resistance), and culturing at 37 ℃ and 250rpm for 60 min; (6) taking 50ul of bacterial liquid, and uniformly spreading the bacterial liquid on an LB solid culture substrate containing resistance; (7) plates were placed upside down in a 37 ℃ oven for overnight incubation.

< bioengineering bacteria Performance test >

Comparison of Hg (II) reducing performance of bioengineering bacteria and control bacteria

(1) Picking single colonies from LB solid culture plates of experimental bacteria for transforming merR op-ompA-merA-pSB1A2 expression vectors and control bacteria for transforming merR op-ompA-pSB1A2 plasmids, respectively inoculating the single colonies into 3mL of liquid LB culture medium (containing 150ug/mL carbenicillin), and culturing at the constant temperature of 37 ℃ and 250rpm overnight;

(2) the overnight cultured broth was cultured as follows 1: expanding the culture at a ratio of 100 in 100ml of liquid LB culture medium, and culturing at 37 ℃ and a constant temperature of 250rpm until OD600 is 1;

(3) adding mercury ions with the same concentration (20uM) into the bacterial liquid grown to OD600 ═ 1, and continuing culturing at 37 ℃ and 250rpm (3 repeated experiments are set for the experimental bacteria and the control bacteria);

(4) respectively sucking 100ul of the two bacterial liquids into a 1.5ml EP tube at the time points of 0 hour, 2 hours, 4 hours, 6 hours and 8 hours, adding 100ul of concentrated nitric acid, and carrying out nitration reaction in a water bath at 65 ℃ for 1 hour;

(5) and (3) sucking 100ul of nitrified bacteria liquid sample into a test tube, using distilled water to fix the volume to 5ml, and then using inductively coupled plasma (ICP-MS) to detect the concentration of mercury ions in the bacteria liquid.

In order to verify that the MerA protein expressed on the surface of the bacterial cell membrane through the regulation of the metal regulatory protein MerR can convert toxic Hg (II) into metal Hg (0) with low toxicity and volatility, certain concentrations of Hg (II) (20uM) are respectively given to the bioengineering bacteria and the control bacteria in the experiment, and the Hg (II) content in the bioengineering bacteria and the control bacteria at different times (0, 2, 4, 6 and 8h) is finally measured according to the steps (1) to (5). As shown in FIG. 6, experimental results show that the bioengineering bacteria can reduce Hg (II) in the water environment by regulating and expressing the OmpA-MerA recombinant protein through the MerR protein, thereby effectively reducing the Hg (II) content in the water environment, while the control bacteria only expressing the OmpA protein can not reduce the Hg (II) content in the water environment.

Secondly, research on reduction performance of bioengineering bacteria on Hg (II) with different concentrations

(1) Picking single colonies from LB solid culture plates of experimental bacteria for transforming merR op-ompA-merA-pSB1A2 plasmid, respectively inoculating the single colonies into 3mL of liquid LB culture medium (containing 150ug/mL carbenicillin), and culturing at 37 ℃ and 250rpm overnight;

(2) carrying out amplification culture on the overnight-cultured bacterial liquid in 100ml of liquid LB culture medium according to the proportion of 1:100, and carrying out constant-temperature culture at 37 ℃ and 250rpm until OD600 is 1;

(3) the bacterial solution grown until OD600 ═ 1 was fractionatedPackaging (3 mL/tube), and adding final concentration of 10uM, 25uM, 50uM, 100uM, 250uM, and 500uM Hg into the packaged bacterial solution²⁺The culture was continued at 37 ℃ and 250rpm (3 replicates were set up);

(4) respectively sucking 100ul of bacterial liquids containing different mercury concentrations into a 1.5ml EP tube for 0h, 2h and 6h, respectively adding 100ul of concentrated nitric acid, and carrying out nitration reaction in a water bath at 65 ℃ for 1 h;

(5) and (3) sucking 100ul of nitrified bacteria liquid sample into a test tube, using distilled water to fix the volume to 5ml, and then using inductively coupled plasma (ICP-MS) to detect mercury ions in the bacteria liquid.

Respectively giving Hg (II) (10uM, 25uM, 50uM, 100uM, 250uM and 500uM) with certain concentration to the bioengineering bacteria, and finally measuring the reduction rate of the bioengineering bacteria to Hg (II) under different concentrations according to the steps (1) to (5), as is obvious from figure 7, when the adding time of 10uM Hg (II) is 2 hours, the reduction rate is as high as 37.06%; the degradation rate of Hg (II) is gradually reduced along with the increase of the concentration of Hg (II), the reduction rate is 26.64 percent when 500uM Hg (II) is added for 2 hours, the reduction rate of Hg (II) is further increased along with the increase of time no matter at high concentration or low concentration, and the reduction rate is up to 59 percent when 10uM Hg (II) is added for 6 hours; when the addition time of 500uM Hg (II) is 6 hours, the reduction rate is 42.39%, and experimental results show that the bioengineering bacteria can efficiently reduce Hg (II) in a water environment, so that high-concentration mercury pollution treatment is realized.

