CN114410559B - Heavy metal-passivated geobacillus engineering strain and construction method thereof - Google Patents

Abstract

The invention discloses a heavy metal-passivated geobacillus engineering strain and a construction method thereof, wherein the heavy metal-passivated geobacillus engineering strain is Geobacter sulfurreducensHMP-1 and is preserved in the Guangdong province microorganism strain preservation center, and the preservation number is as follows: GDMCC No.62189. The heavy metal passivated geobacillus engineering strain pilus surface is marked with cysteine, and the cysteine in the pilus is combined with heavy metal ions to quickly form metal sulfide precipitate, so that the heavy metal removal rate reaches more than 70% under the condition of not attaching any substrate or highly enriching heavy metal plants, and the heavy metal passivated geobacillus engineering strain pilus can be effectively used for improving Cd in the environment ²⁺ The residual problem is solved, and the defects of unstable heavy metal repairing effect, more limited conditions in the repairing process and the like of the existing microorganism are overcome.

Description

Heavy metal-passivated geobacillus engineering strain and construction method thereof

Technical Field

The invention belongs to the field of bioremediation of heavy metals, and particularly relates to an geobacillus engineering strain for passivating heavy metals and a construction method thereof.

Background

The investigation shows that the heavy metal pollutants are the main pollutants in the global soil at present, the soil environment condition directly determines the yield and quality of agricultural products, and part of the heavy metal pollutants can directly harm the human health even through the biological enrichment effect, so that the treatment of the soil metal pollutants cannot be cared for. In the big background of the increasing development of the sustainable agriculture at present, the search for efficient, stable and environment-friendly heavy metal pollution restoration technology is also an important point and a difficult point for guaranteeing the safety of agricultural products.

At present, the microorganism passivation technology becomes a restoration means of heavy metal pollution which is preferentially considered in various fields in recent years due to the characteristics of low restoration cost, remarkable effect, simple operation, no influence on physical and chemical properties of soil and the like. Some microorganisms can deactivate heavy metals through reactions such as biological adsorption, precipitation, transformation, complexation, alkalization and the like, and in recent years, a plurality of microbial agents aiming at heavy metal restoration have been developed, but in general, the technology is not mature, and the selectable strains are single and have unsatisfactory effects.

Heavy metal sulphide treatment has long been an effective means of removing and extracting valuable heavy metals. The Chinese patent with application publication number CN 107287129A discloses a sulfate reducing bacterium capable of passivating heavy metals, which is separated from a paddy field and is preserved in China center for type culture collection, wherein the preservation number is: CCTCC NO: M2015243, the strain name is sulfate reducing bacteria SRB-L. The principle of the strain for repairing heavy metals is as follows: s produced by reduction of sulfate ^2- And heavy metal sulfide precipitate is formed with heavy metal to achieve the effect of passivating the heavy metal. However, the effectiveness of the action of such microorganisms is affected by the substrate (sulfate) and heavy metal contamination cannot be remediated when the available sulfate concentration in the environment is low or absent.

The Chinese patent with application publication number CN 110076193A discloses a Pseudomonas tender strain for heavy metal pollution remediation, which is preserved in the China general microbiological culture Collection center, with the preservation number: 15613. the strain belongs to plant growth promoting bacteria, and the heavy metal repairing mechanism is as follows: the growth of the heavy metal-enriched plants is promoted by generating a plant growth agent, and the method belongs to the process of indirectly repairing the heavy metals. The restoration capability of the microorganism to heavy metals depends on restoration strength of plants highly enriched with heavy metals, so that restoration effect is unstable and application is greatly limited.

Therefore, the heavy metal-inactivating microorganisms disclosed in the related art have not yet had a strain which is highly efficient, stable and directly acts with heavy metals to reduce the toxicity of the heavy metals.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the heavy metal-passivated Geobacillus engineering strain Geobacter sulfurreducensHMP-1 constructed by the method can overcome the defects of unstable effect of repairing heavy metal by the existing microorganism, more limited conditions in the repairing process and the like, achieves the aim of reducing heavy metal toxicity by converting heavy metal ions into heavy metal sulfide precipitate, and has extremely high application value.

In a first aspect of the invention, an engineering strain of heavy metal-passivated geobacillus is provided.

The heavy metal passivated geobacillus engineering strain is thiobacillus HMP-1 (Geobacter sulfurreducens HMP-1), is classified and named Geobacter sulfurreducens, and is preserved in the Guangdong province microorganism strain collection, and the preservation number is: GDMCC No.62189.

