CN114686411B - Mixture of engineering bacteria, kit for sensing and adsorbing heavy metal pollutants and treatment method - Google Patents

Abstract

The invention provides a mixture of engineering bacteria, a kit for sensing and adsorbing heavy metal pollutants and a treatment method. A mixture of engineering bacteria comprises a first engineering bacteria and a second engineering bacteria, wherein the first engineering bacteria comprises Pb ²⁺ Sensing plasmid, pb ²⁺ The induction plasmid comprises a lead ion induction unit and a first envelope fiber expression unit; the second engineering bacteria comprise Hg ²⁺ Sensing plasmid, hg ²⁺ The sensing plasmid comprises a mercury ion sensing unit and a second envelope fiber expression unit, and the engineering bacteria are selected from bacillus subtilis; the first and/or second envelope fiber expression units comprise an amyloid expression cassette. According to the invention, bacillus subtilis which senses and adsorbs heavy metal ions is constructed by utilizing a synthetic biology technology, fluorescent protein is used as signal protein by utilizing the gel property of a biological film of the bacillus subtilis, and heavy metal is adsorbed and bound by amyloid protein, so that intelligent sensing and real-time repair of the heavy metal ions are realized.

Description

Mixture of engineering bacteria, kit for sensing and adsorbing heavy metal pollutants and treatment method

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a mixture of engineering bacteria, a kit for sensing and adsorbing heavy metal pollutants and a treatment method.

Background

Heavy metals mainly refer to metals with remarkable biotoxicity such as lead, mercury, cadmium and the like. With the development of industry, heavy metal pollution is more and more serious and is difficult to degrade in the environment. Besides the harm to the environment, the food has a certain harm to human bodies, and the animals and plants can not degrade heavy metals, so that the food is enriched to cause harm to the human bodies. Mercury is a particularly concealed contaminant because its accumulation in the ecological niche increases upwardly through the food chain, and humans often suffer damage to brain tissue, kidneys and lung tissue by ingestion of contaminated food. Lead is widely used in paints, pipes and pipe materials, gasoline, batteries, ammunition and cosmetics, and is found in all aspects of our environment. However, lead, like other heavy metals, accumulates through the food chain with serious risks due to its non-degradability and physiological toxicity. Lead poisoning is a non-negligible risk to human health, particularly to children's health, and is a public health problem worldwide. With the rapid development of industry, agriculture and economy, heavy metal pollution in various water environments is increasingly aggravated, and effective detection and repair technology is urgently needed for treating the heavy metal pollution.

The current heavy metal repair methods mainly comprise physical repair, chemical repair and biological repair. The traditional physical repair and chemical repair for treating heavy metal pollution have the problems of higher treatment cost, great labor consumption, easiness in secondary pollution and the like. Bioremediation is a relatively economical method that effectively eliminates the adverse effects of contamination, leaving the process and site of remediation free of toxic or recalcitrant compounds. Bioremediation includes phytoremediation, animal remediation and microbial remediation. At present, a combination strategy of phytoremediation and microbial remediation is relatively large. The microbial repair time is short, the effect is quick, and especially the biological film has tolerance to heavy metals, so that the method is a potential biological repair method.

There are various methods for repairing heavy metals by microorganisms, such as binding, immobilization, oxidation, conversion, volatilization, etc. of heavy metals. The process of microorganism repair can be more effectively carried out in a specific area by a method of designing microorganisms or genetic circuits thereof, and the repair effect of the microorganisms on heavy metals is researched by sensing the activities and growth of the microorganisms in a polluted place, metabolic potential, reaction mechanisms to environmental changes and the like.

It has been reported that p-Pb was constructed in 2014 ²⁺ Induction of E.coli whole cell detectors (WEI W, LIU X, SUN P, et al simple well-cell biodetection and bioremediation of heavy metals based on an engineered lead-specific open [ J ]]Environmental Science and Technology,2014, 48:3363-3371). 2017 constructs Hg ²⁺ Ion-sensing and adsorbing E.coli (TAY P, NGUYEN P, and JOSHI N.A synthetic circuit for mercury bioremediation using self-assembling functional amyloids [ J)].ACS Synthetic Biology,2017,6(10)：1841-1850)。

Although engineering bacteria for respectively sensing lead or mercury by escherichia coli are reported before, the engineering bacteria are single pollutant biosensors, and the tolerance and the repair capability are not high.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, an object of the present invention is to provide a mixture of engineering bacteria, a kit for sensing and adsorbing heavy metal contaminants and a treatment method for solving the problems of the prior art.

To achieve the above and other related objects, the present invention is achieved by the following technical means. A mixture of engineering bacteria comprises a first engineering bacteria and a second engineering bacteria, wherein the first engineering bacteria comprises Pb ²⁺ Sensing plasmid, pb ²⁺ The induction plasmid comprises a lead ion induction unit and a first envelope fiber expression unit; the second engineering bacteria comprise Hg ²⁺ Sensing plasmid, hg ²⁺ The sensing plasmid comprises a mercury ion sensing unit and a second envelope fiber expression unit, and the engineering bacteria are selected from bacillus subtilis;

the first envelope fiber expression unit and/or the second envelope fiber expression unit comprises an amyloid expression frame, and the amino acid sequence of the amyloid protein comprises a sequence shown as SEQ ID No. 5.

Preferably, the bacillus subtilis is bacillus subtilis 002 (CGMCC NO 12600).

The bacillus subtilis 002 is stored in China general microbiological culture collection center (CGMCC) in the year 2016, month 6 and number: 12600, address: no.1 and No.3 of the north cinquefoil of the morning sun area of beijing city.

Preferably, the lead ion sensing unit comprises a modulator, and the polynucleotide sequence of the modulator comprises a sequence shown as SEQ ID No. 1.

Preferably, the lead ion sensing unit further comprises a first promoter, the polynucleotide sequence of which comprises the sequence shown in SEQ ID No. 2.

Preferably, the mercury ion sensing unit comprises a suppressor, and the polynucleotide sequence of the suppressor comprises a sequence shown in SEQ ID No. 3.

Preferably, the mercury ion sensing unit further comprises a second promoter, the polynucleotide sequence of which comprises the sequence shown in SEQ ID No. 4.

Preferably, the amyloid expression cassette further comprises a signal protein and a tag protein, the amyloid protein being located at the N-terminus of the expression cassette and the tag protein being located at the C-terminus of the expression cassette, the signal protein being between the amyloid protein and the tag protein.

Preferably, the signal protein is selected from mCherry.

Preferably, the amino acid sequence of the signal protein comprises the sequence shown as SEQ ID No. 6.

Preferably, the tag protein is a Histag.

The second object of the invention is to provide a kit for sensing and adsorbing heavy metal contaminants, which comprises the engineering bacteria and/or the culture thereof. In the invention, the lead ion sensing unit, the first envelope fiber expression unit or the mercury ion sensing unit and the second envelope fiber expression unit are secreted and expressed and self-assembled into fibers on the biological envelope to form the adjustable nano biological film.

It is a further object of the present invention to provide the use of the engineering bacteria and/or their cultures as described above as sensing and adsorbing agents in the bioremediation of heavy metal pollution.

The invention aims at providing a treatment method for sensing and adsorbing heavy metal pollutants, which comprises the following steps:

providing a liquid containing heavy metal ions;

and adding the mixture of the engineering bacteria into the liquid.

