CN111454978B - Surface display engineering bacterium for specifically adsorbing heavy metal lead and construction method and application thereof - Google Patents
Surface display engineering bacterium for specifically adsorbing heavy metal lead and construction method and application thereof Download PDFInfo
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Abstract
The invention relates to a surface display engineering bacterium for specifically adsorbing heavy metal lead, a construction method and application thereof; the method is characterized in that an N-terminal sequence of the ice crystal nucleoprotein is used as an anchoring carrier, and the lead binding protein is displayed on the surface of an escherichia coli cell outer membrane through flexible connecting peptide to construct the specific lead binding protein surface display genetic engineering bacteria. Comprises PbrR surface display engineering bacteria BL21 (pR-FL), pbrR691 surface display engineering bacteria BL21 (pR 691-FL) and PbrD surface display engineering bacteria BL21 (pD-FL). The genetically engineered bacteria provided by the invention can efficiently adsorb lead ions in a water environment, and has absolute selection specificity on the lead ions under the coexistence of mixed metal ions. The surface display cell adsorbent is simple to culture, easy to obtain, short in adsorption period, safe, effective and very convenient, and provides a new technology and a new idea for removing and recovering heavy metals in the future.
Description
Technical Field
The invention relates to a surface display engineering bacterium for specifically adsorbing heavy metal lead, a construction method and application thereof; relates to a construction method of a specific lead binding protein surface display engineering bacterium, provides an application of the engineering bacterium in heavy metal adsorption by utilizing the surface display engineering bacterium for lead ion adsorption constructed by the method, and belongs to the field of molecular biology and genetic engineering bacterium modification.
Background
The rapid development of the world economy makes the pollution discharge problem in the process of industrialization progress more and more serious, and makes the heavy metal pollution problem more and more receive global attention. Lead is a highly toxic heavy metal naturally present in the earth's crust. The lead pollution causes great harm to the environment: firstly, the growth of crops in lead-containing soil may result in poor growth or a substantial yield reduction; secondly, lead is not degradable, has neurotoxicity, is a heavy metal element seriously harmful to human health, and causes damage to health when the amount in the body exceeds a certain level. Although the traditional physical and chemical method has a certain effect on the treatment of heavy metal pollution, the method has extremely high requirements on equipment, high repair cost and complex repair process, and also requires a large amount of manpower and material resources, and the method lacks specificity in the selection of heavy metals. The bioremediation has the advantages of high removal efficiency, low price, simple operation, high adsorption efficiency, small influence on the environment and the like, particularly, in recent years, the surface display technology appears, and the specific protein is fixed on a cell outer membrane by a genetic engineering means, so that the purpose that one or a certain type of heavy metal ions are combined outside cells and removed is achieved, and the bioremediation is widely applied to the field of heavy metal adsorption and removal.
The research shows that the copper-metal-tolerant bacteria CH34 (cupriavidius metallidurans CH 34) is the only bacteria which are proved to contain a special lead ion regulation mechanism at present, almost all genes related to lead ion binding are positioned on a pbr operon, and the expression of all related genes (pbrTRABCD) in the operon is regulated by a PbrR protein from MerR family. Although c. Metallidurans CH34 has specific recognition and enrichment capacity for lead ions, the bacterium is not readily available, has limited information research on its genome and proteome, and the cell has Pb 2+ The adsorption capacity of the lead ion is not high, lead ions need to cross cell membranes to enter cells to realize fixation, the damage to the cells is large, and the lead ion is not suitable for being applied in a high-concentration lead-containing environment.
The continuous progress of recombinant gene technology promotes the development of genetic engineering, and the idea of genetic engineering is used to transform strains to become a means for achieving the aim of orientation. Escherichia coli (Escherichia coli) is taken as a typical laboratory model strain, has clear metabolic pathway and no endogenous plasmid, can be used as excellent chassis cells for genetic engineering transformation to construct surface display engineered strains, and is used as a surface cell adsorbent for removing heavy metal lead. Researchers display the fusion protein of PbrR protein and LPP-OmpA on the surface of Escherichia coli cells, so that the lead ion enrichment capacity of engineering bacteria is improved compared with that of the original strain, but the adsorption capacity is very limited, most of lead specific adsorption depends on PbrR, and the protein is single in selectivity. Therefore, in the present stage, a research library of specific lead ion binding proteins needs to be expanded, more genetic engineering bacteria with specific lead binding capacity are provided, and higher capacity adsorption of lead ions in the environment is realized.
