CN114410559A - Geobacillus engineering strain for passivating heavy metals and construction method thereof - Google Patents
Geobacillus engineering strain for passivating heavy metals and construction method thereof Download PDFInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
Abstract
The invention discloses a heavy metal-passivating Geobactirium engineering strain and a construction method thereof, wherein the heavy metal-passivating Geobactirium engineering strain is Geobactirium sulfureatecrensen HMP-1, is preserved in Guangdong province microorganism strain preservation center, and has a preservation number of: GDMCC NO. 62189. Cysteine is marked on the surface of the pilus of the heavy metal-inactivated Bacillus engineering strain, and the cysteine in the pilus is combined with heavy metal ions to quickly form metal sulfide precipitate, so that the pilus is independent of the heavy metal ionsThe heavy metal removal rate reaches more than 70 percent under the condition of attaching any substrate or highly enriching heavy metal plants, thereby effectively improving Cd in the environment2+The problem of residue is solved, and the defects that the existing microorganism has unstable heavy metal repairing effect and many limited conditions exist in the repairing process are overcome.
Description
Technical Field
The invention belongs to the field of bioremediation of heavy metals, and particularly relates to a geobacillus engineering strain for passivating heavy metals and a construction method thereof.
Background
According to survey, the heavy metal pollutants are the main pollutants in the soil all over the world at present, the soil environment condition directly determines the yield and the quality of agricultural products, and part of the heavy metal pollutants can even directly harm human health through the biological enrichment effect, so that the treatment of the soil metal pollutants cannot be careless. Under the current big background that sustainable agriculture development is increasing day by day, finding a high-efficiency, stable and environment-friendly heavy metal pollution remediation technology is also a key and difficult point for ensuring the safety of agricultural products.
At present, the microbial passivation technology is a heavy metal pollution remediation means which is considered preferentially in various fields in recent years due to the characteristics of low remediation cost, remarkable effect, simplicity in operation, no influence on soil physicochemical properties and the like. Some microorganisms can passivate heavy metals through reactions such as bioadsorption, precipitation, transformation, complexation and alkalization, and in recent years, a plurality of microbial agents for heavy metal remediation are produced, but in general, the technology is not mature, and the selected strains are single and the effect is not satisfactory.
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 bacteria capable of passivating heavy metals, which is separated from a paddy field and is preserved in China center for type culture Collection with the preservation numbers: CCTCC NO: M2015243, and 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 sulphate2-Forming heavy metal sulfide precipitate with heavy metal to achieve the effect of passivating heavy metal. However, the efficacy of such microorganisms is affected by the substrate (sulfate) and cannot remediate heavy metal contamination when the concentration of sulfate available in the environment is low or absent.
The application publication number is CN 110076193A, and discloses a Pseudomonas parfains strain for heavy metal pollution remediation, which is preserved in the China general microbiological culture Collection center (CGMCC), with the preservation number: 15613. the strain belongs to plant growth promoting bacteria, and the mechanism for repairing heavy metal is as follows: the growth of the plant with high enrichment of heavy metal is promoted by generating the plant growth agent, belonging to the process of indirectly repairing heavy metal. The repair capability of the microorganisms to heavy metals depends on the repair strength of plants with high enrichment of heavy metals, so that the repair effect is unstable and the application is greatly limited.
Therefore, the heavy metal-inactivating microorganisms disclosed in the related art do not have a strain that is highly efficient, stable, and directly acts on heavy metals to reduce the toxicity of heavy metals.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides the Geobacter sulfuriducens engineering strain for passivating the heavy metal and the construction method thereof, the Geobacter sulfuriducens HMP-1 constructed and obtained in the invention can overcome the defects of unstable heavy metal repairing effect, more limited conditions in the repairing process and the like of the existing microorganisms, the aim of reducing the toxicity of the heavy metal is realized by converting heavy metal ions into heavy metal sulfide precipitates, and the application value is extremely high.
In a first aspect of the invention, a heavy metal-inactivated Geobacillus engineering strain is provided.