According to a first aspect of the invention, in some embodiments of the invention, the heavy metal inactivated geobacillus engineering strain comprises cysteine on pili.

In the invention, the inventor realizes the technical effect of marking cysteine on the surface of the pili of the geobacillus thioreductase by means of genetic engineering, and realizes the technical purpose by combining the cysteine in the pili with heavy metal ions. The sulfhydryl (-HS) of cysteine in the pili can directly form metal sulfide sediment with heavy metal ions, so that the heavy metal removal rate reaches more than 70% under the condition of not attaching any substrate or enriching heavy metal plants. Meanwhile, the gene modification ensures that the heavy metal tolerance capacity is high, and the Cd with the concentration of 20mg/L can be effectively tolerated ²⁺ 。

In a second aspect of the invention, a construction method of the heavy metal passivated geobacillus engineering strain is provided.

The construction method of the heavy metal passivated geobacillus engineering strain comprises the following steps:

(1) Inserting a cysteine gene into the empty vector pUC19 to obtain a recombinant plasmid pUC-HMP;

(2) The recombinant plasmid pUC-HMP is transformed into competent Geobacter sulfurreducens to obtain the recombinant plasmid.

According to a second aspect of the present invention, in some embodiments of the present invention, the recombinant plasmid pUC-HMP further comprises a marker gene, a promoter gene and a sequence upstream and downstream of the target insertion site.

The sequence upstream and downstream of the target insertion site is used for achieving gene insertion at a specific location and for subsequent amplification detection.

In some preferred embodiments of the invention, the marker genes include a selection gene and a reporter gene.

In some more preferred embodiments of the invention, the marker gene is a selection gene.

In some embodiments of the invention, the selection gene is a gentamicin resistance gene.

Of course, the person skilled in the art can reasonably select other marker genes to replace gentamicin resistance genes according to actual use requirements, so as to achieve the screening purpose.

According to a second aspect of the invention, in some embodiments of the invention, the promoter is a PompJ strong promoter gene.

Of course, the person skilled in the art can reasonably select other promoter genes to replace the PompJ strong promoter gene according to the actual use requirement, so as to achieve the same purpose.

According to a second aspect of the invention, in some embodiments of the invention, the Geobacter sulfurreducens is Geobacter sulfurreducens PCA.

In some preferred embodiments of the present invention, the nucleotide sequence of the recombinant plasmid pUC-HMP is shown in SEQ ID NO. 1, and the plasmid map is shown in FIG. 1.

In some preferred embodiments of the present invention, the construction method of the heavy metal passivated geobacillus engineering strain specifically comprises: the empty vector pUC19 is taken as a vector plasmid, and is mixed with 5 XIn-Fusion HD Enzyme Premix, cysteine gene, gentamicin resistance gene, pompJ strong promoter gene and DNA sequences (the length of the upstream and downstream DNA sequences is 500 bp) at the upstream and downstream insertion sites of a target, and then the mixture is reacted for 15min at 50 ℃ In a PCR instrument, so that the recombinant plasmid pUC-HMP is obtained. pUC-HMP was linearized using the restriction endonuclease ScaI. Linearized pUC-HMP was transformed into Geobacter sulfurreducensPCA competent cells. And (3) screening by using an NBAF solid plate containing 50g/mL gentamicin.

In a third aspect, the invention provides an application of the heavy metal passivated geobacillus engineering strain in heavy metal passivation.

According to a third aspect of the invention, in some embodiments of the invention, the heavy metal is Cd ²⁺ 。

In the invention, the inventor obtains the heavy metal passivated geobacillus engineering strain by means of genetic engineering, cysteine is marked on the surface of pili of the heavy metal passivated geobacillus engineering strain, and the cysteine in pili is combined with heavy metal ions to quickly form metal sulfide precipitate, so that the heavy metal removal rate reaches more than 70% under the condition of not attaching any substrate or highly enriching heavy metal plants. Meanwhile, the gene modification ensures that the heavy metal tolerance capacity is high, and the Cd with the concentration of 20mg/L can be effectively tolerated ²⁺ 。

In a fourth aspect, the invention provides an application of the heavy metal passivated geobacillus engineering strain in environmental treatment.