Compared with the prior art, the invention has the following beneficial effects:

1) According to the invention, lead ion-induced bacillus subtilis and mercury ion-induced bacillus subtilis are constructed by utilizing a synthetic biology technology, and the expression of amyloid protein is started after fluorescent induction of heavy metal pollutants by utilizing a biosensor prepared by utilizing the gel property of a bacterial biofilm, and the intelligent induction and real-time repair of heavy metal ions are realized by taking fluorescent protein as a reporter protein and adsorbing and binding heavy metal by using the amyloid protein.

2) Compared with free bacteria, the biological film has stronger environmental tolerance to heavy metal ions and other pollutants. And bacillus subtilis has the advantages of environmental friendliness and stronger secretion capacity.

Drawings

FIG. 1 shows a map of a recombinant plasmid constructed according to the present invention

FIG. 2 shows TEM images of the first engineered bacterium (PbrR-TMCH) of example 1 and the strain (PbrR-01) of comparative example 1 of the present invention

FIG. 3 shows graphs of the first engineering bacterium (PbrR-TMCH) of example 1 and the strain (PbrR-01) of comparative example 1 of the present invention

Wherein a is a fluorescence microscope image, b is a drawing for absorbing lead ions, c and d are SEM images

FIG. 4 shows graphs of the second engineered bacterium (MerR-TMCH) of example 2 and the strain (MerR-01) of comparative example 2 of the present invention

Wherein a is a fluorescence microscopy image, b is a drawing for absorbing mercury ions, c and d are TEM, and the insertion in c and d is an SEM image

FIG. 5 is a SEM image showing the co-culture of the first engineered bacterium (PbrR-TMCH) of example 1 and the second engineered bacterium (MerR-TMCH) of example 2 of the present invention

FIG. 6 shows a fluorescence microscopic image and an adsorption image of heavy metal ions in the co-culture of the first engineered bacterium (PbrR-TMCH) of example 1 and the second engineered bacterium (MerR-TMCH) of example 2 of the present invention

Wherein a is a fluorescence microscope image, and b is a heavy metal ion adsorption image

FIG. 7 shows a schematic diagram of the present invention

Detailed Description

Further advantages and effects of the present invention will become apparent to those skilled in the art from the disclosure of the present invention, which is described by the following specific examples.

Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention. The test methods in the following examples, in which specific conditions are not noted, are generally conducted under conventional conditions or under conditions recommended by the respective manufacturers.

Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.

In this application, the plasmid is pHT01, purchased from MOBITEC;

in this application, the lead ion sensing unit includes a regulator PbrR and a first promoter PpbrA, provided by su Jin Weizhi biotechnology limited;

in the present application, the mercury ion sensing unit comprises a suppressor MerR and a second promoter Pmer, provided by the biotechnology company Jin Weizhi, su;

in the present application, the amyloid TasA is provided by the biotechnology company Jin Weizhi, su, and the amyloid is derived from the amyloid of bacillus subtilis;

in this application, the fluorescent protein mCherry is provided by the biotechnology company, jin Weizhi, su;

in the application, the engineering bacteria are bacillus subtilis 002 (CGMCC NO. 12600);

in the present application, the E.coli DH 5. Alpha. Competent cells (CW 08085) were provided by Jiangsu kang, century biotechnology Co., ltd;

in the present application, sacI/KpnI double cleavage and KpnI/SmaI are purchased from Fermentas company;

in this application, T4 DNA ligase, model 2011A, purchased from TAKARA;

in this application, plasmid miniprep kit (DP 103), purchased from the company digen biochemical technology (beijing);

in this application, LB medium (LD 001) was purchased from Taiwan Biotechnology Co.

In the following examples, unless otherwise specified, the phenotype of the engineered bacterial biofilm was observed in MSgg medium, which was formulated as follows: 100mM 3- (morpholino) propanesulfonic acid, 0.5% glycerol, 0.5% monosodium glutamate monohydrate, 5mM potassium phosphate, 50. Mu.g/ml L-tryptophan, 50. Mu.g/ml L-threonine, 2. Mu.M thiamine hydrochloride (vitamin B1), mgCl ₂ ·6H2O 2mM，CaCl ₂ 700μM，FeCl ₃ ·6H ₂ O 50μM，MnCl ₂ 50μM，ZnCl ₂ 1 mu M, wherein an amino acid solution, thiamine hydrochloride and an ionic solution are prepared into mother solution by distilled water, filtered and sterilized, stored in a refrigerator at the temperature of 4 ℃, and the rest dissolved in deionized water and sterilized by high-temperature steam. The substances are diluted to the above concentrations when in use. The method for constructing and extracting plasmids, the method for transferring bacillus subtilis by adopting the Spizizen transformation method and the method for detecting the biological film can be seen in the content of patent CN106754600A 'bacillus subtilis, biological film and construction and application'.

Example 1

In this embodiment, a first engineering bacterium (PbrR-TMCH) carrying a recombinant plasmid Pbr01-TMCH is constructed, and the specific steps are as follows:

1) Construction of recombinant plasmid Pbr01 containing lead ion sensing Unit

Plasmid pHT01 and the fragment SacI-PbrR-PpbrA-KpnI containing the lead ion sensing unit were digested with SacI/KpnI, the product was subjected to rubber cutting recovery, then ligated with T4 DNA ligase, and added to the EP tube in the desired ratio according to the length and concentration of each fragment, and the reaction system was incubated at 16℃for ligation for 4 hours.

Transferring the connection product into E.coli DH5 alpha competent cells, selecting monoclonal with carbenicillin resistance, extracting plasmid DNA by using a plasmid small extraction kit according to the operation instructions, and carrying out double enzyme digestion identification by using SacI and KpnI. The positive plasmid obtained by identification is sequenced to obtain the correct integrated plasmid Pbr01, the plasmid map is shown in figure 1, wherein the regulator PbrR and the first promoter PpbrA in the lead ion sensing unit are respectively connected to the plasmid pHT01 through SacI restriction enzyme cutting sites and KpnI restriction enzyme cutting sites.

The sequence of the plasmid pHT01 is shown in a sequence table SEQ ID No:7, the nucleotide sequences corresponding to the regulator PbrR of the inserted lead ion sensing unit and the first promoter PpbrA are respectively shown in SEQ ID No:1 and SEQ ID No:2.

2) Construction of an integrative plasmid Pbr01-TMCH containing lead ion sensing units and envelope fiber units

The fragment TasA-mCherry-histag containing the envelope fiber expression unit is digested by KpnI/SmaI double enzyme, inserted into the corresponding site of recombinant plasmid Pbr01, recovered by cutting the fragment with enzyme, then connected by T4 DNA ligase, and the reaction conditions are operated according to the specification, and the connection conditions are set at 16 ℃ for 4 hours.

Transferring the connection product into competent cells of escherichia coli DH5 alpha, selecting monoclonal with carbenicillin resistance, extracting plasmid DNA by using an extraction plasmid kit according to the operation instructions, and carrying out double enzyme digestion identification by using KpnI and SmaI. The positive plasmid obtained by identification is sequenced to obtain the correct integrated plasmid Pbr01-TMCH, the plasmid map is shown in figure 1, wherein the amyloid TasA is connected with a first promoter PpbrA in a lead ion induction unit through a KpnI enzyme cutting site, the tag protein Histag is connected with the recombinant plasmid Pcr 01 through a SmaI enzyme cutting site, the fluorescent protein mCherry is positioned between the amyloid TasA and the tag protein Histag, and the amyloid TasA and the fluorescent protein mCherry are connected by using a linker of eight amino acids.