The heavy metal passivation geobacillus engineering strain is Geobacter sulfurreducens HMP-1(Geobacter sulfurreducens HMP-1), is classified and named as Geobacter sulfurreducens, is preserved in Guangdong province microbial strain preservation center, and has a preservation number: GDMCC NO. 62189.
According to the first aspect of the present invention, in some embodiments of the present invention, the trichome of the engineered strain of geobacter with heavy metal inactivation contains cysteine.
In the invention, the technical effect of marking cysteine on the surface of pilus of thioredoxin is realized by means of genetic engineering, and the technical purpose is realized by combining the cysteine in the pilus with heavy metal ions. Specifically, the sulfhydryl (-HS) of cysteine in the pilus can directly and rapidly form metal sulfide precipitate with heavy metal ions, so that the pilus does not need any substrate or any substrateThe removal rate of heavy metals reaches more than 70 percent under the condition of highly enriching the heavy metal plants. Meanwhile, the gene modification enables the heavy metal to obtain strong heavy metal tolerance capability and can effectively tolerate 20mg/L Cd2+。
In a second aspect of the invention, a method for constructing a heavy metal-inactivated Geobacillus engineered strain according to the first aspect of the invention is provided.
The construction method of the heavy metal passivation geobacillus engineering strain comprises the following steps:
(1) inserting the cysteine gene into an empty vector pUC19 to obtain a recombinant plasmid pUC-HMP;
(2) and (3) converting the recombinant plasmid pUC-HMP into competent Geobacter sulfurfuglucens to obtain the recombinant plasmid pUC-HMP.
According to the 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 upstream and downstream sequences of the target insertion site.
The target insertion site upstream and downstream sequences are used for realizing gene insertion of specific positions and subsequent amplification detection.
In some preferred embodiments of the invention, the marker gene comprises 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, those skilled in the art can reasonably select other marker genes to replace the gentamicin resistance gene according to the actual use requirements so as to achieve the purpose of screening.
According to a second aspect of the invention, in some embodiments of the invention, the promoter gene is a PompJ strong promoter gene.
Of course, those skilled in the art can reasonably select other promoter genes to replace the PompJ strong promoter gene according to the actual use requirement to achieve the same purpose.
According to a second aspect of the invention, in some embodiments of the invention, the Geobacter sulfuridurens is a Geobacter sulfuridurens 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-inactivated geobacillus engineering strain specifically comprises: mixing an empty vector pUC19 serving as a vector plasmid with 5 XIn-Fusion HD Enzyme Premix, a cysteine gene, a gentamicin resistance gene, a PompJ strong promoter gene and upstream and downstream DNA sequences (the lengths of the upstream and downstream DNA sequences are all 500bp) of a target insertion site, and reacting for 15min at 50 ℃ In a PCR instrument to obtain a recombinant plasmid pUC-HMP. pUC-HMP was linearized using the restriction enzyme ScaI. The linearized pUC-HMP was transformed into competent cells of Geobacter sulfureatencA. And (3) screening by using an NBAF solid plate containing 50g/mL gentamicin.
In a third aspect of the invention, the application of the heavy metal-inactivated Geobacillus engineered strain of the first aspect of the invention in heavy metal inactivation is provided.
According to a third aspect of the invention, in some embodiments of the invention, the heavy metal is Cd2+。
In the invention, the inventor obtains a heavy metal-inactivated Geobacillus engineering strain by means of genetic engineering, wherein cysteine is marked on the surface of the pilus of the heavy metal-inactivated Geobacillus engineering strain, 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 of the heavy metal-inactivated Geobacillus engineering strain can reach more than 70% under the condition of not attaching any substrate or highly enriching heavy metal plants. Meanwhile, the gene modification enables the heavy metal to obtain strong heavy metal tolerance capability and can effectively tolerate 20mg/L Cd2+。
In a fourth aspect of the invention, the application of the heavy metal-inactivated Geobacillus sp.engineering strain in environmental management is provided.