Preferably, the research selects three non-membrane proteins PbrR, pbrR691 and PbrD from the Cu-tolerant metallothionein bacteria, takes an N-terminal sequence of ice crystal nucleoprotein INP as an anchoring protein, respectively constructs three surface display engineering bacteria for specifically adsorbing heavy metal lead in E.coli BL21 (DE 3) in a form of fusion protein through flexible connecting peptide, has excellent lead ion binding capacity, avoids toxic action caused by lead ions entering cells, and solves the bottleneck problems of single lead binding protein selection, low adsorption efficiency and the like in the existing research.
Disclosure of Invention
The invention aims to provide a construction method and an application method of a specific lead binding protein surface display engineering strain, so that the surface display engineering strain constructed by the method has high-capacity adsorption and specific selectivity on lead ions. The method is characterized in that an N-terminal sequence of the ice crystal nucleoprotein is used as an anchoring carrier, and the lead binding protein is displayed on the surface of an escherichia coli cell outer membrane through flexible connecting peptide to construct the specific lead binding protein surface display genetic engineering bacteria. Application method of engineering strain in lead-containing solution, wherein engineering strain can be in single lead ion solution with different concentrations or mixed with metal ionExerts excellent adsorption effect in the sub-solution and shows good adsorption effect on Pb 2+ High adsorption capacity and good selection specificity.
The technical scheme of the invention is as follows:
the construction of the specific lead binding protein surface display engineering strain of the invention comprises the following steps:
(1) Respectively amplifying nucleotide sequences coding the anchoring protein INPN and the lead binding protein by Polymerase Chain Reaction (PCR), removing a stop codon at the tail end of the anchoring protein INPN during amplification, introducing flexible connecting peptide FL (GGGGS), and introducing a hexahistidine Tag (His-Tag) in front of the stop codon of a lead binding protein coding sequence;
(2) Fusing the gene sequence of the anchoring protein INPN and the lead binding protein in an enzyme digestion connection mode to construct a fusion protein module;
(3) Cloning the fusion protein module onto a pET-28a (+) plasmid vector; and introducing the cloned plasmid vector into a host chassis cell BL21 (DE 3), and screening a positive clone strain with a target screening plasmid through colony PCR, namely the specific lead binding protein surface display engineering strain.
Preferably, the lead binding protein used in step (1) is selected from the three non-membrane proteins PbrR, pbrR691 or PbrD of cupreovirus metallothionein CH 34;
the fusion protein modules in the step (2) are respectively PbrR fusion modules INPN-FL-PbrR, pbrR691 fusion modules INPN-FL-PbrR691 or PbrD fusion modules INPN-FL-PbrD, as shown in figure 1;
the nucleotide sequence of the encoded ice nucleoprotein N-terminal structural domain INPN in the step (1) is shown as SEQ ID No.1, and the nucleotide sequences of the lead binding proteins PbrR, pbrR691 and PbrD are respectively shown as SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 4;
the nucleotide sequence of the cloning vector pET28a (+) plasmid in the step (3) is shown as SEQ ID No.5, and the target screening plasmids are respectively: recombinant plasmid pR-FL (nucleotide sequence is shown as SEQ ID No. 6) containing PbrR fusion module, recombinant plasmid pR691-FL (nucleotide sequence is shown as SEQ ID No. 7) containing PbrR691 fusion module, and recombinant plasmid pD-FL (nucleotide sequence is shown as SEQ ID No. 8) containing PbrD fusion module. All recombinant plasmid maps are shown in FIG. 2.
The flexible connecting peptide FL consists of four glycine and one serine residue stretches (GGGGS).
Through the steps, the surface display engineering bacteria for specifically adsorbing heavy metal lead, which are constructed by the invention, comprise PbrR surface display engineering bacteria BL21 (pR-FL), and the nucleotide sequence of the contained recombinant plasmid pR-FL is SEQ ID No.6; the PbrR691 surface display engineering bacterium BL21 (pR 691-FL) contains a recombinant plasmid pR691-FL with the nucleotide sequence of SEQ ID No.7; the PbrD surface display engineering bacterium BL21 (pD-FL) contains recombinant plasmid pD-FL nucleotide sequence as SEQ ID No.8.