Third, research on specificity performance of biological engineering bacteria on reduction of Hg (II)

(3) the bacterial liquid grown to OD600 ═ 1 was dispensed (3 mL/tube), Hg (II), Cu (II), Zn (II), Pb (II), Ni (II), Cd (II), Cr (III) and Fe (III) were added to the dispensed bacterial liquid to a final concentration of 1uM or 10uM, and the culture was continued at 37 ℃ and 250rpm (3 replicate experiments were set);

(4) respectively sucking 100ul of bacterial liquid containing different metal concentrations into a 1.5ml EP tube within 0h and 6h, respectively adding 100ul of concentrated nitric acid, and carrying out nitration reaction in a water bath at 65 ℃ for 1 h;

Adding 8 kinds of metal ions Hg (II), Cu (II), Zn (II), Pb (II), Ni (II), Cd (II), Cr (III) and Fe (III) into the culture medium of the bioengineering bacteria respectively, and comparing the reduction capability of the bioengineering bacteria to different metal ions to reflect the selectivity of the bioengineering bacteria to Hg (II). As can be seen from FIG. 8, the bioengineering bacteria only specifically reduce Hg (II) to effectively reduce the concentration of Hg (II) in the aqueous environment, while the concentrations of the other metal ions Cu (II), Zn (II), Pb (II), Ni (II), Cd (II), Cr (III) and Fe (III) in the aqueous environment are substantially unchanged after the treatment by the bioengineering bacteria. Research results show that the bioengineering bacteria have better selectivity to Hg (II) and can specifically reduce the content of mercury ions in the water environment.

Therefore, the plasmid merR op-ompA-merA-pSB1A2 is successfully constructed by utilizing genetic engineering means such as PCR amplification, enzyme digestion identification, connection, transformation and the like, the bioengineering bacteria containing the plasmid can regulate the expression of mercury ion reductase MerA on the surface of a microbial cell through a transcription activation mechanism of a metal regulatory protein merR, so that mercury ions in a water environment are degraded, and the control bacteria only expressing the OmpA protein can not reduce the content of Hg (II) in the water environment, which indicates that the MerA protein can convert toxic Hg (II) into metal Hg (0) with low toxicity and volatility. Experiments show that the bioengineering bacteria expressing the mercury ion reductase MerA on the cell surface have higher reduction rate no matter low-concentration mercury ions or high-concentration mercury ions, and the reduction rate tends to increase along with time. Meanwhile, the bioengineering bacteria expressing the mercury ion reductase MerA on the cell surface only reduce mercury ions in the water environment, do not reduce other metal ions, and show high selectivity to Hg (II). The bioengineering bacteria of the invention are used for treating Hg (II) pollution in water environment, and have the advantages of ecological environmental protection, good selectivity, high sensitivity, low cost and reusability.

Further, the reagents used in the present invention are shown in table 10 below.

Watch 10

The equipment used in the present invention is shown in Table 11 below.

TABLE 11

The formulation of the medium used in the present invention is shown in Table 12 below.

TABLE 12

The electrophoresis solution used in the present invention is 50 × TAE electrophoresis solution (1L), specifically: 242g of Tris base was weighed, dissolved completely in deionized water, 57.1ml of glacial acetic acid and 100ml of 0.5M NaOH (pH 8.0) were added thereto, and the volume was adjusted to 1000ml, and the working solution concentration was 1 ×. The formulation of the 6 × DNA loading buffer (1ml) used is shown in table 13 below.

Watch 13

In the present invention, an agarose gel electrophoresis experiment is used. Agarose gel electrophoresis experiments are mainly used to separate and identify DNA molecules. The DNA molecules are mainly composed of ribose, phosphate and base, and the structure of ribose-phosphate backbone is repetitive, so that the same number of double-stranded DNA molecules have the same electrostatic charge, and nucleic acid, which is an amphoteric molecule, has a negative overall charge at pH around 8.0, and will move to the positive electrode at the same speed under electrophoresis, so that the migration rate of DNA molecules depends on the molecular weight and structure of DNA molecules themselves under the same electric field strength. For DNA molecules of the same configuration, the migration velocity is inversely proportional to the relative molecular weight. For DNA molecules with the same molecular weight and different configurations, an agarose gel electrophoresis experiment can also obtain effective separation, and the migration rate of the DNA molecules with different configurations in a certain electric field is as follows: covalently closed circular supercoiled DNA > linear DNA > open circular double stranded circular DNA. In general, agarose gel electrophoresis experiments are suitable for separating DNA fragments ranging in size from 0.2kb to 50 kb. Taking a 1% agarose gel electrophoresis experiment as an example, the main operation process is as follows: weighing 2g of agarose, adding 4ml of 50 XTAE electrophoresis buffer solution, uniformly mixing, fixing the volume to 200ml by using deionized water, heating in a microwave oven until the agarose is completely dissolved, cooling at room temperature, cooling to about 60 ℃, quickly pouring into a mold, and adding 2-4ul of ethidium bromide. After uniform mixing, continuing cooling until the colloid is solidified, transferring the prepared gel into an electrophoresis system from a mould, and adding 1 XTAE electrophoresis buffer solution until the gel surface is slightly submerged. Then, a DNA sample was added to the lane of the agarose gel, and the gel plate after the addition was immediately subjected to electrophoresis by applying a current thereto at a voltage of 100V, so that the sample was moved from the negative electrode (black) to the positive electrode (red). The electrophoresis was stopped when bromophenol blue in the sample moved to about 1/3 deg.f below the gel plate. Carefully remove the Gel, observe under ultraviolet lamp, DNA present shows red fluorescence band, use Gel Doc XR Gel imaging system to photograph and preserve.