In the invention, the inventor obtains the heavy metal passivated geobacillus engineering strain by means of genetic engineering, cysteine is marked on the surface of pili of the heavy metal passivated geobacillus engineering strain, and the cysteine in pili is combined with heavy metal ions to quickly form metal sulfide precipitate, so that the heavy metal removal rate reaches more than 70% under the condition of not attaching any substrate or highly enriching heavy metal plants. Meanwhile, the gene modification ensures that the heavy metal tolerance capacity is high, and the Cd with the concentration of 20mg/L can be effectively tolerated ²⁺ Thereby being effectively used for improving Cd in the environment ²⁺ Residual problems.

The beneficial effects of the invention are as follows:

the inventor obtains a heavy metal passivated geobacillus engineering strain Geobacter sulfurreducens HMP-1 by means of genetic engineering, cysteine is marked on the surface of pili of the heavy metal passivated geobacillus engineering strain, and the heavy metal passivated geobacillus engineering strain is combined with heavy metal ions through the cysteine in pili to quickly form metal sulfide sediment, so that the heavy metal removal rate reaches more than 70 percent under the condition of not attaching any substrate or highly enriching heavy metal plants. At the same time, the gene is modified to make itObtain stronger heavy metal tolerance capability and can effectively tolerate 20mg/L Cd ²⁺ Thereby being effectively used for improving Cd in the environment ²⁺ The residual problem is solved, and the defects of unstable heavy metal repairing effect, more limited conditions in the repairing process and the like of the existing microorganism are overcome.

Drawings

FIG. 1 is a plasmid map of a recombinant plasmid pUC-HMP in an example of the present invention.

FIG. 2 is a gel electrophoresis diagram of amplification Geobacter sulfurreducens HMP-1 using validation primers verF1/R1 and verF2/R2, wherein DNA ladder is used as marker.

FIG. 3 shows the effective state Cd of Geobacter sulfurreducens HMP-1 at different concentrations ²⁺ Growth curve under conditions.

FIG. 4 is a fluorescent plot of Geobacter sulfurreducens HMP-1 with active Cd forming CdS semiconductor minerals.

FIG. 5 is a scanning electron microscope image of Geobacter sulfurreducensHMP-1 combined with active Cd

FIG. 6 is a fluorescent plot of the formation of CdS semiconductor minerals from Geobacter sulfurreducens HMP-1 cell membrane solution and active Cd.

Detailed Description

In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to the following specific embodiments. It should be understood that the detailed description is presented herein for purposes of illustration only and is not intended to limit the invention.

The experimental materials and reagents used, unless otherwise specified, are those conventionally available commercially.

Construction method of heavy metal-passivated geobacillus engineering strain

The specific construction steps are as follows:

(1) Construction of pUC-HMP:

the recombinant plasmid pUC-HMP was obtained by mixing pUC19 (TaKaRa In Japan) as a vector plasmid with 5 XIn-Fusion HD Enzyme Premix (TaKaRa In Japan), a cysteine gene, a gentamicin resistance gene, a PompJ strong promoter gene and DNA sequences upstream and downstream of the target insertion site (both of which were 500bp In length) and then reacting at 50℃for 15 minutes In a PCR instrument.

Wherein, the cysteine gene, the gentamicin resistance gene, the PompJ strong promoter gene and the DNA sequence at the upstream and downstream of the target insertion site are synthesized by biological companies.

The nucleotide sequence of the constructed recombinant plasmid pUC-HMP is as follows:

wherein the underlined part is a cysteine gene.

The plasmid map of the recombinant plasmid pUC-HMP is shown in FIG. 1.

(2) pUC-HMP was linearized using restriction enzyme ScaI (New England BioLabs, MA, USA) and 10-15. Mu.g of the linearized pUC-HMP was then concentrated to 10. Mu.L.

The plasmid concentration method comprises the following steps: 1) 15 μg of the linearized plasmid is taken in a 1.5mL centrifuge tube, 10 μl of 3M sodium acetate (pH=5.2) is added, and the mixture is mixed by shaking; 2) Adding 250 μL of glacial ethanol (precooled at 4 ℃), shaking, mixing, standing at-20deg.C for >30min, and centrifuging at 15000g for 30min at 4deg.C; 3) Discarding the supernatant, adding 1mL 70% glacial ethanol (pre-cooling at 4deg.C), centrifuging at 15000g at 4deg.C for 30min; 4) Discarding the supernatant, adding 500 μl of glacial ethanol (pre-cooling at 4deg.C), centrifuging at 15000g at 4deg.C for 30min; 5) The supernatant was discarded, left at room temperature for 10min, ethanol was evaporated, 10. Mu.L of sterile water was added to dissolve the plasmid, and the concentration was determined and deemed optimal when the concentration was greater than 1. Mu.g/. Mu.L.