The invention also provides an application of the surface display engineering bacteria in a lead-containing environment, and the application method comprises the following steps:
(1) Strain activation: picking single colony of engineering strain BL21 (pR-FL), BL21 (pR 691-FL) or BL21 (pD-FL), inoculating to 3-5ml LB liquid culture medium, placing on 37 ℃ constant temperature shaking table for overnight culture to activate thallus; transferring the activated seed culture solution into a shake flask containing 100mL of non-antibiotic liquid culture medium according to the inoculation proportion of 1-3%;
(2) To-be-cultured solution OD 600 After = 0.6-0.8, respectively adding IPTG inducer with the final concentration of 0.2-0.5mM into each shake flask, and simultaneously adding heavy metal ions with the final concentration of 100-2000 mu M into each shake flask; placing the culture solution added with the inducer and the metal ions in a constant temperature shaking table at the temperature of 22-26 ℃ and the rpm of 200-220 to induce protein expression;
(3) After the bacterial liquid is cultured for 12 hours, centrifuging for 5-10min under the condition of 5000-8000rpm, collecting all thalli, washing at least three times by using sterile deionized water, placing thalli precipitates in an oven at 60-65 ℃ for continuously heating for 24 hours, fully drying the thalli and weighing the dry weight (CDW) of the thalli;
(4) And (3) measuring the metal content: nitrifying bacteria by a microwave digestion method, removing impurities, and determining the content of metal adsorbed by the engineering strain by an atomic absorption spectrometry or an inductively coupled plasma mass spectrometry;
the metal adsorption capacity (μmol/g CDW) = total metal content (μmol)/dry cell weight (g) measured.
The LB medium used for the strain culture in the invention comprises the following components (based on 1L of the medium): 10g of NaCl, 10g of tryptone and 5g of yeast powder, and adjusting the pH value of the culture medium to 7.2-7.4 after all the components are dissolved. And autoclaved at 121 ℃ for 20min.
The advantages of the invention are as follows: by utilizing a cell surface display technology, the specific lead binding protein is displayed on the cell surface of escherichia coli through INPN, three strains of specific lead binding protein surface display engineering bacteria are provided, the lead ion adsorption performance is extremely high, the specific selection of lead ions can be realized in a plurality of metal mixed solutions, and the removal of heavy metal lead in the solutions can be realized through the removal of bacteria. The method solves the problems of single selection and low adsorption efficiency of the current lead binding protein, can prevent lead ions from entering cells, relieves the toxicity of heavy metals on the growth of the cells, and has important significance on in-situ remediation of the environment polluted by the heavy metals.
The genetic engineering bacteria provided by the invention can efficiently adsorb lead ions in a water environment, and has absolute selection specificity on the lead ions under the coexistence of mixed metal ions. The surface display cell adsorbent is simple to culture, easy to obtain, short in adsorption period, safe, effective and very convenient, and provides a new technology and a new idea for removing and recovering heavy metals in the future.
Drawings
FIG. 1 is a flow chart of the building of INPN-FL-PbrR, INPN-FL-PbrR691 and INPN-FL-PbrR modules;
FIG. 2 is a map of recombinant plasmid pR-FL/pR691-FL/pD-FL
FIG. 3 shows Western blot detection results after the cell fractionation of engineering bacteria
FIG. 4 shows the adsorption effect of engineering bacteria in single lead ion solutions of different concentrations
FIG. 5 shows the adsorption effect of engineering bacteria in mixed heavy metal solutions of different concentrations
Detailed Description
The invention is further illustrated by the following examples in conjunction with the accompanying drawings:
the operations of plasmid extraction, PCR product purification, agarose gel recovery and the like related to the embodiment of the invention are all carried out according to a corresponding kit provided by Bomader biotechnology, inc.; all restriction enzymes and T4 DNA ligase used were purchased from Thermo Fisher.