In the present invention, the inventors have obtained by means of genetic engineeringThe surface of the heavy metal-inactivated Geobacillus engineering strain pilus 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 of the heavy metal-inactivated Geobacillus engineering strain can reach more than 70% under the condition of not attaching any substrate or highly enriching heavy metal plants. Meanwhile, the gene modification enables the heavy metal to obtain strong heavy metal tolerance capability and can effectively tolerate 20mg/L Cd2+Thereby being effectively used for improving Cd in the environment2+And (4) residual problems.
The invention has the beneficial effects that:
the inventor obtains a heavy metal-passivated Geobacter sulfuriduens engineering strain HMP-1 by means of genetic engineering, cysteine is marked on the surface of the pilus of the heavy metal-passivated Geobacter engineering strain, 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 of the heavy metal-passivated Geobacter engineering strain reaches more than 70% under the condition of not attaching any substrate or highly enriching heavy metal plants. Meanwhile, the gene modification enables the heavy metal to obtain strong heavy metal tolerance capability and can effectively tolerate 20mg/L Cd2+Thereby being effectively used for improving Cd in the environment2+The problem of residue is solved, and the defects that the existing microorganism has unstable heavy metal repairing effect and many limited conditions exist in the repairing process are overcome.
Drawings
FIG. 1 is a plasmid map of a recombinant plasmid pUC-HMP in the example of the present invention.
FIG. 2 is a gel electrophoresis image of Geobacter sulfuriduens HMP-1 amplified using verification primers verF1/R1 and verF2/R2, wherein DNA ladder is used as a marker.
FIG. 3 shows effective Cd of Geobacter sulfuriducens HMP-1 in different concentrations2+Growth curve under conditions.
FIG. 4 is a fluorescence diagram of a CdS semiconductor mineral formed by Geobacter sulfurglucens HMP-1 and effective state Cd.
FIG. 5 is a scanning electron micrograph of Geobacter sulfureatenseHMP-1 combined with available Cd
FIG. 6 is a fluorescence diagram of a CdS semiconductor mineral formed by a Geobacter sulfurducens HMP-1 cell membrane solution and an effective state 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 specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
The experimental materials and reagents used are, unless otherwise specified, all consumables and reagents which are conventionally available from commercial sources.
Construction method of geobacillus engineering strain for passivating heavy metals
The specific construction steps are as follows:
(1) construction of pUC-HMP:
an empty vector pUC19 (TaKaRa, Japan) is used as a vector plasmid, and is mixed with 5 XIn-Fusion HD Enzyme Premix (TaKaRa, Japan), a cysteine gene, a gentamicin resistance gene, a PompJ strong promoter gene and upstream and downstream DNA sequences (the lengths of the upstream and downstream DNA sequences are all 500bp) of a target insertion site, and then the mixture is reacted for 15min at 50 ℃ In a PCR instrument, so that a recombinant plasmid pUC-HMP is obtained.
Wherein, the cysteine gene, the gentamicin resistance gene, the PompJ strong promoter gene and the DNA sequences at the upper and lower reaches of the target insertion site have the synthesis of biological companies.
The nucleotide sequence of the constructed recombinant plasmid pUC-HMP is as follows:
among them, the cysteine gene is underlined.
The plasmid map of the recombinant plasmid pUC-HMP is shown in FIG. 1.
(2) pUC-HMP was linearized using the restriction enzyme ScaI (New England BioLabs, MA, USA), and 10-15. mu.g of the linearized pUC-HMP was concentrated to 10. mu.L.
The plasmid concentration method comprises the following steps: 1) 15 mu g of the linearized plasmid is put into a 1.5mL centrifuge tube, 10 mu L of 3M sodium acetate (pH 5.2) is added, and the mixture is shaken and mixed evenly; 2) adding 250 μ L of glacial ethanol (precooling at 4 deg.C), shaking, mixing, standing at-20 deg.C for 30min, and centrifuging at 4 deg.C for 30min at 15000 g; 3) discarding the supernatant, adding 1mL 70% glacial ethanol (precooling at 4 ℃), and centrifuging at 15000g for 30min at 4 ℃; 4) discarding the supernatant, adding 500 μ L of glacial ethanol (precooling at 4 deg.C), and centrifuging at 15000g for 30min at 4 deg.C; 5) discard the supernatant, leave at room temperature for 10min to ensure the ethanol is completely volatilized, add 10. mu.L of sterile water to dissolve the plasmid, and determine the concentration, wherein the concentration is considered to be optimal when the concentration is more than 1. mu.g/. mu.L.