(3) 1-2. Mu.L of concentrated linearized pUC-HMP was transformed into 40. Mu. LGeobacter sulfurreducensPCA (purchased from American type culture Collection, strain ATCC-51573) competent cells by means of a transient electric shock method by means of an electrotransfer apparatus (Micropulser, bio-Rad, USA) and placed in NBAF liquid medium for 16-18h (in this step, 1% final concentration of yeast was added in advance to the NBAF medium). After 18h of cultivation, the bacterial liquid was concentrated to 2mL in an anaerobic tank. 200. Mu.L of the mixture was plated on NBAF solid plates containing 50g/mL gentamicin, and cultured anaerobically. After the pink single colony grows out of the flat plate, a single colony is selected, verified, and the geobacillus engineering strain Geobacter sulfurreducensHMP-1 is obtained after verification;

the conversion parameters of the instantaneous electric shock method are as follows: the shock parameter was 1.47Kv/cm for 5ms.

The NBAF media formulations for the strains cultured in this example are shown in Table 1.

TABLE 1 NBAF media formulation

The pH of the NBAF medium was adjusted to 6.5-7.0.

The NB salts in the NBAF medium are specifically: 25g NH/liter 100 XNB salt solution ₄ Cl，42g KH ₂ PO ₄ ，22g K ₂ HPO ₄ ，38g KCl，36g NaCl。

The NB minerals in NBAF medium were specifically: each liter of NB mineral solution contains 2.14g nitrilotriacetic acid (NTA))，0.1g MnCl ₂ ·4H ₂ O，0.3g FeSO ₄ ·7H ₂ O，0.17g CoCl ₂ ·6H ₂ O，0.2g ZnSO ₄ ·7H ₂ O，0.03g CuCl ₂ ·2H ₂ O，0.005g AlK(SO ₄ ) ₂ ·2H ₂ O，0.005g H ₃ BO ₃ ，0.09g Na ₂ MnO ₄ ·2H ₂ O，0.11g NiSO ₄ ·6H ₂ O，0.02g Na ₂ WO ₄ ·2H ₂ O。

The DL vitamins in the NBAF medium are specifically: the DL vitamin solution contains 0.005g vitamin B5,0.0001g vitamin B12,0.005g p-aminobenzoic acid, 0.005g alpha-lipoic acid, 0.005g nicotinic acid, 0.005g vitamin B1,0.005g riboflavin, 0.01g vitamin B6 and 0.002g folic acid.

The prepared NBAF medium was treated with a gas mixture (CO) ₂ ：N ₂ =80%/20%) was aerated to remove oxygen from the medium.

The solid NBAF is prepared by adding 1.5% agar into the NBAF liquid culture medium, and performing aeration treatment to remove oxygen in the culture medium.

In the examples, the strain verification was performed by PCR method, and the verification primers were verF1/R1 and verF2/R2.

Wherein, the nucleotide sequence of the verification primer is:

verF1/R1：

the upstream primer VerF1:5'-ACGAACCGAACAGGCTTATGTC-3' (SEQ ID NO: 2);

downstream primer VerR1:5'-AAACCTATCCGCCCGAAAGT-3' (SEQ ID NO: 3).

verF2/R2：

The upstream primer VerF2:5'-ATGATCAAACCTATCCGCCCGAAAG-3' (SEQ ID NO: 4);

downstream primer VerR2:5'-ATTGGGTCAGTCTCGTAGTTGTCGG-3' (SEQ ID NO: 5).

The results are shown in FIG. 2.

Using DNA ladder as a marker, geobacter sulfurreducens PCA as a control, it was found that only Geobacter sulfurreducensHMP-1 was amplified by PCR using verF1/R1 and verF2/R2, and the amplified product length was expected, indicating that Geobacter sulfurreducensHMP-1 construction was successful.