Example 1 construction of lead-binding protein surface display engineering bacteria
(1) PCR amplification of the gene fragment: specific primers of the amplified fragment are designed, and high fidelity polymerase is utilized to respectively amplify the ice crystal nucleoprotein N-terminal sequence INPN (primers N-F and N-R) shown in SEQ ID No.1, the PbrR coding sequence (primers R-F and R-R) shown in SEQ ID No.2, the PbrR691 coding sequence (primers R691-F and R691-R) shown in SEQ ID No.3 and the PbrD coding sequence (primers D-F and D-R) shown in SEQ ID No. 4. Removing the termination codon of INPN, introducing flexible connecting peptide FL (GGGGS) after the termination codon, and respectively introducing enzyme cutting sites NcoI and BamHI at the upstream and downstream of the sequence; bamHI enzyme cutting sites are respectively introduced into the upstream of the coding genes of PbrR, pbrR691 and PbrD, a hexahistidine Tag (His-Tag) is introduced before a downstream termination code, and an EcoRI enzyme cutting site is introduced after a termination codon.
The primer sequences used for the amplification of the target fragment are shown below, wherein the underlined parts are the restriction enzyme sites:
N-F：CATGCCATGGGCATGACCCTGGATAAAGCACTGG；
N-R：CGGGATCCGGAACCACCACCACCGGTCTGCAGATTCTGCGGTGTGG；
R-F：CGGGATCCATGAACATTCAGATTGGTGAACTGGC
R-R：CCGGAATTCTTAATGATGATGGTGATGATGATCGCTCGGATGTGCGGTGGTGCCAC
R691-F：CGGGATCCATGATGCGTATTGGTGAACTGGG
R691-R：CCGGAATTCTTAATGATGATGGTGATGATGTGCAGGTTCTGCCAGGCTATTCAG
D-F：CGGGATCCATGATGCCGGTTTATCTGGCCGATC
D-R：CCGGAATTCTTAATGATGATGGTGATGATGACGACATGCATAGGCACCATCATTAC
the PCR system for sequence amplification is 50 μ L, which comprises ddH20 18 μ L,2 XPhanta Max Buffer 25 μ L, dNTP Mix1 μ L, phanta Max Super-Fidelity DNA polymerase 1 μ L, and upstream primer, downstream primer and template each 1 μ L;
the PCR conditions of the sequence amplification are pre-denaturation at 95 ℃, denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, and extension at 72 ℃ for 60s, and the PCR is completed by complete extension at 72 ℃ for 5min after 35 cycles.
(2) Construction of the fusion protein Module: after the PCR amplification products are verified by agarose gel electrophoresis, gel bands corresponding to INPN-FL (552 bp), pbrR (435 bp), pbrR691 (396 bp) and PbrD (723 bp) are obtained, and the target bands are recovered according to the specification of a gel recovery kit. Then, carrying out enzyme digestion on the gene fragment recovered from the gel: INPN-FL was cleaved with NcoI and BamHI, and PbrR/PbrR691/PbrD were cleaved with BamHI and EcoRI, respectively. Purifying the digestion product according to the instruction of the PCR product purification kit, determining the concentration of each DNA solution, and respectively mixing the INPN-FL gene sequence with the PbrR/PbrR691/PbrD protein coding sequence according to the ratio of 1:1 to prepare a T4 ligase ligation system. The ligation reaction procedure was: at 22 ℃ for 30min;70 ℃ for 5min. The obtained DNA product was again gel-cast and the fusion gene fragment was recovered to obtain the PbrR fusion module INPN-FL-PbrR, the PbrR691 fusion module INPN-FL-PbrR691 and the PbrD fusion module INPN-FL-PbrR, as shown in FIG. 1.
(3) The plasmid pET-28a (+) and the fusion gene fragment after purification and recovery are cut by restriction enzymes NcoI and EcoRI, and the linear plasmid and the fusion gene after purification and recovery of the PCR product are obtained according to the ratio of 1:5, preparing a T4 ligase ligation system for ligation. The ligation products were transformed into competent cells e.coli BL21 (DE 3), positive clones were screened by colony PCR, recombinant plasmids were extracted and sent to beijing jinzhi biotechnology limited for sequencing. Obtaining a lead binding protein recombinant plasmid expression vector (figure 2) without any mutation, which comprises a recombinant plasmid pR-FL containing a PbrR fusion module, a nucleotide sequence shown as SEQ ID No.6, a recombinant plasmid pR691-FL containing a PbrR691 fusion module, a nucleotide sequence shown as SEQ ID No.7, a recombinant plasmid pD-FL containing a PbrD fusion module, and a nucleotide sequence shown as SEQ ID No.8. The obtained positive clone bacterial strains are specific lead binding protein surface display engineering bacteria which are PbrR surface display engineering bacteria BL21 (pR-FL) containing recombinant plasmids pR-FL, pbrR691 surface display engineering bacteria BL21 (pR 691-FL) containing recombinant plasmids pR691-FL and PbrD surface display engineering bacteria BL21 (pD-FL) containing recombinant plasmids pD-FL.