(3) 1-2. mu.L of concentrated linearized pUC-HMP was transformed into competent cells of 40. mu.L of LGeobacter surfurtucens PCA (purchased from American type culture Collection, Strain No. ATCC-51573) by an electrokinetic converter (MicroPulser, Bio-Rad, USA) by transient electric shock, and cultured in NBAF liquid medium for 16-18h (in this step, yeast was added to the NBAF medium in advance to a final concentration of 1%). After 18h of incubation, the broth was concentrated to 2mL in an anaerobic chamber. 200. mu.L of the suspension was spread on a NBAF solid plate containing 50g/mL gentamicin, and cultured anaerobically. Picking out a single colony after the plate grows pink single colony, verifying the single colony, and obtaining a Geobacter sulfureateducens HMP-1 without error verification;
wherein, the conversion parameters of the instantaneous electric shock method are as follows: the shock parameter was 1.47Kv/cm and the time was 5 ms.
The NBAF medium formulation involved in the culture of the strain in this example is shown in Table 1.
TABLE 1 NBAF Medium formulation
The pH of NBAF medium was adjusted to 6.5-7.0.
The NB salt in the NBAF culture medium is specifically: the 100 XNB salt solution contained 25g NH per liter4Cl，42g KH2PO4，22g K2HPO4，38g KCl，36g NaCl。
The NB minerals in NBAF medium are specifically: each liter of NB mineral solution contained 2.14g nitrilotriacetic acid (NTA), 0.1g MnCl2·4H2O，0.3g FeSO4·7H2O，0.17g CoCl2·6H2O，0.2g ZnSO4·7H2O，0.03g CuCl2·2H2O，0.005g AlK(SO4)2·2H2O，0.005g H3BO3，0.09g Na2MnO4·2H2O，0.11g NiSO4·6H2O，0.02g Na2WO4·2H2O。
The DL vitamins in NBAF culture medium are specifically: each liter of 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 culture medium utilizes mixed gas (CO)2：N 280%/20%) was aerated to remove oxygen from the medium.
The solid NBAF is prepared by adding 1.5% of agar into the NBAF liquid culture medium, and simultaneously carrying out aeration treatment to remove oxygen in the culture medium.
In the examples, the strain was verified by PCR using primers verF1/R1 and verF 2/R2.
Wherein the nucleotide sequence of the verification primer is as follows:
verF1/R1：
the upstream primer VerF 1: 5'-ACGAACCGAACAGGCTTATGTC-3' (SEQ ID NO: 2);
the downstream primer VerR 1: 5'-AAACCTATCCGCCCGAAAGT-3' (SEQ ID NO: 3).
verF2/R2：
The upstream primer VerF 2: 5'-ATGATCAAACCTATCCGCCCGAAAG-3' (SEQ ID NO: 4);
the downstream primer VerR 2: 5'-ATTGGGTCAGTCTCGTAGTTGTCGG-3' (SEQ ID NO: 5).
The results are shown in FIG. 2.
By using the DNA ladder as a marker and the Geobacter sulfurducens PCA as a control, the fact that only the Geobacter sulfurducens HMP-1 generates an amplification product after PCR amplification is carried out by using verF1/R1 and verF2/R2, and the length of the amplification product is in accordance with the expectation can be found, which indicates that the construction of the Geobacter sulfurducens HMP-1 is successful.
In this embodiment, the preparation method of the Geobacter sulfureateducens PCA competent cell comprises the following steps: geobacter sulfureatecens cultured to logarithmic phase is precooled at 4 ℃ and then centrifuged (4500 Xg 12min) to collect thalli, the centrifugal tube is shaken clockwise by 4 ℃ precooled electrotransfer buffer solution to wash the thalli twice (4500 Xg 12min), finally 1mL of electrotransfer solution is used for resuspending the thalli and transferring the thalli to a 2mL anaerobic centrifugal tube, 8000 Xg is centrifuged for 5min, 20 mu L of electrotransfer solution is added to resuspend the thalli after supernatant is discarded, 40 mu L of thalli is absorbed and placed in a 2mL new anaerobic centrifugal tube and is used for converting linearized plasmids, and the operations are all carried out under anaerobic conditions.