In this example, the preparation method of Geobacter sulfurreducens PCA competent cells is as follows: the cells were cultured to a logarithmic phase of Geobacter sulfurreducens PCA 4 ℃and centrifuged (4500 Xg 12 min) after precooling and collected, washed twice with 4℃precooled electrotransfer buffer (4500 Xg 12 min) by shaking the tube clockwise, finally resuspended and transferred to a 2mL anaerobic centrifuge tube with 1mL electrotransfer solution, centrifuged 8000 Xg for 5min, the supernatant was discarded, 20. Mu.L of electrotransfer solution was added to resuspend the cells, 40. Mu.L was aspirated and placed in a 2mL new anaerobic centrifuge tube, and used to transform linearized plasmids, all under anaerobic conditions.

The formula of the electrotransport buffer solution is as follows: final concentration of 175mM sucrose, 1mM MgCl ₂ And 1mM Heps (ph=7).

The constructed geobacillus engineering strain Geobacter sulfurreducensHMP-1 (classified and named Geobacter sulfurreducens) is preserved in the Guangdong province microorganism strain collection center (GDMCC for short) at the address: building 5 of Guangzhou city, first, china, no. 100, college of China, no. 59, with a preservation date of 2022, 1 month and 5 days, and a preservation number: GDMCC No.62189.

Application of Geobacter sulfurreducensHMP-1 in Cd metal passivation enrichment

To demonstrate the practical use and effect of Geobacter sulfurreducens HMP-1 prepared in the above examples, the inventors have demonstrated the following experiments.

The specific test steps are as follows:

geobacter sulfurreducens HMP-1 (inoculated at 3% by volume when OD600 is 0.6) prepared in the above example was taken and Cd was added in an amount of 10 to 30mg/L in total ²⁺ Solution (control group is Cd free) ²⁺ Geobacter sulfurreducens HMP-1) of the solution, and the change of the OD600 absorbance of each group within 0 to 90min was detected.

The results are shown in FIG. 3.

It was found that Cd was added at different concentrations ²⁺ The Geobacter sulfurreducens HMP-1 of the solution showed significant absorbance change after 20min, indicating Cd in the solution ²⁺ The concentration was changed mainly due to the promotion of Cd by cysteine on Geobacter sulfurreducens HMP-1 pili ²⁺ CdS semiconductor nano-minerals are formed, thereby reducing Cd in the solution ²⁺ Concentration. The inventors have also found that wild type Geobacter sulfurreducens was not modified in absorbance, indicating that wild type Geobacter sulfurreducens does not have Cd ²⁺ Passivation effect.

To further verify the above test results, the inventors individually determined Geobacter sulfurreducens HMP-1 to be Cd-free ²⁺ And Cd-containing ²⁺ The culture is carried out on a culture medium with the concentration of 20mg/L, and the CdS semiconductor nano-mineral has fluorescence, so that the CdS semiconductor nano-mineral and the CdS semiconductor nano-mineral are subjected to fluorescence detection under the wavelength of 300-360 nm.

The results are shown in FIG. 4.

It can be found that in the absence of Cd ²⁺ Geobacter sulfurreducens HMP-1 cultured on the medium of (C) does not fluoresce, but does not contain Cd ²⁺ The Geobacter sulfurreducens HMP-1 cultured on the medium showed significant fluorescence. And found to contain Cd ²⁺ The growth condition of Geobacter sulfurreducens HMP-1 cultured on the culture medium is good, which indicates that the culture medium has stronger tolerance to heavy metal (Cd), and the highest tolerance concentration can reach at least 20mg/L.

Further observation was performed on Geobacter sulfurreducens HMP-1 in the medium using a Scanning Electron Microscope (SEM).

As a result, geobacter sulfurreducens HMP-1 has a rich Cd surface ²⁺ Particles (FIG. 5), demonstrating that Geobacter sulfurreducens HMP-1 is effective in binding Cd ²⁺ Is enriched on the surface of the pilus.

For further verification Geobacter sulfurreducens HMP-1 for Cd ²⁺ The inventors carried out cell membrane separation treatment on Geobacter sulfurreducens HMP-1, and specific detection steps are as follows: after Geobacter sulfurreducens HMP-1 was cultured in NBAF liquid medium (100 mL) to the logarithmic phase,centrifugation was performed at 8000g for 10 minutes at 4℃and the supernatant was discarded, and resuspended in 10mL of 0.9% sterile physiological saline. Cells were completely disrupted using a cell disruptor (JY 92-IIDN, ningbo Xinzhi Biotechnology Co., ltd.). Then centrifuged again at 10000g for 10 minutes at 4℃and the supernatant collected to remove cell debris. Transferring the supernatant to an ultracentrifuge, centrifuging at 4 ℃ for 1 hour with 200000g, discarding the supernatant, and adding 2mL of 0.9% sterile physiological saline for resuspension to obtain a purified cell membrane fraction. Directly mixing the purified Geobacter sulfurreducens HMP-1 cell membrane with 2mL of 40mg/L Cd ²⁺ The solutions were mixed and fluorescence was detected at a wavelength of 300-360 nm.