Example 2 expression and localization of fusion proteins
(1) Strain activation: picking single colony of engineering strain BL21 (pR-FL), BL21 (pR 691-FL) or BL21 (pD-FL), inoculating to 5ml LB liquid culture medium, placing on 37 ℃ constant temperature shaking table for overnight culture to activate thallus;
(2) Transferring the activated seed culture solution into a shake flask containing 100mL of non-antibiotic liquid culture medium according to an inoculation proportion of 1%, and waiting for OD of bacterial liquid 600 = 0.6-0.8, adding IPTG to a final concentration of 0.2mM, setting the shaker temperature to 22 ℃, collecting all cell precipitates after 12h of induction, washing the cell precipitates with PBS solution for three times, and then resuspending the cell precipitates with 10mL of PBS;
(3) The cells were disrupted with a sonicator for 10 minutes (5 seconds per sonication, 5 seconds pause), and then centrifuged at 8000rpm for 10 minutes to remove incompletely disrupted cell debris. The centrifuged supernatant was collected, transferred to a dedicated centrifuge tube of a Beckmann ultracentrifuge, and centrifuged at 39000rpm for 1h, with cytoplasmic fractions present in the supernatant and membrane fractions in the pellet fraction. With a solution containing 0.01mM magnesium chloride (MgCl) 2 ) And 2% TritonX-100 in PBS buffer and added to incubate at room temperature for 30min to dissolve the membrane components sufficiently, after which it was centrifuged again at 39000rpm for 1h. The final precipitate is the outer cell membrane component of the engineering bacteria, and the supernatant is the inner cell membrane component of the engineering bacteria.
(4) The different cell components were analyzed by Western blotting.
The predicted molecular weights of PbrR, pbrR691 and PbrD are 16.4kD,15.2kD and 26.7kD, respectively. Adding INPN (19 kD), a linker peptide (G4S) and a histidine tag (His) 6 Tag, and finally the expected molecular weights of the respective fusion proteins are 36.6kD (INPN-FL-PbrR), 35.5kD (INPN-FL-PbrR 691) and 46.9kD (INPN-FL-PbrD), respectively.
As shown in FIG. 3, the results of cell fractionation revealed that the band of interest was present only in the outer membrane fraction, and that no band of the target protein was present in both the inner membrane fraction and the cytoplasm fraction, confirming the specific localization of the fusion protein on the outer membrane of the cell. We have demonstrated that we have successfully displayed the proteins of interest for lead adsorption PbrR, pbrR691 or PbrD on the outer cell membrane of E.coli.