The formulation of the electrotransfer buffer solution is as follows: the final concentration was 175mM sucrose, 1mM MgCl2And 1mM Heps (pH 7).
The constructed geobacillus engineering strain Geobacter sulfureatenseHMP-1 (classified and named Geobacter sulfureatenses) is stored in Guangdong province microbial culture collection center (GDMCC for short), and the address is as follows: the preservation date of No. 59 building 5 of No. 100 college of the Piezo-Lizhou city, 1 month and 5 days of 2022, the preservation number is: GDMCC NO. 62189.
Application of Geobacter sulfureatenseHMP-1 in Cd metal passivation and enrichment
In order to demonstrate the practical use and effects of the Geobacter sulfuriduens HMP-1 prepared in the above examples, the inventors verified and illustrated by the following experiments.
The specific test steps are as follows:
taking the Geobacter sulfureates HMP-1 prepared in the embodiment (when the OD600 is 0.6, inoculating is carried out according to the volume ratio of 3 percent), and respectively adding Cd with the total content of 10-30 mg/L2+Solution (control group without Cd addition)2+And (3) detecting the change condition of the OD600 absorbance of each group within 0-90 min respectively by using Geobacter sulfureaters HMP-1 of the solution.
The results are shown in FIG. 3.
It can be found that different concentrations of Cd were added2+The obvious absorbance change of the Geobacter sulfureatedchmp-1 in the solution appears after 20min, which shows that the Cd in the solution2+The concentration of Cd in the bacterial suspension is changed mainly due to cysteine on the pilus of Geobacter sulfureatedeus HMP-12+CdS semiconductor nano-minerals are formed, thereby reducing Cd in the solution2+And (4) concentration. The inventor also uses wild Geobacter sulfurducens as a control, but does not find the phenomenon of generating absorbance change, which indicates that the wild Geobacter sulfurducens does not contain Cd2+The effect of passivation.
To further verify the above test results, the inventors separately prepared Geobacter sulfuriducens HMP-1 without Cd2+And Cd content2+The CdS semiconductor nano-mineral is cultured on a culture medium (with the concentration of 20mg/L), and the CdS semiconductor nano-mineral have fluorescence, so that the CdS semiconductor nano-mineral and the CdS semiconductor nano-mineral are subjected to fluorescence detection at the wavelength of 300-360 nm.
The results are shown in FIG. 4.
It can be found that in the absence of Cd2+The cultured Geobacter sulfuriduens HMP-1 does not produce fluorescence, but contains Cd2+The Geobacter sulfuriduens HMP-1 cultured on the medium shows obvious fluorescence. And found in the presence of Cd2+The growth condition of the Geobacter sulfuridunens HMP-1 cultured on the culture medium is good, which shows that the Geobacter sulfuridunens HMP-1 has stronger heavy metal (Cd) tolerance, and the highest tolerance concentration can reach at least 20 mg/L.
The culture medium was further observed for Geobacter sulfuriduccuns HMP-1 by Scanning Electron Microscopy (SEM).
The result shows that the surface of Geobacter sulfureatenduns HMP-1 is rich in Cd2+The particles (figure 5) prove that the Geobacter sulfureatenseHMP-1 can effectively convert Cd2+Is enriched on the surface of pilus.