The amino acid sequence of the amyloid TasA is shown in a sequence table SEQ ID No.5, the amino acid sequence of the fluorescent protein mCherry is shown in a sequence table SEQ ID No.6, and the sequence of the integrating plasmid Pbr01-TMCH is shown in the sequence table SEQ ID No.: 8.

3) Construction and culture of first engineering bacterium (PbrR-TMCH)

The properly sequenced integrative plasmid Pbr01-TMCH was transformed into Bacillus subtilis 002 by the Spizizen transformation method and incubated overnight at 37℃with LB plates with 5. Mu.g/L chloramphenicol resistance. Extracting genome, and identifying whether the genome contains target gene fragments by colony PCR, if so, proving that plasmid transformation is successful, and obtaining first engineering bacteria (PbrR-TMCH) carrying integrated plasmid Pbr 01-TMCH.

The preparation method of the LB culture medium comprises the following steps: 2.5g of LB medium is dissolved in 100ml of distilled water, 1.5g of agar powder is added, high-temperature steam sterilization is carried out, and when cooling to about 50 ℃, chloramphenicol solution is added to a final concentration.

4) The first engineering bacterium (PbrR-TMCH) senses and adsorbs Pb ²⁺ Is to be tested in (2)

Inoculating the first engineering bacterium (PbrR-TMCH) carrying the integrated plasmid PbrR-TMCH into a strain containing Pb according to the ratio of 1:100 ²⁺ And (3) standing and culturing at 30 ℃ in MSgg culture media with the concentration of 1 mu M, 5 mu M, 10 mu M and 20 mu M respectively, observing fluorescence and biofilm generation conditions, and measuring the heavy metal adsorption effect after 3-5 days.

Example 2

In this example, a second engineered bacterium (MerR-TMCH) carrying the integrative plasmid Mer01-TMCH was constructed as follows:

1) Construction of recombinant plasmid Mer01 containing Mercury ion sensing Unit

Plasmid pHT01 and fragment SacI-MerR-Pmer-KpnI containing mercury ion sensing unit were cut with SacI/KpnI double enzyme respectively, the product was subjected to rubber cutting recovery, then ligated with T4 DNA ligase, added to EP tube in the desired ratio according to the length and concentration of each fragment, and the reaction system was incubated at 16℃for ligation for 4 hours.

Transferring the connection product into E.coli DH5 alpha competent cells, selecting monoclonal with carbenicillin resistance, extracting plasmid DNA by using a plasmid small extraction kit according to the operation instructions, and carrying out double enzyme digestion identification by using SacI and KpnI. The positive plasmid obtained by identification is sequenced to obtain the correct recombinant plasmid Mer01, the map of the recombinant plasmid Mer01 is shown in figure 1, wherein the regulator MerR and the second promoter Pmer in the mercury ion sensing unit are respectively connected to the plasmid pHT01 through SacI enzyme cutting sites and KpnI enzyme cutting sites.

The sequence of the plasmid pHT01 is shown in a sequence table SEQ ID No:7, the nucleotide sequences corresponding to the inhibitor MerR of the inserted mercury ion sensing unit and the second promoter Pmer are respectively shown in SEQ ID No:3 and SEQ ID No:4.

2) Construction of an integration plasmid Mer01-TMCH containing Mercury ion sensing units and envelope fiber units

The membrane fiber unit fragment TasA-mCherry-histag is digested by KpnI/SmaI double enzyme, inserted into the corresponding site of the recombinant plasmid Mer01, recovered by cutting gel in the enzyme cutting section, and the T4 DNA ligase reaction condition is operated according to the specification, and the ligation condition is placed at 16 ℃ for 4 hours.

Transferring the connection product into competent cells of escherichia coli DH5 alpha, selecting monoclonal with carbenicillin resistance, extracting plasmid DNA by using an extraction plasmid kit according to the operation instructions, and carrying out double enzyme digestion identification by using KpnI and SmaI. The positive plasmid obtained by identification is sequenced to obtain the correct integrated plasmid Mer01-TMCH, the plasmid map is shown in figure 1, wherein the amyloid TasA is connected with a second promoter Pmer in a mercury ion sensing unit through a KpnI enzyme cleavage site, the tag protein Histag is connected with the recombinant plasmid Mer01 through a SmaI enzyme cleavage site, the fluorescent protein mCherry is positioned between the amyloid TasA and the tag protein Histag, and the amyloid TasA and the fluorescent protein mCherry are connected by using a linker of eight amino acids.

The amino acid sequence of the amyloid TasA is shown in a sequence table SEQ ID No.5, the amino acid sequence of the fluorescent protein mCherry is shown in a sequence table SEQ ID No.6, and the sequence of the integrative plasmid Mer01-TMCH is shown in the sequence table SEQ ID No.: 9.

3) Construction and culture of a second engineering bacterium (Merr-TMCH)

The properly sequenced integrative plasmid Mer01-TMCH was transformed into Bacillus subtilis 002 by Spizizizer transformation and incubated overnight at 37℃with LB plates resistant to 5. Mu.g/L chloramphenicol. Extracting genome, and identifying whether the genome contains target gene fragments by colony PCR, if so, proving that plasmid transformation is successful, and obtaining a second engineering bacterium (MerR-TMCH) carrying an integrated plasmid Mer 01-TMCH.

The preparation method of the LB culture medium comprises the following steps: 2.5g of LB medium is dissolved in 100ml of distilled water, 1.5g of agar powder is added, high-temperature steam sterilization is carried out, and when cooling to about 50 ℃, chloramphenicol solution is added to a final concentration.

4) The second engineering bacterium (MerR-TMCH) senses and adsorbs Hg ²⁺ Is to be tested in (2)

Inoculating the second engineering bacteria (MerR-TMCH) carrying the integrated plasmid Mer01-TMCH into the vector containing Hg according to the ratio of 1:100 ²⁺ And (3) standing and culturing in MSgg culture medium with the concentration of 0.5 mu M at 30 ℃, observing fluorescence and biofilm generation conditions, and measuring the heavy metal adsorption effect after 3-5 days.

Example 3

Mixing and culturing a first engineering bacterium (PbrR-TMCH) carrying an integrated plasmid Pbr01-TMCH and a second engineering bacterium (MerR-TMCH) carrying an integrated plasmid Mer01-TMCH, inoculating the mixed culture to Pb-containing bacteria ²⁺ At a concentration of 10. Mu.M and Hg ²⁺ And (3) standing and culturing in MSgg culture medium with the concentration of 0.5 mu M at 30 ℃, observing fluorescence and biofilm generation conditions, and measuring the heavy metal adsorption effect after 3-5 days.

Comparative example 1

The recombinant plasmid Pbr01 obtained in example 1 was sequenced correctly and then directly transformed into Bacillus subtilis 002 to obtain a strain carrying the recombinant plasmid PbrR01 (PbrR-01), which was subjected to a single-clone overnight culture and inoculated with Pb at a ratio of 1:100 ²⁺ And (3) standing and culturing at 30 ℃ in MSgg culture media with the concentration of 1 mu M, 5 mu M, 10 mu M and 20 mu M respectively, observing fluorescence and biofilm generation conditions, and measuring the heavy metal adsorption effect after 3-5 days.