The invention uses the rubber cutting recovery experiment. The DNA fragments cut out in the agarose gel electrophoresis experiment are recovered by using an Omega Bio-Tek gel cutting recovery kit, and the main steps are as follows: 1) the gel placed in a clean, sterile EP tube was weighed, the net weight of the gel calculated, and an appropriate volume of Binding Buffer XP2 was added to the EP tube at a rate of 100ul Binding Buffer XP2 per 100mg weight of gel. 2) The EP tube is placed in a water bath at 55-60 deg.C until the gel is completely melted, during which the gel can be turned upside down to accelerate the melting of the gel. 3) After the gel is completely melted, the solution is transferred into a container with a collecting pipe by a pipette

The DNA Mini column was centrifuged at 10000Xg for 1 minute at room temperature, and the centrifuged solution was discarded. 4) Go to again

300ul Binding Buffer XP2 was added to the DNA Mini column and centrifuged at 10000Xg for 1 minute at room temperature to discard the centrifuged solution. 5) To the direction of

700ul of SPW Wash Buffer was added to the DNA Mini column, and the filtrate was discarded after centrifugation at room temperature for 1 min. 6) Repeating the step 5), centrifuging at room temperature for 3min, and drying

DNA Mini column. 7) The sample collection tubes were replaced with sterile EP tubes and 20-30 ul of sterile MilliQ H2O was added for infiltration

And (3) carrying out high-speed centrifugation on the DNA Mini column after standing for 2min at room temperature to elute a DNA sample, measuring the concentration by using a NanoDrop1000 micro-protein nucleic acid tester, and storing to-4 ℃.

While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.

The sequence of the merR in the invention is as follows:

TTACGGCATAGCAGAACCAGCCAGTGAAGCACCACCTTGCAGCGAAGCGATCAGCG GACACGACACGTTACCGCGACGAGCATGACATGCGCAAACCAGTTCTGACAGCACT GCTTCCATACGTGCCAGGTCTGCCATCTTTTCACGGACATCCTTCAGTTTGTGTTCGG CCAGAGAGCTCGCTTCTTCGCAATGCGTACCGTCTTCCAGGCGCAGCAGTTCTGCGA TTTCATCCAGGGAGAAGCCCAGACGCTGAGCACTTTTGACAAAGCGAACACGGGTC ACGTCTGCTTCACCGTAGCGACGAATAGAGCCATACGGCTTATCCGGTTCCAGCAGC AGGCCTTTGCGTTGATAGAAGCGGATCGTTTCCACATTCACACCGGCAGCCTTAGCG AAGACACCAATCGTCAGGTTTTCCAGGTTATTTTCCAT

the sequence of the op (operator) in the invention is as follows:

ATCGCTTGACTCCGTACATGAGTACGGAAGTAAGGTTACGCTATCCAATTTCAATTCG AAAGGACAAGCGC

the sequence of ompA in the present invention is as follows:

ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTAGCG CAGGCCGCTCCGAAAGATAACACCTGGTACACTGGTGCTAAACTGGGCTGGTCCCA GTACCATGACACTGGTTTCATCAACAACAATGGCCCGACCCATGAAAACCAACTGGG CGCTGGTGCTTTTGGTGGTTACCAGGTTAACCCGTATGTTGGCTTTGAAATGGGTTAC GACTGGTTAGGTCGTATGCCGTACAAAGGCAGCGTTGAAAACGGTGCATACAAAGCT CAGGGCGTTCAACTGACCGCTAAGCTGGGTTACCCAATCACTGACGACCTGGACATC TACACTCGTCTGGGTGGCATGGTATGGCGTGCAGACACTAAATCCAACGTTTATGGTA AAAACCACGACACCGGCGTTTCTCCGGTCTTCGCTGGCGGTGTTGAGTACGCGATCA CTCCTGAAATCGCTACCCGTCTGGAATACCAGTGGACCAACAACATCGGTGACGCAC ACACCATCGGCACTCGTCCGGACAACGGAGGATCC

the sequence of the merA of the invention is as follows:

ATGACCCATCTGAAAATTACCGGTATGACCTGTGATAGCTGTGCAGCACATGTTAAAG AAGCCCTGGAAAAAGTTCCGGGTGTTCAGAGCGCACTGGTTTCATATCCGAAAGGTA CCGCACAGCTGGCAATTGTTCCGGGTACATCACCGGATGCACTGACCGCAGCAGTTG CAGGTCTGGGTTATAAAGCTACCCTGGCAGATGCCCCGCTGGCCGATAATCGTGTGG GTCTGCTGGATAAAGTTCGTGGTTGGATGGCAGCAGCCGAAAAACATAGCGGTAATG AACCGCCAGTTCAGGTTGCAGTGATTGGCTCAGGTGGTGCAGCAATGGCAGCAGCA CTGAAAGCTGTGGAACAGGGTGCACAGGTTACCCTGATTGAACGTGGTACAATTGGT GGCACATGTGTGAATGTGGGTTGTGTTCCGAGCAAAATTATGATTCGTGCGGCACATA TTGCCCATCTGCGCCGCGAATCTCCGTTTGATGGCGGTATTGCAGCCACCGTTCCGAC CATTGATCGTAGTAAATTACTGGCACAGCAGCAGGCGCGTGTTGATGAACTGCGCCA TGCAAAATATGAAGGTATTCTGGGTGGCAATCCGGCTATTACCGTGGTTCATGGTGAA GCTCGTTTTAAAGATGATCAGTCTCTGACCGTGCGCCTGAATGAAGGTGGTGAACGT GTTGTTATGTTTGATCGTTGTCTGGTGGCAACAGGCGCCTCACCGGCTGTTCCGCCGA TTCCGGGTCTGAAAGAAAGTCCGTATTGGACGTCTACTGAAGCACTGGCATCAGATA CCATTCCGGAACGTCTGGCGGTGATTGGTAGTAGCGTTGTTGCCCTGGAACTGGCGC AGGCCTTTGCACGTCTGGGTTCTAAAGTTACCGTGCTGGCCCGTAATACACTGTTCTT TCGCGAAGATCCGGCCATTGGTGAAGCCGTTACAGCTGCATTTCGCGCAGAAGGTAT TGAAGTTCTGGAACATACCCAGGCAAGCCAGGTTGCGCATATGGATGGTGAATTTGT TCTGACAACCACCCATGGTGAACTGCGCGCCGATAAACTGTTGGTGGCGACCGGCC GTACCCCGAATACACGTTCTCTGGCACTGGATGCAGCAGGTGTGACAGTTAATGCGC AGGGTGCCATTGTGATTGATCAGGGTATGCGCACAAGCAATCCGAATATTTATGCGGC CGGTGATTGTACTGATCAGCCGCAGTTTGTGTATGTTGCGGCAGCGGCAGGCACCCG CGCCGCAATTAATATGACCGGTGGTGATGCTGCGCTGGATCTGACCGCAATGCCGGC CGTGGTGTTTACCGATCCGCAGGTTGCGACAGTTGGTTATAGTGAAGCGGAAGCACA TCATGATGGAATTGAAACCGATAGCCGCACCCTGACCCTGGATAATGTACCGCGTGC ACTGGCTAATTTTGATACACGCGGTTTTATTAAACTGGTGATTGAAGAAGGCTCACAT CGTCTGATTGGTGTGCAGGCAGTTGCCCCGGAAGCAGGTGAACTGATTCAGACCGC AGCACTGGCTATTCGTAATCGTATGACCGTGCAGGAACTGGCAGATCAGCTGTTTCC GTATCTGACCATGGTGGAAGGCCTGAAACTGGCGGCACAGACCTTTAATAAAGATGT TAAACAGCTGAGCTGTTGTGCCGGTTAA

the sequence of the recombinant gene merR op-ompA-merA is as follows:

TCTAGATTACGGCATAGCAGAACCAGCCAGTGAAGCACCACCTTGCAGCGAAGCGAT CAGCGGACACGACACGTTACCGCGACGAGCATGACATGCGCAAACCAGTTCTGACA GCACTGCTTCCATACGTGCCAGGTCTGCCATCTTTTCACGGACATCCTTCAGTTTGTG TTCGGCCAGAGAGCTCGCTTCTTCGCAATGCGTACCGTCTTCCAGGCGCAGCAGTTC TGCGATTTCATCCAGGGAGAAGCCCAGACGCTGAGCACTTTTGACAAAGCGAACAC GGGTCACGTCTGCTTCACCGTAGCGACGAATAGAGCCATACGGCTTATCCGGTTCCA GCAGCAGGCCTTTGCGTTGATAGAAGCGGATCGTTTCCACATTCACACCGGCAGCCT TAGCGAAGACACCAATCGTCAGGTTTTCCAGGTTATTTTCCATATCGCTTGACTCCGT ACATGAGTACGGAAGTAAGGTTACGCTATCCAATTTCAATTCGAAAGGACAAGCGC ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTAGCG CAGGCCGCTCCGAAAGATAACACCTGGTACACTGGTGCTAAACTGGGCTGGTCCCA GTACCATGACACTGGTTTCATCAACAACAATGGCCCGACCCATGAAAACCAACTGGG CGCTGGTGCTTTTGGTGGTTACCAGGTTAACCCGTATGTTGGCTTTGAAATGGGTTAC GACTGGTTAGGTCGTATGCCGTACAAAGGCAGCGTTGAAAACGGTGCATACAAAGCT CAGGGCGTTCAACTGACCGCTAAGCTGGGTTACCCAATCACTGACGACCTGGACATC TACACTCGTCTGGGTGGCATGGTATGGCGTGCAGACACTAAATCCAACGTTTATGGTA AAAACCACGACACCGGCGTTTCTCCGGTCTTCGCTGGCGGTGTTGAGTACGCGATCA CTCCTGAAATCGCTACCCGTCTGGAATACCAGTGGACCAACAACATCGGTGACGCAC ACACCATCGGCACTCGTCCGGACAACGGAGGATCCACTAGTATGACCCATCTGAAAA TTACCGGTATGACCTGTGATAGCTGTGCAGCACATGTTAAAGAAGCCCTGGAAAAAG TTCCGGGTGTTCAGAGCGCACTGGTTTCATATCCGAAAGGTACCGCACAGCTGGCAA TTGTTCCGGGTACATCACCGGATGCACTGACCGCAGCAGTTGCAGGTCTGGGTTATA AAGCTACCCTGGCAGATGCCCCGCTGGCCGATAATCGTGTGGGTCTGCTGGATAAAG TTCGTGGTTGGATGGCAGCAGCCGAAAAACATAGCGGTAATGAACCGCCAGTTCAG GTTGCAGTGATTGGCTCAGGTGGTGCAGCAATGGCAGCAGCACTGAAAGCTGTGGA ACAGGGTGCACAGGTTACCCTGATTGAACGTGGTACAATTGGTGGCACATGTGTGAA TGTGGGTTGTGTTCCGAGCAAAATTATGATTCGTGCGGCACATATTGCCCATCTGCGC CGCGAATCTCCGTTTGATGGCGGTATTGCAGCCACCGTTCCGACCATTGATCGTAGTA AATTACTGGCACAGCAGCAGGCGCGTGTTGATGAACTGCGCCATGCAAAATATGAAG GTATTCTGGGTGGCAATCCGGCTATTACCGTGGTTCATGGTGAAGCTCGTTTTAAAGA TGATCAGTCTCTGACCGTGCGCCTGAATGAAGGTGGTGAACGTGTTGTTATGTTTGAT CGTTGTCTGGTGGCAACAGGCGCCTCACCGGCTGTTCCGCCGATTCCGGGTCTGAAA GAAAGTCCGTATTGGACGTCTACTGAAGCACTGGCATCAGATACCATTCCGGAACGT CTGGCGGTGATTGGTAGTAGCGTTGTTGCCCTGGAACTGGCGCAGGCCTTTGCACGT CTGGGTTCTAAAGTTACCGTGCTGGCCCGTAATACACTGTTCTTTCGCGAAGATCCGG CCATTGGTGAAGCCGTTACAGCTGCATTTCGCGCAGAAGGTATTGAAGTTCTGGAAC ATACCCAGGCAAGCCAGGTTGCGCATATGGATGGTGAATTTGTTCTGACAACCACCC ATGGTGAACTGCGCGCCGATAAACTGTTGGTGGCGACCGGCCGTACCCCGAATACAC GTTCTCTGGCACTGGATGCAGCAGGTGTGACAGTTAATGCGCAGGGTGCCATTGTGA TTGATCAGGGTATGCGCACAAGCAATCCGAATATTTATGCGGCCGGTGATTGTACTGA TCAGCCGCAGTTTGTGTATGTTGCGGCAGCGGCAGGCACCCGCGCCGCAATTAATAT GACCGGTGGTGATGCTGCGCTGGATCTGACCGCAATGCCGGCCGTGGTGTTTACCGA TCCGCAGGTTGCGACAGTTGGTTATAGTGAAGCGGAAGCACATCATGATGGAATTGA AACCGATAGCCGCACCCTGACCCTGGATAATGTACCGCGTGCACTGGCTAATTTTGAT ACACGCGGTTTTATTAAACTGGTGATTGAAGAAGGCTCACATCGTCTGATTGGTGTGC AGGCAGTTGCCCCGGAAGCAGGTGAACTGATTCAGACCGCAGCACTGGCTATTCGTA ATCGTATGACCGTGCAGGAACTGGCAGATCAGCTGTTTCCGTATCTGACCATGGTGG AAGGCCTGAAACTGGCGGCACAGACCTTTAATAAAGATGTTAAACAGCTGAGCTGTT GTGCCGGTTAAACTAGT

the base sequence of the XbaI restriction enzyme cutting site is as follows: TCTAGA

The base sequence of the SpeI enzyme cutting site in the invention is as follows: ACTAGT.

SEQUENCE LISTING

<110> university of south America

<120> bioengineering bacteria for reducing bivalent mercury ions and preparation method and application thereof