To further verify that Geobacter sulfureates HMP-1 is used for Cd2+The enrichment position of the Geobacter sulfureatenseHMP-1 is subjected to cell membrane separation treatment by the inventor, and the specific detection steps are as follows: after Geobacter sulfurglucens HMP-1 was cultured in NBAF liquid medium (100mL) to logarithmic phase, it was centrifuged at 4 ℃ at 8000g for 10 minutes, and the supernatant was discarded and resuspended in 10mL of 0.9% sterile physiological saline. The cells were completely disrupted using a cell disrupter (JY92-IIDN, Ningbo Xinzhi Biotech Co., Ltd.). Then, the mixture was centrifuged again at 10000g for 10 minutes at 4 ℃ and the supernatant was collected to remove cell debris. Transferring the supernatant to an ultracentrifuge, centrifuging at 4 deg.C and 200000g for 1 hr, discarding the supernatant, and adding 2mL of 0.9% sterile physiological saline for resuspension to obtain purified cell membrane fraction. Directly mixing the purified Geobacter sulfureates HMP-1 cell membrane with 2mL of 40mg/L Cd2+The solutions were mixed and fluorescence was detected at 300-360nm wavelength.
The results are shown in FIG. 6.
The purified Geobacter sulfureates HMP-1 cell membrane is directly mixed with 2mL of 40mg/L Cd2+The solution generates strong fluorescence after being mixed, which indicates that the Geobacter sulfurducens HMP-1 leads Cd to pass through the cell membrane2+Converted into CdS semiconductor nano-mineral, thereby realizing Cd2+Passivation and enrichment of.
In summary, the Geobacter sulfureatescens HMP-1 constructed in the above embodiments has strong heavy metal (Cd) tolerance, and Cd can be adsorbed by surface pilus thereof2+Converted into CdS semiconductor nano-mineral, thereby realizing Cd2+Passivation of (3). And Cd2+The particles are mainly concentrated on the surface of Geobacter sulfureaters HMP-1, so that the heavy metal pollutants can be enriched conveniently.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> Fujian agriculture and forestry university
<120> a ground bacterium engineering strain for passivating heavy metals and a construction method thereof
<130>
<160> 5
<170> PatentIn version 3.5
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<211> 5768
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ttaaggatta aacggataat tggccaatta cccccatacc ccaacacaag cagcaaaaag 2160
aagaaaggag acacttatgc ttcagaaact cagaaacagg aaaggtttca cccttatcga 2220
gctgctgatc gtcgttgcga tcatcggtat tctcgctgca attgcgattc cgcagttctc 2280
ggcgtatcgt gtcaaggcgt acaacagcgc ggcgtcaagc gacttgagaa acctgaagac 2340
tgctcttgag tccgcatttg ctgatgatca aacctatccg cccgaaagtt gttgtcgggg 2400
tgttgttaat tgattaaata catactggag gaaaccatga aaaagatcat tactatagtt 2460
gctatgttgc tcgcaatgca gggaatagcc attgccgccg ggaaaattcc tacaacaacg 2520
atgggtggca aggactttac tttcaaacct tctactaacg tgagtgtttc ctacttcact 2580
acgaacggtg caacttctac cgcgggaact gtcaataccg attatgctgt caataccaaa 2640
aactcttctg gtaaccgggt gttcacctca accaataata catctaacat ctggtacatc 2700
gaaaatgatg catggaaagg taaggcagtt tcagatagcg atgttaccgc cttgggaacc 2760
ggtgacgtag gcaagtctga tttttctggt accgagtgga agtcgcagta gtcatatcgg 2820
ataactgatt gaaaaagggg ggcatgttta tgcccccctt tttcaatcat gagcttttat 2880
gaataaaaag ataaaatcca tcaatgattt aaaccgcggt gcaacatctg gtgatgactt 2940
gttggattgg gtcagtctcg tagttgtcgg aatagagggc agacattgcg gaacgtgcca 3000
gtcagccgga ccacgagcct ccttcccctc tttaatgtat acacgttatt atttccgcag 3060
cctccatgct acaataagca acggtattca ccgacatcga gttgtcggcc tagtcggagt 3120
ggtggagcgc cccatggacg taccctcgaa aacgtggcgc ggatgcgccg ttcttagtcc 3180
cggcctttcc gcacggcgcc aggacgtgct ttccctctac ctggacgccg gctacccgac 3240
caccctgatc ctctccctgg ctacgccgga gggcggtctt gacgaccggc tcgaagcagc 3300
tctgtcgatt accttccgga acttggtgaa ggttctcccc gagctgctca acctcaagaa 3360
atctggtgac aaggacgata catatgctat tatgcacgtc cctttggacg cgattgaggt 3420
taaaagacgc tgtattgccc tggagatggg aacacctttt tcgcagctca tcgatctgga 3480
tgtgtacgac ggcagcggaa cccctctaga gtcgacctgc aggcatgcaa gcttggcgta 3540
atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat 3600
acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt 3660
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
<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 (10)
1. The heavy metal passivation geobacillus engineering strain is characterized by being Geobacter sulfurreducenseHMP-1 (Geobacter sulfurreducenseHMP-1), classified and named as Geobacter sulfurreducenses, deposited in Guangdong province microbial strain collection center, and deposited in a number of: GDMCC NO. 62189.