Example 3 surface display of lead ion adsorption by genetically engineered bacteria
(1) Picking single colony of engineering strain BL21 (pR-FL), BL21 (pR 691-FL) or BL21 (pD-FL), inoculating to 3ml LB liquid culture medium, placing on 37 ℃ constant temperature shaking table for overnight culture to activate thallus; the activated seed culture solution is transferred to a shake flask containing 100mL of non-resistant liquid culture medium according to the inoculation proportion of 2%. To-be-cultured solution OD 600 After = 0.6-0.8, IPTG inducer with a final concentration of 0.5mM and Pb with different concentrations were added to each flask 2+ To final concentrations of 100, 300, 500, 1000, 1500 and 2000. Mu.M, respectively; placing the culture solution added with the inducer and the metal ions in a constant temperature shaking table at 26 ℃ and 200rpm to induce protein expression;
(2) After the bacterial liquid is cultured for 12 hours, centrifuging for 10min under the condition of 5000rpm, collecting all thalli, washing for three times by using sterile deionized water, placing thalli precipitates in a 65 ℃ oven for continuously heating for 24 hours, fully drying the thalli and weighing the dry weight (CDW) of the thalli;
(3) And (3) measuring the metal content: digesting nitrifying bacteria by microwaves, removing impurities, and measuring the content of metal adsorbed by each engineering strain by an atomic absorption spectrometry; pb of the Strain 2+ Adsorption capacity (. Mu. Mol/g CDW) = total metal content measured (. Mu. Mol)/dry cell weight (g)
The adsorption results are shown in figure 4, and the adsorption capacity of all recombinant strains on lead ions is superior to that of the wild type starting strain BL21. When the lead ion concentration is not high, the adsorption capacity of the strain is the same as the tendency of the increase in the lead concentration. However, too high a heavy metal concentration may inhibit the growth and protein expression of bacteria, thereby significantly reducing the lead ion adsorption capacity of the strain. When Pb is present in the medium 2+ At a concentration of 500. Mu.M, the maximum adsorption value of the strain BL21 (pR 691-FL) was 732.8. Mu. Mol/g CDW, which was 2.5 times that of the control group, and the maximum adsorption value of BL21 (pD-FL) was obtainedThe value was 710.1. Mu. Mol/g CDW, which is 2.42 times that of the control group. When the lead concentration is 1000. Mu.M, the maximum lead ion adsorption amount of the engineering bacteria BL21 (pR-FL) is 694.3. Mu. Mol/g CDW, which is 1.7 times the adsorption amount of the wild type strain BL21. The three strains of the lead binding protein surface display engineering bacteria constructed in the research are proved to have obvious adsorption effect on lead ions.
Example 4 surface display of Selective specific adsorption of genetically engineered bacteria to lead ions
(1) Picking single colony of engineering strain BL21 (pR-FL), BL21 (pR 691-FL) or BL21 (pD-FL), inoculating to 4ml LB liquid culture medium, placing on 37 ℃ constant temperature shaking table for overnight culture to activate thallus; the activated seed culture solution is transferred to a shake flask containing 100mL of non-resistant liquid culture medium according to the inoculation proportion of 2%. To-be-cultured solution OD 600 After = 0.6-0.8, IPTG inducer was added to each flask to a final concentration of 0.2mM and mixed metal ions containing lead ions (Pb) to final concentrations of 100, 500 and 1000. Mu.M, respectively 2+ ) Cadmium ion (Cd) 2+ ) Arsenic ion (As) 3+ ) And mercury ions (Hg) 2+ ). Placing the culture solution added with the inducer and the metal ions in a constant temperature shaking table at 22 ℃ and 210rpm to induce protein expression;
(2) After the bacterial liquid is cultured for 12h, centrifuging for 8min at 7000rpm, collecting all thalli, washing with sterile deionized water for three times, placing thalli precipitates in a 60 ℃ oven for 24h, fully drying the thalli and weighing the dry weight (CDW) of the thalli;
(3) And (3) measuring the metal content: digesting nitrifying bacteria by microwave, removing impurities, and determining the content of metal adsorbed by each engineering strain by inductively coupled plasma mass spectrometry; pb of the Strain 2+ Adsorption capacity (. Mu. Mol/g CDW) = total metal content measured (. Mu. Mol)/dry cell weight (g)
As shown in FIG. 5, the adsorption results showed that the solutions containing mixed metal ions of different concentrations contained Pb in a significant amount 2+ The adsorption of the engineering bacteria, and the existence of other metal ions, do not affect the adsorption capacity of the three target protein surface display engineering bacteria on lead, which shows that the PbrR, pbrR691 or PbrD surface display engineering bacteria constructed by the research have Pb on Pb 2+ All have higher selectionAnd (4) specificity selection. Pb of BL21 (pR-FL) pair when the concentration of mixed metal ions is 100. Mu.M 2+ Respectively is Cd 2+ And Hg 2+ 5.2 times and 13 times; the adsorption selectivity of BL21 (pR 691-FL) on lead ions is Cd 2+ And Hg 2+ 5.8 times and 16.5 times; the adsorption selectivity of BL21 (pD-FL) on lead ions is Cd 2+ And Hg 2+ 5.7 times and 12.4 times of that of the strain, but the engineering bacteria do not adsorb As 3+ The ability of the cell to perform. When the concentration of the mixed metal ions is increased to 500 mu M, the adsorption capacity of all engineering bacteria is increased; when the concentration of the mixed metal ions reaches 1000 mu M, the adsorption amount of the lead ions on the three engineering bacteria is not greatly changed, probably because the target protein reaches an adsorption saturation state. Three engineering bacteria BL21 (pR-FL), BL21 (pR 691-FL) or BL21 (pD-FL) for Pb 2+ The maximum binding capacities of the two materials are 942.1, 754.3 and 864.9 mu mol/g CDW respectively, and the two materials have higher Pb 2+ The adsorption capacity and the selection specificity are good, and the application value is good.