Comparative example 2

The recombinant plasmid Mer01 obtained in example 2 was sequenced correctly and transformed directly into Bacillus subtilis 002 to obtain a strain (Mer-01) carrying the recombinant plasmid MerR01, which was cultured overnight and inoculated at a 1:100 ratio into a vector containing Hg ²⁺ 3 in MSgg Medium at a concentration of 0.5. Mu.MAnd (3) carrying out stationary culture at 0 ℃, observing fluorescence and biofilm generation conditions, and measuring the heavy metal adsorption effect after 3-5 days.

Comparative example 3

The strain carrying the recombinant plasmid PbrR01 (PbrR-01) and the strain carrying the recombinant plasmid MerR01 (Mer-01) are mixed and cultured, and inoculated into a strain containing Pb according to the ratio of 1:100 ²⁺ At a concentration of 10. Mu.M and Hg ²⁺ And (3) standing and culturing in MSgg culture medium with the concentration of 0.5 mu M at 30 ℃, observing fluorescence and biofilm generation conditions, and measuring the heavy metal adsorption effect after 3-5 days.

The induction and adsorption treatments of heavy metal contaminants were performed in examples 1 to 3 and comparative examples 1 to 3, respectively, with the following results:

1) Induction and adsorption of lead ion contaminants

The strain of comparative example 1 (PbrR-01) and the first engineered bacterium of example 1 (PbrR-TMCH) were observed with a Transmission Electron Microscope (TEM). As shown in FIG. 2, the first engineering bacterium (PbrR-TMCH) constructed by the invention does not express Pb0 when no lead ions exist; in Pb ²⁺ Amyloid fibrils were produced at concentrations of 1. Mu.M, 5. Mu.M, 10. Mu.M, and 20. Mu.M, respectively. While the strain of comparative example 1 (PbrR-01) was in Pb ²⁺ At a concentration of 20. Mu.M, amyloid fibrils were not expressed either.

The strain of comparative example 1 (PbrR-01) and the first engineered bacterium of example 1 (PbrR-TMCH) were placed in Pb ²⁺ At a concentration of 10ppb, observation was carried out by a Scanning Electron Microscope (SEM). As a result, as shown in FIG. 3, the biofilm of the first engineering bacterium showed red fluorescence and adsorbed heavy metals (FIGS. 3a and b), and the biofilm was expressed in large amounts (FIG. 3 c), whereas strain 1 (PbrR-01) of comparative example 1 did not have significant biofilm production (FIG. 3 d).

2) Induction and adsorption of mercury ion pollutants

The second engineered bacterium (Merr-TMCH) obtained in example 2 was observed with a fluorescence microscope. The results are shown in FIG. 4a, at Hg ²⁺ At a concentration of 0.5. Mu.M, the biofilm of the second engineered bacterium (MerR-TMCH) showed red fluorescence.

The second engineering bacterium (Merr-TMCH) obtained in example 2 and the strain (Mer-01) of comparative example 2 were subjected to mercury contamination conditions and the mercury ion content was measured using a high-resolution plasma mass spectrometer (ICP-MS). As a result, as shown in FIG. 4b, the second engineering bacterium can significantly adsorb heavy metals in the solution, and the concentration of mercury ions in the solution (0.2 ppb) is 1/170 of the concentration of comparative example 2 (34 ppb).

The second engineering bacterium (Merr-TMCH) obtained in example 2 and the strain (Mer-01) of comparative example 2 were placed under mercury-contaminated conditions and biofilm observation was performed by TEM and SEM. As a result, as shown in FIGS. 4c and 4d, the second engineering bacteria had a large amount of biofilm expression after sensing mercury ions in the environment, whereas comparative example 2 did not have significant biofilm production (inset in FIGS. 4c and 4 d).

3) Induction and adsorption of lead ion and mercury ion composite pollutant

Experiments were performed to sense and adsorb heavy metal contaminants for example 3 and comparative example 3.

As a result of SEM observation of the expression of the biofilm, as shown in FIG. 5, the mixture of the first engineering bacterium (PbrR-TMCH) and the second engineering bacterium (MerR-TMCH) was able to induce mercury and lead ions simultaneously and produce a large amount of fiber coating.

The fluorescence was observed by a fluorescence microscope, and the result is shown in FIG. 6, in which the first engineering bacterium (PbrR-TMCH) and the second engineering bacterium (MerR-TMCH) were cultured in a mixed manner, and the resulting mixture showed a clear red fluorescence.

The heavy metal adsorption condition is measured by an inductively coupled plasma spectrometer (ICP-AS), and the result is shown in fig. 6b, which shows that the mixture of the first engineering bacteria and the second engineering bacteria can effectively sense the composite pollutant of lead and mercury and has obvious adsorption effect on the composite pollutant.

The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.

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Sequence listing

<110> Shanghai university of science and technology

<120> a mixture of engineering bacteria, kit for sensing and adsorbing heavy metal contaminants, and treatment method

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 438

<212> DNA

<213> regulator PbrR (Artificial Sequence)

<400> 1

atgaacatcc agattggcga actggcgaaa cgcacagcat gcccggtggt gacgatccgc 60

ttttacgaac aagaaggcct tctgccgccg cccggtagaa gcagaggcaa ctttcgttta 120

tatggcgaag aacacgtgga acgtttacag tttatccgcc attgccgcag ccttgatatg 180

ccgctgagcg atgtgagaac gctgctgagc tatcgcaaac gcccggatca agattgcggc 240

gaggtgaata tgctgctgga tgaacacatc cgccaagttg aaagccgcat tggcgcgctg 300

ctggaactga aacatcattt agtggaactg cgcgaagcat gctctggtgc aagaccggcg 360

cagtcatgcg gcattctgca aggtttaagc gattgcgtgt gcgatacaag aggcacaacg 420

gcgcatccga gcgattaa 438

<210> 2

<211> 86

<212> DNA

<213> first promoter PpbrA (Artificial Sequence)

<400> 2

ggcaacccct tgtgtgtatt catctcgcgt tgccgattta acaccctcta gttactatag 60

agtcaagaca tctcccatcc gacgcc 86

<210> 3

<211> 435

<212> DNA

<213> suppressor MerR (Artificial Sequence)

<400> 3

atggaaaaca atttagagaa tttaacgatt ggcgtgtttg cgaaagcggc gggcgtgaac 60

gtggaaacga tccgctttta ccagcgcaaa ggtttactgc gcgaaccgga taaaccgtat 120

ggaagcatcc gcagatatgg cgaagcggac gtggtgcgcg tgaaatttgt gaaaagcgcg 180

cagcgtttag gcttttcttt agatgaaatc gcggaactgc tgcgtttaga tgatggcaca 240

cattgcgaag aagcttcttc tttagcggaa cataaactga aagacgtgcg cgagaaaatg 300

gcagatctgg cgcgcatgga aacggtgctg agcgaactgg tgtgcgcatg ccatgcgcgc 360

aaaggcaacg tgagctgccc gctgattgca tctttacaag gtgaagctgg tttagcaaga 420

agcgcgatgc cgtaa 435

<210> 4

<211> 82

<212> DNA

<213> second promoter Pmer (Artificial Sequence)

<400> 4

atcgcttgac tccgtacatg agtacggaag taaggttacg ctatccaatc caaattcaaa 60

agaggagaaa ttaactggta cc 82

<210> 5

<211> 261

<212> PRT

<213> amyloid TasA (Artificial Sequence)