2. The heavy metal-inactivated engineered strain of Geobacillus according to claim 1, wherein the pilus of the heavy metal-inactivated engineered strain of Geobacillus contains cysteine.
3. The method for constructing the heavy metal-inactivated Geobacillus engineering strain of claim 1 or 2, comprising the following steps:
(1) inserting the cysteine gene into an empty vector pUC19 to obtain a recombinant plasmid pUC-HMP;
(2) and (3) converting the recombinant plasmid pUC-HMP into competent Geobacter sulfurfuglucens to obtain the recombinant plasmid pUC-HMP.
4. The method according to claim 3, wherein the recombinant plasmid pUC-HMP further comprises a marker gene, a promoter gene and upstream and downstream sequences of a target insertion site.
5. Construction process according to claim 4, characterized in that the marker genes comprise a selection gene, preferably a selection gene, more preferably a gentamicin resistance gene, and a reporter gene.
6. The method of claim 4, wherein the promoter gene is a PompJ strong promoter gene.
7. The method according to claim 3, wherein the Geobacter sulfurducens is Geobacter sulfureatensePCA.
8. The method according to claim 3 or 4, wherein the nucleotide sequence of the recombinant plasmid pUC-HMP is represented by SEQ ID NO 1.
9. The use of the engineered strain of Geobacillus heavy metal-inactivated according to claim 1 or 2 for heavy metal inactivation.
10. The use of the engineered strain of Geobacillus heavy metal-inactivated according to claim 1 or 2 in environmental management.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114410527A (en) * | 2022-01-25 | 2022-04-29 | 福建农林大学 | Broad-spectrum soil heavy metal passivation microbial inoculum and preparation method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107287129A (en) * | 2016-04-01 | 2017-10-24 | 兰州大学 | One plant can make heavy metal settle sulfate reducing bacteria and its application |
| CN111411064A (en) * | 2020-03-11 | 2020-07-14 | 福建农林大学 | Method for in-situ fluorescence labeling of geobacillus |
| WO2020191281A1 (en) * | 2019-03-20 | 2020-09-24 | University Of Massachusetts | Microbial nanowires modified to contain peptides and methods of making |
| CN114410527A (en) * | 2022-01-25 | 2022-04-29 | 福建农林大学 | Broad-spectrum soil heavy metal passivation microbial inoculum and preparation method and application thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107287129A (en) * | 2016-04-01 | 2017-10-24 | 兰州大学 | One plant can make heavy metal settle sulfate reducing bacteria and its application |
| WO2020191281A1 (en) * | 2019-03-20 | 2020-09-24 | University Of Massachusetts | Microbial nanowires modified to contain peptides and methods of making |
| CN111411064A (en) * | 2020-03-11 | 2020-07-14 | 福建农林大学 | Method for in-situ fluorescence labeling of geobacillus |
| CN114410527A (en) * | 2022-01-25 | 2022-04-29 | 福建农林大学 | Broad-spectrum soil heavy metal passivation microbial inoculum and preparation method and application thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114410527A (en) * | 2022-01-25 | 2022-04-29 | 福建农林大学 | Broad-spectrum soil heavy metal passivation microbial inoculum and preparation method and application thereof |
| CN114410527B (en) * | 2022-01-25 | 2023-09-12 | 福建农林大学 | Broad-spectrum soil heavy metal passivation microbial inoculum and preparation method and application thereof |
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