<130> CN18NN9856I

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 435

<212> DNA

<213> Artificial sequence

<400> 1

ttacggcata gcagaaccag ccagtgaagc accaccttgc agcgaagcga tcagcggaca 60

cgacacgtta ccgcgacgag catgacatgc gcaaaccagt tctgacagca ctgcttccat 120

acgtgccagg tctgccatct tttcacggac atccttcagt ttgtgttcgg ccagagagct 180

cgcttcttcg caatgcgtac cgtcttccag gcgcagcagt tctgcgattt catccaggga 240

gaagcccaga cgctgagcac ttttgacaaa gcgaacacgg gtcacgtctg cttcaccgta 300

gcgacgaata gagccatacg gcttatccgg ttccagcagc aggcctttgc gttgatagaa 360

gcggatcgtt tccacattca caccggcagc cttagcgaag acaccaatcg tcaggttttc 420

caggttattt tccat 435

<210> 2

<211> 71

<212> DNA

<213> Artificial sequence

<400> 2

atcgcttgac tccgtacatg agtacggaag taaggttacg ctatccaatt tcaattcgaa 60

aggacaagcg c 71

<210> 3

<211> 549

<212> DNA

<213> Artificial sequence

<400> 3

atgaaaaaga cagctatcgc gattgcagtg gcactggctg gtttcgctac cgtagcgcag 60

gccgctccga aagataacac ctggtacact ggtgctaaac tgggctggtc ccagtaccat 120

gacactggtt tcatcaacaa caatggcccg acccatgaaa accaactggg cgctggtgct 180

tttggtggtt accaggttaa cccgtatgtt ggctttgaaa tgggttacga ctggttaggt 240

cgtatgccgt acaaaggcag cgttgaaaac ggtgcataca aagctcaggg cgttcaactg 300

accgctaagc tgggttaccc aatcactgac gacctggaca tctacactcg tctgggtggc 360

atggtatggc gtgcagacac taaatccaac gtttatggta aaaaccacga caccggcgtt 420

tctccggtct tcgctggcgg tgttgagtac gcgatcactc ctgaaatcgc tacccgtctg 480

gaataccagt ggaccaacaa catcggtgac gcacacacca tcggcactcg tccggacaac 540

ggaggatcc 549

<210> 4

<211> 1686

<212> DNA

<213> Artificial sequence

<400> 4

atgacccatc tgaaaattac cggtatgacc tgtgatagct gtgcagcaca tgttaaagaa 60

gccctggaaa aagttccggg tgttcagagc gcactggttt catatccgaa aggtaccgca 120

cagctggcaa ttgttccggg tacatcaccg gatgcactga ccgcagcagt tgcaggtctg 180

ggttataaag ctaccctggc agatgccccg ctggccgata atcgtgtggg tctgctggat 240

aaagttcgtg gttggatggc agcagccgaa aaacatagcg gtaatgaacc gccagttcag 300

gttgcagtga ttggctcagg tggtgcagca atggcagcag cactgaaagc tgtggaacag 360

ggtgcacagg ttaccctgat tgaacgtggt acaattggtg gcacatgtgt gaatgtgggt 420

tgtgttccga gcaaaattat gattcgtgcg gcacatattg cccatctgcg ccgcgaatct 480

ccgtttgatg gcggtattgc agccaccgtt ccgaccattg atcgtagtaa attactggca 540

cagcagcagg cgcgtgttga tgaactgcgc catgcaaaat atgaaggtat tctgggtggc 600

aatccggcta ttaccgtggt tcatggtgaa gctcgtttta aagatgatca gtctctgacc 660

gtgcgcctga atgaaggtgg tgaacgtgtt gttatgtttg atcgttgtct ggtggcaaca 720

ggcgcctcac cggctgttcc gccgattccg ggtctgaaag aaagtccgta ttggacgtct 780

actgaagcac tggcatcaga taccattccg gaacgtctgg cggtgattgg tagtagcgtt 840

gttgccctgg aactggcgca ggcctttgca cgtctgggtt ctaaagttac cgtgctggcc 900

cgtaatacac tgttctttcg cgaagatccg gccattggtg aagccgttac agctgcattt 960

cgcgcagaag gtattgaagt tctggaacat acccaggcaa gccaggttgc gcatatggat 1020

ggtgaatttg ttctgacaac cacccatggt gaactgcgcg ccgataaact gttggtggcg 1080

accggccgta ccccgaatac acgttctctg gcactggatg cagcaggtgt gacagttaat 1140

gcgcagggtg ccattgtgat tgatcagggt atgcgcacaa gcaatccgaa tatttatgcg 1200

gccggtgatt gtactgatca gccgcagttt gtgtatgttg cggcagcggc aggcacccgc 1260

gccgcaatta atatgaccgg tggtgatgct gcgctggatc tgaccgcaat gccggccgtg 1320

gtgtttaccg atccgcaggt tgcgacagtt ggttatagtg aagcggaagc acatcatgat 1380

ggaattgaaa ccgatagccg caccctgacc ctggataatg taccgcgtgc actggctaat 1440

tttgatacac gcggttttat taaactggtg attgaagaag gctcacatcg tctgattggt 1500

gtgcaggcag ttgccccgga agcaggtgaa ctgattcaga ccgcagcact ggctattcgt 1560

aatcgtatga ccgtgcagga actggcagat cagctgtttc cgtatctgac catggtggaa 1620

ggcctgaaac tggcggcaca gacctttaat aaagatgtta aacagctgag ctgttgtgcc 1680

ggttaa 1686

<210> 5

<211> 2759

<212> DNA

<213> Artificial sequence

<400> 5

tctagattac ggcatagcag aaccagccag tgaagcacca ccttgcagcg aagcgatcag 60