<400> 5

Met Gly Met Lys Lys Lys Leu Ser Leu Gly Val Ala Ser Ala Ala Leu

1 5 10 15

Gly Leu Ala Leu Val Gly Gly Gly Thr Trp Ala Ala Phe Asn Asp Ile

20 25 30

Lys Ser Lys Asp Ala Thr Phe Ala Ser Gly Thr Leu Asp Leu Ser Ala

35 40 45

Lys Glu Asn Ser Ala Ser Val Asn Leu Ser Asn Leu Lys Pro Gly Asp

50 55 60

Lys Leu Thr Lys Asp Phe Gln Phe Glu Asn Asn Gly Ser Leu Ala Ile

65 70 75 80

Lys Glu Val Leu Met Ala Leu Asn Tyr Gly Asp Phe Lys Ala Asn Gly

85 90 95

Gly Ser Asn Thr Ser Pro Glu Asp Phe Leu Ser Gln Phe Glu Val Thr

100 105 110

Leu Leu Thr Val Gly Lys Glu Gly Gly Asn Gly Tyr Pro Lys Asn Ile

115 120 125

Ile Leu Asp Asp Ala Asn Leu Lys Asp Leu Tyr Leu Met Ser Ala Lys

130 135 140

Asn Asp Ala Ala Ala Ala Glu Lys Ile Lys Lys Gln Ile Asp Pro Lys

145 150 155 160

Phe Leu Asn Ala Ser Gly Lys Val Asn Val Ala Thr Ile Asp Gly Lys

165 170 175

Thr Ala Pro Glu Tyr Asp Gly Val Pro Lys Thr Pro Thr Asp Phe Asp

180 185 190

Gln Val Gln Met Glu Ile Gln Phe Lys Asp Asp Lys Thr Lys Asp Glu

195 200 205

Lys Gly Leu Met Val Gln Asn Lys Tyr Gln Gly Asn Ser Ile Lys Leu

210 215 220

Gln Phe Ser Phe Glu Ala Thr Gln Trp Asn Gly Leu Thr Ile Lys Lys

225 230 235 240

Asp His Thr Asp Lys Asp Gly Tyr Val Lys Glu Asn Glu Lys Ala His

245 250 255

Ser Glu Asp Lys Asn

260

<210> 6

<211> 239

<212> PRT

<213> fluorescent protein mCherry (Artificial Sequence)

<400> 6

Val Ser Lys Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe Met

1 5 10 15

Arg Phe Lys Val His Met Glu Gly Ser Val Asn Gly His Glu Phe Glu

20 25 30

Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala

35 40 45

Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile

50 55 60

Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His Pro

65 70 75 80

Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe Lys

85 90 95

Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val Thr

100 105 110

Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys Leu

115 120 125

Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys Thr

130 135 140

Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly Ala

145 150 155 160

Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly His

165 170 175

Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val Gln

180 185 190

Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr Ser His

195 200 205

Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly Arg

210 215 220

His Ser Thr Gly Gly Met Asp Glu Leu Tyr Lys Ala Gly Thr Ser

225 230 235

<210> 7

<211> 542

<212> DNA

<213> plasmid pHT01 (Artificial Sequence)

<400> 7

gagctcttaa tcgctcggat gcgccgttgt gcctcttgta tcgcacacgc aatcgcttaa 60

accttgcaga atgccgcatg actgcgccgg tcttgcacca gagcatgctt cgcgcagttc 120

cactaaatga tgtttcagtt ccagcagcgc gccaatgcgg ctttcaactt ggcggatgtg 180

ttcatccagc agcatattca cctcgccgca atcttgatcc gggcgtttgc gatagctcag 240

cagcgttctc acatcgctca gcggcatatc aaggctgcgg caatggcgga taaactgtaa 300

acgttccacg tgttcttcgc catataaacg aaagttgcct ctgcttctac cgggcggcgg 360

cagaaggcct tcttgttcgt aaaagcggat cgtcaccacc gggcatgctg tgcgtttcgc 420

cagttcgcca atctggatgt tcatggcgtc ggatgggaga tgtcttgact ctatagtaac 480

tagagggtgt taaatcggca acgcgagatg aatacacaca aggggttgcc ggtaccggat 540

cc 542

<210> 8

<211> 8501

<212> DNA

<213> integrative plasmid Pbr01-TMCH (Artificial Sequence)