The results are shown in FIG. 6.

The Geobacter sulfurreducens HMP-1 cell membrane obtained by purification is directly combined with 2mL of 40mg/L Cd ²⁺ The solution mixed produces strong fluorescence, indicating Geobacter sulfurreducens HMP-1 is a method for binding Cd through the cell membrane ²⁺ Is converted into CdS semiconductor nano-mineral, thereby realizing Cd ²⁺ Passivation and enrichment of (c).

In conclusion, geobacter sulfurreducens HMP-1 constructed in the above examples has a strong tolerance to heavy metals (Cd) and is capable of binding Cd via its surface pili ²⁺ Is converted into CdS semiconductor nano-mineral, thereby realizing Cd ²⁺ Is not limited. And Cd is ²⁺ The particles are mainly concentrated on the surface of Geobacter sulfurreducens HMP-1, so that the concentration of heavy metal pollutants is facilitated.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Fujian university of agriculture and forestry

<120> an engineering strain of geobacillus for passivating heavy metals and construction method thereof

<130>

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 5768

<212> DNA

<213> artificial sequence

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aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta 3720

atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc 3780

gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 3840

ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa 3900

aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 3960

ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 4020

aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 4080

gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc 4140

tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg 4200

tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga 4260

gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag 4320

cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta 4380

cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag 4440

agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg 4500

caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac 4560

ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc 4620

aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag 4680

tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc 4740

agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac 4800

gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc 4860

accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg 4920

tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag 4980

tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc 5040

acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac 5100

atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag 5160

aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac 5220

tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg 5280

agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc 5340

gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact 5400

ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg 5460

atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa 5520

tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt 5580

tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg 5640

tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga 5700

cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc 5760

ctttcgtc 5768

<210> 2

<211> 22

<212> DNA

<213> artificial sequence

<400> 2

acgaaccgaa caggcttatg tc 22

<210> 3

<211> 20

<212> DNA

<213> artificial sequence

<400> 3

aaacctatcc gcccgaaagt 20

<210> 4

<211> 25

<212> DNA

<213> artificial sequence

<400> 4

atgatcaaac ctatccgccc gaaag 25

<210> 5

<211> 25

<212> DNA

<213> artificial sequence

<400> 5

attgggtcag tctcgtagtt gtcgg 25

Claims (9)

1. The heavy metal-passivated geobacillus engineering strain is characterized by being sulfur-reduced geobacillus HMP-1 (Geobacter sulfurreducens HMP-1), is classified and named Geobacter sulfurreducens and is deposited in the Guangdong province microorganism strain collection center, and the collection number is: GDMCC No.62189.

2. The heavy metal-passivated geobacillus engineering strain according to claim 1, wherein the heavy metal-passivated geobacillus engineering strain has cysteine on pili.

3. The construction method of the heavy metal passivated geobacillus engineering strain of claim 1 or 2, comprising the following steps:

(1) Inserting a cysteine gene into the empty vector pUC19 to obtain a recombinant plasmid pUC-HMP;

(2) Transforming the recombinant plasmid pUC-HMP into competent Geobacter sulfurreducens to obtain;

the nucleotide sequence of the recombinant plasmid pUC-HMP is shown as SEQ ID NO: 1.

4. The method according to claim 3, wherein the recombinant plasmid pUC-HMP further comprises a marker gene, a promoter gene and a sequence upstream and downstream of the target insertion site.

5. The method according to claim 4, wherein the marker gene is a selection gene and the selection gene is a gentamicin resistance gene.

6. The method according to claim 4, wherein the promoter is a PompJ strong promoter gene.

7. A method of construction according to claim 3, wherein Geobacter sulfurreducens is Geobacter sulfurreducens PCA.

8. The use of the heavy metal passivated geobacillus engineering strain according to claim 1 or 2 in heavy metal passivation.

9. Use of the heavy metal passivated geobacillus engineering strain according to claim 1 or 2 in environmental management.