cggacacgac acgttaccgc gacgagcatg acatgcgcaa accagttctg acagcactgc 120

ttccatacgt gccaggtctg ccatcttttc acggacatcc ttcagtttgt gttcggccag 180

agagctcgct tcttcgcaat gcgtaccgtc ttccaggcgc agcagttctg cgatttcatc 240

cagggagaag cccagacgct gagcactttt gacaaagcga acacgggtca cgtctgcttc 300

accgtagcga cgaatagagc catacggctt atccggttcc agcagcaggc ctttgcgttg 360

atagaagcgg atcgtttcca cattcacacc ggcagcctta gcgaagacac caatcgtcag 420

gttttccagg ttattttcca tatcgcttga ctccgtacat gagtacggaa gtaaggttac 480

gctatccaat ttcaattcga aaggacaagc gcatgaaaaa gacagctatc gcgattgcag 540

tggcactggc tggtttcgct accgtagcgc aggccgctcc gaaagataac acctggtaca 600

ctggtgctaa actgggctgg tcccagtacc atgacactgg tttcatcaac aacaatggcc 660

cgacccatga aaaccaactg ggcgctggtg cttttggtgg ttaccaggtt aacccgtatg 720

ttggctttga aatgggttac gactggttag gtcgtatgcc gtacaaaggc agcgttgaaa 780

acggtgcata caaagctcag ggcgttcaac tgaccgctaa gctgggttac ccaatcactg 840

acgacctgga catctacact cgtctgggtg gcatggtatg gcgtgcagac actaaatcca 900

acgtttatgg taaaaaccac gacaccggcg tttctccggt cttcgctggc ggtgttgagt 960

acgcgatcac tcctgaaatc gctacccgtc tggaatacca gtggaccaac aacatcggtg 1020

acgcacacac catcggcact cgtccggaca acggaggatc cactagtatg acccatctga 1080

aaattaccgg tatgacctgt gatagctgtg cagcacatgt taaagaagcc ctggaaaaag 1140

ttccgggtgt tcagagcgca ctggtttcat atccgaaagg taccgcacag ctggcaattg 1200

ttccgggtac atcaccggat gcactgaccg cagcagttgc aggtctgggt tataaagcta 1260

ccctggcaga tgccccgctg gccgataatc gtgtgggtct gctggataaa gttcgtggtt 1320

ggatggcagc agccgaaaaa catagcggta atgaaccgcc agttcaggtt gcagtgattg 1380

gctcaggtgg tgcagcaatg gcagcagcac tgaaagctgt ggaacagggt gcacaggtta 1440

ccctgattga acgtggtaca attggtggca catgtgtgaa tgtgggttgt gttccgagca 1500

aaattatgat tcgtgcggca catattgccc atctgcgccg cgaatctccg tttgatggcg 1560

gtattgcagc caccgttccg accattgatc gtagtaaatt actggcacag cagcaggcgc 1620

gtgttgatga actgcgccat gcaaaatatg aaggtattct gggtggcaat ccggctatta 1680

ccgtggttca tggtgaagct cgttttaaag atgatcagtc tctgaccgtg cgcctgaatg 1740

aaggtggtga acgtgttgtt atgtttgatc gttgtctggt ggcaacaggc gcctcaccgg 1800

ctgttccgcc gattccgggt ctgaaagaaa gtccgtattg gacgtctact gaagcactgg 1860

catcagatac cattccggaa cgtctggcgg tgattggtag tagcgttgtt gccctggaac 1920

tggcgcaggc ctttgcacgt ctgggttcta aagttaccgt gctggcccgt aatacactgt 1980

tctttcgcga agatccggcc attggtgaag ccgttacagc tgcatttcgc gcagaaggta 2040

ttgaagttct ggaacatacc caggcaagcc aggttgcgca tatggatggt gaatttgttc 2100

tgacaaccac ccatggtgaa ctgcgcgccg ataaactgtt ggtggcgacc ggccgtaccc 2160

cgaatacacg ttctctggca ctggatgcag caggtgtgac agttaatgcg cagggtgcca 2220

ttgtgattga tcagggtatg cgcacaagca atccgaatat ttatgcggcc ggtgattgta 2280

ctgatcagcc gcagtttgtg tatgttgcgg cagcggcagg cacccgcgcc gcaattaata 2340

tgaccggtgg tgatgctgcg ctggatctga ccgcaatgcc ggccgtggtg tttaccgatc 2400

cgcaggttgc gacagttggt tatagtgaag cggaagcaca tcatgatgga attgaaaccg 2460

atagccgcac cctgaccctg gataatgtac cgcgtgcact ggctaatttt gatacacgcg 2520

gttttattaa actggtgatt gaagaaggct cacatcgtct gattggtgtg caggcagttg 2580

ccccggaagc aggtgaactg attcagaccg cagcactggc tattcgtaat cgtatgaccg 2640

tgcaggaact ggcagatcag ctgtttccgt atctgaccat ggtggaaggc ctgaaactgg 2700

cggcacagac ctttaataaa gatgttaaac agctgagctg ttgtgccggt taaactagt 2759

Claims

1. The bioengineering bacterium for reducing the bivalent mercury ions is characterized by comprising an expression vectormerR op-ompA-merA-pSB1A2, comprising a recombinant genemerR op-ompA-merAAnd the pSB1A2 plasmid backbone,merRthe gene sequence of (A) is shown in SEQ ID NO.1,opthe gene sequence of (A) is shown in SEQ ID NO.2,ompAthe gene sequence of (A) is shown in SEQ ID NO.3,merAthe gene sequence of (A) is shown in SEQ ID NO.4, and the bioengineering bacteria is escherichia coli.