<400> 8

ttaagttatt ggtatgactg gttttaagcg caaaaaaagt tgctttttcg tacctattaa 60

tgtatcgttt tagaaaaccg actgtaaaaa gtacagtcgg cattatctca tattataaaa 120

gccagtcatt aggcctatct gacaattcct gaatagagtt cataaacaat cctgcatgat 180

aaccatcaca aacagaatga tgtacctgta aagatagcgg taaatatatt gaattacctt 240

tattaatgaa ttttcctgct gtaataatgg gtagaaggta attactatta ttattgatat 300

ttaagttaaa cccagtaaat gaagtccatg gaataataga aagagaaaaa gcattttcag 360

gtataggtgt tttgggaaac aatttccccg aaccattata tttctctaca tcagaaaggt 420

ataaatcata aaactctttg aagtcattct ttacaggagt ccaaatacca gagaatgttt 480

tagatacacc atcaaaaatt gtataaagtg gctctaactt atcccaataa cctaactctc 540

cgtcgctatt gtaaccagtt ctaaaagctg tatttgagtt tatcaccctt gtcactaaga 600

aaataaatgc agggtaaaat ttatatcctt cttgttttat gtttcggtat aaaacactaa 660

tatcaatttc tgtggttata ctaaaagtcg tttgttggtt caaataatga ttaaatatct 720

cttttctctt ccaattgtct aaatcaattt tattaaagtt catttgatat gcctcctaaa 780

tttttatcta aagtgaattt aggaggctta cttgtctgct ttcttcatta gaatcaatcc 840

ttttttaaaa gtcaatatta ctgtaacata aatatatatt ttaaaaatat cccactttat 900

ccaattttcg tttgttgaac taatgggtgc tttagttgaa gaataaaaga ccacattaaa 960

aaatgtggtc ttttgtgttt ttttaaagga tttgagcgta gcgaaaaatc cttttctttc 1020

ttatcttgat aataagggta actattgccg atcgtccatt ccgacagcat cgccagtcac 1080

tatggcgtgc tgctagcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc 1140

ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt 1200

aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt 1260

cgagctctta atcgctcgga tgcgccgttg tgcctcttgt atcgcacacg caatcgctta 1320

aaccttgcag aatgccgcat gactgcgccg gtcttgcacc agagcatgct tcgcgcagtt 1380

ccactaaatg atgtttcagt tccagcagcg cgccaatgcg gctttcaact tggcggatgt 1440

gttcatccag cagcatattc acctcgccgc aatcttgatc cgggcgtttg cgatagctca 1500

gcagcgttct cacatcgctc agcggcatat caaggctgcg gcaatggcgg ataaactgta 1560

aacgttccac gtgttcttcg ccatataaac gaaagttgcc tctgcttcta ccgggcggcg 1620

gcagaaggcc ttcttgttcg taaaagcgga tcgtcaccac cgggcatgct gtgcgtttcg 1680

ccagttcgcc aatctggatg ttcatggcgt cggatgggag atgtcttgac tctatagtaa 1740

ctagagggtg ttaaatcggc aacgcgagat gaatacacac aaggggttgc cggtaccgga 1800

tccatgggta tgaaaaagaa attgagttta ggagttgctt ctgcagcact aggattagct 1860

ttagttggag gaggaacatg ggcagcattt aacgacatta aatcaaagga tgctactttt 1920

gcatcaggta cgcttgattt atctgctaaa gagaattcag cgagtgtgaa cttatcaaat 1980

ctaaagccgg gagataagtt gacaaaggat ttccaatttg aaaataacgg atcacttgcg 2040

atcaaagaag ttctaatggc gcttaattat ggagatttta aagcaaacgg cggcagcaat 2100

acatctccag aagatttcct cagccagttt gaagtgacat tgttgacagt tggaaaagag 2160

ggcggcaatg gctacccgaa aaacattatt ttagatgatg cgaaccttaa agacttgtat 2220

ttgatgtctg ctaaaaatga tgcagcggct gctgaaaaaa tcaaaaaaca aattgaccct 2280

aaattcttaa atgcaagcgg taaagtcaat gtagcaacaa ttgatggtaa aaccgctcct 2340

gaatatgatg gtgttccaaa aacaccaact gacttcgatc aggttcaaat ggaaatccaa 2400

ttcaaggatg ataaaacaaa agatgaaaaa gggcttatgg ttcaaaataa atatcaaggc 2460

aactccatta agcttcaatt ctcattcgaa gctacacagt ggaacggctt gacaatcaaa 2520

aaggaccata ctgataaaga tggttacgtg aaagaaaatg aaaaagcgca tagcgaggat 2580

aaaaatgccg gcggcggcgg aggatcagga ggaggaggct caggatccgt gagcaagggc 2640

gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca catggagggc 2700

tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc 2760

acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc 2820

ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc 2880

gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag 2940

gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac 3000

aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacg 3060

atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag 3120

atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt caagaccacc 3180

tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat caagttggac 3240

atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc cgagggccgc 3300

cactccaccg gcggcatgga cgagctgtac aaggcgggta ctagtcacca tcatcaccat 3360

catcattaac ccggggcagc ccgcctaatg agcgggcttt tttcacgtca cgcgtccatg 3420

gagatctttg tctgcaactg aaaagtttat accttacctg gaacaaatgg ttgaaacata 3480

cgaggctaat atcggcttat taggaatagt ccctgtacta ataaaatcag gtggatcagt 3540

tgatcagtat attttggacg aagctcggaa agaatttgga gatgacttgc ttaattccac 3600

aattaaatta agggaaagaa taaagcgatt tgatgttcaa ggaatcacgg aagaagatac 3660

tcatgataaa gaagctctaa aactattcaa taaccttaca atggaattga tcgaaagggt 3720

ggaaggttaa tggtacgaaa attaggggat ctacctagaa agccacaagg cgataggtca 3780

agcttaaaga acccttacat ggatcttaca gattctgaaa gtaaagaaac aacagaggtt 3840

aaacaaacag aaccaaaaag aaaaaaagca ttgttgaaaa caatgaaagt tgatgtttca 3900

atccataata agattaaatc gctgcacgaa attctggcag catccgaagg gaattcatat 3960

tacttagagg atactattga gagagctatt gataagatgg ttgagacatt acctgagagc 4020

caaaaaactt tttatgaata tgaattaaaa aaaagaacca acaaaggctg agacagactc 4080

caaacgagtc tgttttttta aaaaaaatat taggagcatt gaatatatat tagagaatta 4140

agaaagacat gggaataaaa atattttaaa tccagtaaaa atatgataag attatttcag 4200

aatatgaaga actctgtttg tttttgatga aaaaacaaac aaaaaaaatc cacctaacgg 4260

aatctcaatt taactaacag cggccaaact gagaagttaa atttgagaag gggaaaaggc 4320

ggatttatac ttgtatttaa ctatctccat tttaacattt tattaaaccc catacaagtg 4380

aaaatcctct tttacactgt tcctttaggt gatcgcggag ggacattatg agtgaagtaa 4440

acctaaaagg aaatacagat gaattagtgt attatcgaca gcaaaccact ggaaataaaa 4500

tcgccaggaa gagaatcaaa aaagggaaag aagaagttta ttatgttgct gaaacggaag 4560

agaagatatg gacagaagag caaataaaaa acttttcttt agacaaattt ggtacgcata 4620

taccttacat agaaggtcat tatacaatct taaataatta cttctttgat ttttggggct 4680

attttttagg tgctgaagga attgcgctct atgctcacct aactcgttat gcatacggca 4740

gcaaagactt ttgctttcct agtctacaaa caatcgctaa aaaaatggac aagactcctg 4800

ttacagttag aggctacttg aaactgcttg aaaggtacgg ttttatttgg aaggtaaacg 4860

tccgtaataa aaccaaggat aacacagagg aatccccgat ttttaagatt agacgtaagg 4920

ttcctttgct ttcagaagaa cttttaaatg gaaaccctaa tattgaaatt ccagatgacg 4980

aggaagcaca tgtaaagaag gctttaaaaa aggaaaaaga gggtcttcca aaggttttga 5040

aaaaagagca cgatgaattt gttaaaaaaa tgatggatga gtcagaaaca attaatattc 5100

cagaggcctt acaatatgac acaatgtatg aagatatact cagtaaagga gaaattcgaa 5160

aagaaatcaa aaaacaaata cctaatccta caacatcttt tgagagtata tcaatgacaa 5220

ctgaagagga aaaagtcgac agtactttaa aaagcgaaat gcaaaatcgt gtctctaagc 5280

cttcttttga tacctggttt aaaaacacta agatcaaaat tgaaaataaa aattgtttat 5340

tacttgtacc gagtgaattt gcatttgaat ggattaagaa aagatattta gaaacaatta 5400

aaacagtcct tgaagaagct ggatatgttt tcgaaaaaat cgaactaaga aaagtgcaat 5460

aaactgctga agtatttcag cagttttttt tatttagaaa tagtgaaaaa aatataatca 5520

gggaggtatc aatatttaat gagtactgat ttaaatttat ttagactgga attaataatt 5580

aacacgtaga ctaattaaaa tttaatgagg gataaagagg atacaaaaat attaatttca 5640

atccctatta aattttaaca agggggggat taaaatttaa ttagaggttt atccacaaga 5700

aaagacccta ataaaatttt tactagggtt ataacactga ttaatttctt aatgggggag 5760

ggattaaaat ttaatgacaa agaaaacaat cttttaagaa aagcttttaa aagataataa 5820

taaaaagagc tttgcgatta agcaaaactc tttacttttt cattgacatt atcaaattca 5880

tcgatttcaa attgttgttg tatcataaag ttaattctgt tttgcacaac cttttcagga 5940

atataaaaca catctgaggc ttgttttata aactcagggt cgctaaagtc aatgtaacgt 6000

agcatatgat atggtatagc ttccacccaa gttagccttt ctgcttcttc tgaatgtttt 6060

tcatatactt ccatgggtat ctctaaatga ttttcctcat gtagcaaggt atgagcaaaa 6120

agtttatgga attgatagtt cctctctttt tcttcaactt ttttatctaa aacaaacact 6180

ttaacatctg agtcaatgta agcataagat gtttttccag tcataatttc aatcccaaat 6240

cttttagaca gaaattctgg acgtaaatct tttggtgaaa gaattttttt atgtagcaat 6300

atatccgata cagcaccttc taaaagcgtt ggtgaatagg gcattttacc tatctcctct 6360

cattttgtgg aataaaaata gtcatattcg tccatctacc tatcctatta tcgaacagtt 6420

gaacttttta atcaaggatc agtccttttt ttcattattc ttaaactgtg ctcttaactt 6480

taacaactcg atttgttttt ccagatctcg agggtaacta gcctcgccga tcccgcaaga 6540

ggcccggcag tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 6600

tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca 6660

ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt 6720

ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga 6780

tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa 6840

gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct 6900

gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat 6960

acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga 7020

tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc 7080

caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat 7140

gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa 7200

cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac 7260

tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa 7320

agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc 7380

tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc 7440

ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag 7500

acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta 7560

ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa 7620

gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc 7680

gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat 7740

ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga 7800

gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt 7860

ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata 7920

cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac 7980

cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg 8040

ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg 8100

tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag 8160

cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct 8220

ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc 8280

aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt 8340

ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg 8400

tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga 8460

gtcagtgagc gaggaagcgg aagagcgccc aatacgcatg c 8501

<210> 9

<211> 8488

<212> DNA

<213> integrative plasmid Mer01-TMCH (Artificial Sequence)

<400> 9

ttaagttatt ggtatgactg gttttaagcg caaaaaaagt tgctttttcg tacctattaa 60

tgtatcgttt tagaaaaccg actgtaaaaa gtacagtcgg cattatctca tattataaaa 120

gccagtcatt aggcctatct gacaattcct gaatagagtt cataaacaat cctgcatgat 180

aaccatcaca aacagaatga tgtacctgta aagatagcgg taaatatatt gaattacctt 240

tattaatgaa ttttcctgct gtaataatgg gtagaaggta attactatta ttattgatat 300

ttaagttaaa cccagtaaat gaagtccatg gaataataga aagagaaaaa gcattttcag 360

gtataggtgt tttgggaaac aatttccccg aaccattata tttctctaca tcagaaaggt 420

ataaatcata aaactctttg aagtcattct ttacaggagt ccaaatacca gagaatgttt 480

tagatacacc atcaaaaatt gtataaagtg gctctaactt atcccaataa cctaactctc 540

cgtcgctatt gtaaccagtt ctaaaagctg tatttgagtt tatcaccctt gtcactaaga 600

aaataaatgc agggtaaaat ttatatcctt cttgttttat gtttcggtat aaaacactaa 660

tatcaatttc tgtggttata ctaaaagtcg tttgttggtt caaataatga ttaaatatct 720

cttttctctt ccaattgtct aaatcaattt tattaaagtt catttgatat gcctcctaaa 780

tttttatcta aagtgaattt aggaggctta cttgtctgct ttcttcatta gaatcaatcc 840

ttttttaaaa gtcaatatta ctgtaacata aatatatatt ttaaaaatat cccactttat 900

ccaattttcg tttgttgaac taatgggtgc tttagttgaa gaataaaaga ccacattaaa 960

aaatgtggtc ttttgtgttt ttttaaagga tttgagcgta gcgaaaaatc cttttctttc 1020

ttatcttgat aataagggta actattgccg atcgtccatt ccgacagcat cgccagtcac 1080

tatggcgtgc tgctagcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc 1140

ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt 1200

aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt 1260

cgagctctta cggcatcgcg cttcttgcta aaccagcttc accttgtaaa gatgcaatca 1320

gcgggcagct cacgttgcct ttgcgcgcat ggcatgcgca caccagttcg ctcagcaccg 1380

tttccatgcg cgccagatct gccattttct cgcgcacgtc tttcagttta tgttccgcta 1440

aagaagaagc ttcttcgcaa tgtgtgccat catctaaacg cagcagttcc gcgatttcat 1500

ctaaagaaaa gcctaaacgc tgcgcgcttt tcacaaattt cacgcgcacc acgtccgctt 1560

cgccatatct gcggatgctt ccatacggtt tatccggttc gcgcagtaaa cctttgcgct 1620

ggtaaaagcg gatcgtttcc acgttcacgc ccgccgcttt cgcaaacacg ccaatcgtta 1680

aattctctaa attgttttcc atatcgcttg actccgtaca tgagtacgga agtaaggtta 1740

cgctatccaa tccaaattca aaagaggaga aattaactgg taccggatcc atgggtatga 1800

aaaagaaatt gagtttagga gttgcttctg cagcactagg attagcttta gttggaggag 1860

gaacatgggc agcatttaac gacattaaat caaaggatgc tacttttgca tcaggtacgc 1920

ttgatttatc tgctaaagag aattcagcga gtgtgaactt atcaaatcta aagccgggag 1980

ataagttgac aaaggatttc caatttgaaa ataacggatc acttgcgatc aaagaagttc 2040

taatggcgct taattatgga gattttaaag caaacggcgg cagcaataca tctccagaag 2100

atttcctcag ccagtttgaa gtgacattgt tgacagttgg aaaagagggc ggcaatggct 2160

acccgaaaaa cattatttta gatgatgcga accttaaaga cttgtatttg atgtctgcta 2220

aaaatgatgc agcggctgct gaaaaaatca aaaaacaaat tgaccctaaa ttcttaaatg 2280

caagcggtaa agtcaatgta gcaacaattg atggtaaaac cgctcctgaa tatgatggtg 2340

ttccaaaaac accaactgac ttcgatcagg ttcaaatgga aatccaattc aaggatgata 2400

aaacaaaaga tgaaaaaggg cttatggttc aaaataaata tcaaggcaac tccattaagc 2460

ttcaattctc attcgaagct acacagtgga acggcttgac aatcaaaaag gaccatactg 2520

ataaagatgg ttacgtgaaa gaaaatgaaa aagcgcatag cgaggataaa aatgccggcg 2580

gcggcggagg atcaggagga ggaggctcag gatccgtgag caagggcgag gaggataaca 2640

tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc 2700

acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca 2760

agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg tcccctcagt 2820

tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc 2880

tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg 2940

tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc 3000

gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagacgatg ggctgggagg 3060

cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc 3120

tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac aaggccaaga 3180

agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc acctcccaca 3240

acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac tccaccggcg 3300

gcatggacga gctgtacaag gcgggtacta gtcaccatca tcaccatcat cattaacccg 3360

gggcagcccg cctaatgagc gggctttttt cacgtcacgc gtccatggag atctttgtct 3420

gcaactgaaa agtttatacc ttacctggaa caaatggttg aaacatacga ggctaatatc 3480

ggcttattag gaatagtccc tgtactaata aaatcaggtg gatcagttga tcagtatatt 3540

ttggacgaag ctcggaaaga atttggagat gacttgctta attccacaat taaattaagg 3600

gaaagaataa agcgatttga tgttcaagga atcacggaag aagatactca tgataaagaa 3660

gctctaaaac tattcaataa ccttacaatg gaattgatcg aaagggtgga aggttaatgg 3720

tacgaaaatt aggggatcta cctagaaagc cacaaggcga taggtcaagc ttaaagaacc 3780

cttacatgga tcttacagat tctgaaagta aagaaacaac agaggttaaa caaacagaac 3840

caaaaagaaa aaaagcattg ttgaaaacaa tgaaagttga tgtttcaatc cataataaga 3900

ttaaatcgct gcacgaaatt ctggcagcat ccgaagggaa ttcatattac ttagaggata 3960

ctattgagag agctattgat aagatggttg agacattacc tgagagccaa aaaacttttt 4020

atgaatatga attaaaaaaa agaaccaaca aaggctgaga cagactccaa acgagtctgt 4080

ttttttaaaa aaaatattag gagcattgaa tatatattag agaattaaga aagacatggg 4140

aataaaaata ttttaaatcc agtaaaaata tgataagatt atttcagaat atgaagaact 4200

ctgtttgttt ttgatgaaaa aacaaacaaa aaaaatccac ctaacggaat ctcaatttaa 4260

ctaacagcgg ccaaactgag aagttaaatt tgagaagggg aaaaggcgga tttatacttg 4320

tatttaacta tctccatttt aacattttat taaaccccat acaagtgaaa atcctctttt 4380

acactgttcc tttaggtgat cgcggaggga cattatgagt gaagtaaacc taaaaggaaa 4440

tacagatgaa ttagtgtatt atcgacagca aaccactgga aataaaatcg ccaggaagag 4500

aatcaaaaaa gggaaagaag aagtttatta tgttgctgaa acggaagaga agatatggac 4560

agaagagcaa ataaaaaact tttctttaga caaatttggt acgcatatac cttacataga 4620

aggtcattat acaatcttaa ataattactt ctttgatttt tggggctatt ttttaggtgc 4680

tgaaggaatt gcgctctatg ctcacctaac tcgttatgca tacggcagca aagacttttg 4740

ctttcctagt ctacaaacaa tcgctaaaaa aatggacaag actcctgtta cagttagagg 4800

ctacttgaaa ctgcttgaaa ggtacggttt tatttggaag gtaaacgtcc gtaataaaac 4860

caaggataac acagaggaat ccccgatttt taagattaga cgtaaggttc ctttgctttc 4920

agaagaactt ttaaatggaa accctaatat tgaaattcca gatgacgagg aagcacatgt 4980

aaagaaggct ttaaaaaagg aaaaagaggg tcttccaaag gttttgaaaa aagagcacga 5040

tgaatttgtt aaaaaaatga tggatgagtc agaaacaatt aatattccag aggccttaca 5100

atatgacaca atgtatgaag atatactcag taaaggagaa attcgaaaag aaatcaaaaa 5160