2. The method for preparing bioengineering bacteria for reducing divalent mercury ions according to claim 1, comprising the following steps:

step one, respectively realizing by utilizing PCR amplification reactionmerR op、ompA、merAAmplification of the Gene fragment, the amplified fragmentmerR opGene fragments andompAthe gene segments are connected by overlapping extension PCR technology and amplified by PCR amplification reaction to obtain the target gene segmentmerR op-ompA；

Step two, using restriction enzymes XbaI and SpeI to carry out plasmid skeleton alignment on pSB1A2 and target gene fragmentmerR op- ompAPerforming double enzyme digestion to obtain target gene fragmentmerR op-ompAExposing the same cohesive end with the pSB1A2 plasmid skeleton, and connecting the target gene segment with T4DNA ligasemerR op-ompAConnected with pSB1A2 plasmid skeleton to realizemerR op-ompAConstruction of the pSB1A2 plasmid;

step three, using restriction enzyme SpeI pairmerR op-ompAPlasmid pSB1A2 and amplifiedmerAThe gene fragment is subjected to single enzyme digestion and then is subjected to T4DNA ligasemerR op-ompAPlasmid pSB1A2 and amplifiedmerAGene fragment connection to complete expression vectormerR op-ompA- merAConstruction of pSB1A 2;

step four, the expression vector is transformed by a chemical transformation methodmerR op-ompA- merA-pSB1A2 is transformed into competent cells to obtain the bioengineering bacteria.

3. The method for preparing bioengineering bacteria for reducing divalent mercury ions according to claim 2, wherein the target gene fragment in the step onemerR op-ompAThe preparation method specifically comprises the following steps: respectively by total synthesismerR op、 ompA、merAUsing gene sequence as template, making first PCR amplification reaction, then making amplified productmerR opAndompAthe complementary segment of the end of the gene segment is realized by overlapping extension PCR technologymerR opAndompAto obtain a ligated fragmentmerR op-ompAPerforming a second PCR amplification reaction to realize the connection pieceSegment ofmerR op-ompAAmplifying to obtain the target gene fragmentmerR op-ompA。

4. The method according to claim 2, wherein the competent cells in step four areE.coliDH5 α competent cells.

5. The method for preparing bioengineering bacteria for reducing divalent mercury ions according to claim 3, wherein, during the first PCR amplification reaction,merR opthe primers to be added comprise IMerr-OP XF and OP-ompA UP,ompAthe primers to be added include OP-ompA DN, OmpA SR,merAprimers to be added comprise MerA SF and MerA SR; the primers added in the second PCR amplification reaction comprise IMerr-OP XF and OmpA SR;

the sequence of IMerr-OP XF is: GTAGTTCTAGATTACGGCATAGCAGAACCAG, respectively;

the sequence of OP-ompA UP is: AATTTCAATTCGAAAGGACAAGCGCATGAAAAAGACAGCTATCGCGATTG, respectively;

the sequence of OP-ompA DN is: CAATCGCGATAGCTGTCTTTTTCATGCGCTTGTCCTTTCGAATTGAAATT, respectively;

the sequence of OmpA SR is: GTAGTACTAGTGGATCCTCCGTTGTCCG, respectively;

the sequence of MerA SF is: GTAGTACTAGTATGACCCATCTGAAAATTACCG, respectively;

the sequence of the MerA SR is: GTAGTACTAGT TTAACCGGCACAACAGCT are provided.

6. The method for preparing bioengineering bacteria for reducing divalent mercury ions according to claim 2, wherein the reaction temperature for the double enzyme digestion in the second step is 37 ℃ and the reaction time is 1 h.

7. The method for preparing bioengineering bacteria for reducing divalent mercury ions according to claim 2, wherein the reaction temperature of the single enzyme digestion in step three is 37 ℃ and the reaction time is 1 h.

8. The method for preparing bioengineering bacteria for reducing divalent mercury ions according to claim 3, wherein the first PCR amplification reaction comprises 30 cycles, wherein one cycle comprises denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 10s, and extension at 72 ℃ for 5 s; the second PCR amplification reaction comprises 30 cycles, wherein one cycle comprises denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 10s and extension at 72 ℃ for 10s in sequence.

9. Use of bioengineered bacteria for reduction of divalent mercury ions according to claim 1 for remediation of hg (ii) pollution in an aqueous environment.

10. The use of bioengineering bacteria for reducing divalent mercury ions according to claim 9, wherein the bioengineering bacteria is introduced into a water environment polluted by Hg (II) to reduce Hg (II) to Hg (0) to treat the water environment.