acaaatacct aatcctacaa catcttttga gagtatatca atgacaactg aagaggaaaa 5220

agtcgacagt actttaaaaa gcgaaatgca aaatcgtgtc tctaagcctt cttttgatac 5280

ctggtttaaa aacactaaga tcaaaattga aaataaaaat tgtttattac ttgtaccgag 5340

tgaatttgca tttgaatgga ttaagaaaag atatttagaa acaattaaaa cagtccttga 5400

agaagctgga tatgttttcg aaaaaatcga actaagaaaa gtgcaataaa ctgctgaagt 5460

atttcagcag ttttttttat ttagaaatag tgaaaaaaat ataatcaggg aggtatcaat 5520

atttaatgag tactgattta aatttattta gactggaatt aataattaac acgtagacta 5580

attaaaattt aatgagggat aaagaggata caaaaatatt aatttcaatc cctattaaat 5640

tttaacaagg gggggattaa aatttaatta gaggtttatc cacaagaaaa gaccctaata 5700

aaatttttac tagggttata acactgatta atttcttaat gggggaggga ttaaaattta 5760

atgacaaaga aaacaatctt ttaagaaaag cttttaaaag ataataataa aaagagcttt 5820

gcgattaagc aaaactcttt actttttcat tgacattatc aaattcatcg atttcaaatt 5880

gttgttgtat cataaagtta attctgtttt gcacaacctt ttcaggaata taaaacacat 5940

ctgaggcttg ttttataaac tcagggtcgc taaagtcaat gtaacgtagc atatgatatg 6000

gtatagcttc cacccaagtt agcctttctg cttcttctga atgtttttca tatacttcca 6060

tgggtatctc taaatgattt tcctcatgta gcaaggtatg agcaaaaagt ttatggaatt 6120

gatagttcct ctctttttct tcaacttttt tatctaaaac aaacacttta acatctgagt 6180

caatgtaagc ataagatgtt tttccagtca taatttcaat cccaaatctt ttagacagaa 6240

attctggacg taaatctttt ggtgaaagaa tttttttatg tagcaatata tccgatacag 6300

caccttctaa aagcgttggt gaatagggca ttttacctat ctcctctcat tttgtggaat 6360

aaaaatagtc atattcgtcc atctacctat cctattatcg aacagttgaa ctttttaatc 6420

aaggatcagt cctttttttc attattctta aactgtgctc ttaactttaa caactcgatt 6480

tgtttttcca gatctcgagg gtaactagcc tcgccgatcc cgcaagaggc ccggcagtca 6540

ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat 6600

tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa 6660

aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt 6720

tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag 6780

ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt 6840

tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg 6900

gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag 6960

aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta 7020

agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg 7080

acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta 7140

actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac 7200

accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt 7260

actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca 7320

cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag 7380

cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta 7440

gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag 7500

ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt 7560

tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat 7620

aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta 7680

gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa 7740

acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt 7800

tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag 7860

ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta 7920

atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca 7980

agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag 8040

cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa 8100

agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga 8160

acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc 8220

gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc 8280

ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt 8340

gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt 8400

gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag 8460

gaagcggaag agcgcccaat acgcatgc 8488

Claims (4)

1. The engineering bacteria mixture comprises a first engineering bacteria and a second engineering bacteria, and is characterized in that the first engineering bacteria comprises Pb ²⁺ Induction plasmidThe Pb ²⁺ The induction plasmid comprises a lead ion induction unit and a first envelope fiber expression unit; the second engineering bacteria comprise Hg ²⁺ Sensing plasmid, hg ²⁺ The sensing plasmid comprises a mercury ion sensing unit and a second envelope fiber expression unit, and the engineering bacteria are selected from bacillus subtilis;

the first envelope fiber expression unit and the second envelope fiber expression unit comprise an amyloid expression frame, and the amino acid sequence of the amyloid comprises a sequence shown as SEQ ID No. 5;

the lead ion sensing unit comprises a regulator, wherein the polynucleotide sequence of the regulator comprises a sequence shown as SEQ ID No.1, and the lead ion sensing unit further comprises a first promoter, and the polynucleotide sequence of the first promoter comprises a sequence shown as SEQ ID No. 2;

the mercury ion sensing unit comprises a inhibitor, wherein the polynucleotide sequence of the inhibitor comprises a sequence shown as SEQ ID No.3, and the mercury ion sensing unit further comprises a second promoter, and the polynucleotide sequence of the second promoter comprises a sequence shown as SEQ ID No. 4;

the amyloid expression frame also comprises a signal protein and a tag protein, wherein the amyloid is positioned at the N end of the expression frame, the tag protein is positioned at the C end of the expression frame, the signal protein is positioned between the amyloid and the tag protein, the signal protein is selected from mCherry, and the amino acid sequence of the signal protein comprises a sequence shown as SEQ ID No. 6; the tag protein is a Hitag.

2. A kit for sensing and adsorbing heavy metal contaminants, the kit comprising a mixture of engineered bacteria of claim 1.

3. Use of the mixture of engineering bacteria according to claim 1 as sensing and adsorbing agent in bioremediation of heavy metal pollution.

4. A method of treating a metal contaminant by sensing and adsorbing the contaminant, comprising the steps of:

providing a liquid containing heavy metal ions, wherein the heavy metal ions are lead ions or mercury ions;

adding the mixture of engineering bacteria of claim 1 to the liquid.

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