The results of the application tests of the four groups of examples show that the lead binding protein surface display engineering bacteria obtained by the construction method provided by the invention can anchor the target protein on the outer membrane surface of Escherichia coli cells in the form of fusion protein, and after 12h of induced expression at 22-26 ℃, no matter in the presence of single heavy metal ions Pb or in a mixed solution containing other heavy metal ions, the engineering bacteria BL21 (pR-FL), BL21 (pR 691-FL) or BL21 (pD-FL) can be used for 100-2000 mu M Pb 2+ Has higher adsorption capacity, good selection specificity and good application value, and provides a new idea and a new technology for bioremediation of heavy metal polluted environments.
While the invention has been described in further detail with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit of the invention, and such changes and modifications are to be considered within the scope of the invention.
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<110> Tianjin university
<120> surface display engineering bacterium for specifically adsorbing heavy metal lead, and construction method and application thereof
<160> 8
<170> SIPOSequenceListing 1.0
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<211> 537
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atgactctcg acaaggcgtt ggtgctgcgt acctgtgcaa ataacatggc cgatcactgc 60
ggccttatat ggcccgcgtc cggcacggtg gaatccagat actggcagtc aaccaggcgg 120
catgagaatg gtctggtcgg tttactgtgg ggcgctggaa ccagcgcttt tctaagcgtg 180
catgccgatg ctcgatggat tgtctgtgaa gttgccgttg cagacatcat cagtctggaa 240
gagccgggaa tggtcaagtt tccgcgggcc gaggtggttc atgtcggcga caggatcagc 300
gcgtcacact tcatttcggc acgtcaggcc gaccctgcgt caacgtcaac gtcaacgtca 360
acgtcaacgt taacgccaat gcctacggcc atacccacgc ccatgcctgc ggtagcaagt 420
gtcacgttac cggtggccga acaggcccgt catgaagtgt tcgatgtcgc gtcggtcagc 480
gcggctgccg ccccagtaaa caccctgccg gtgacgacgc cgcagaattt gcagacc 537
<210> 2
<211> 438
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atgaatatcc agatcggcga gcttgccaag cgcaccgcat gcccggtggt gaccattcgc 60
ttctacgaac aagaagggct gttgccgccg ccgggccgca gccgggggaa ttttcgcctg 120
tatggcgagg agcacgtgga gcgcttgcag ttcattcgtc actgccggtc tctggatatg 180
ccgttgagcg acgtacggac cttattgagt taccggaagc ggcccgacca ggattgcggt 240
gaagtcaata tgctcttgga tgagcacatc cgtcaggtcg aatctcggat cggagctttg 300
ctcgaactga agcaccattt ggtggaactg cgcgaagcct gttctggtgc caggcccgcc 360
caatcgtgcg ggattctgca gggactgtcg gactgcgtgt gtgatacgcg ggggaccacc 420
gcccatccaa gcgactag 438
<210> 3
<211> 399
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atgatgcgga tcggtgaact gggcaagaag gcagattgct tggtgcagac cgtgcgcttt 60
tacgagtcag aaggcttgct gcccgagcct gcacgtagcg agggcaactt caggctctat 120
gacgaagtcc atttgcagcg cttgctgttc atccgccgct gccgggcgaa ggacatgacg 180
ctggatgaga tccgtcaact gctgaactta cgggatcggc cagagttggg ctgcggcgag 240
gtgaacgcgc tggtcgacgc tcatatcgcg caagtgcgga ccaagatgaa ggaattgcgc 300
gccttggagc gcgagttaat ggatctgcga cgctcctgcg atagcgcccg aacctcgcgc 360
gagtgcggca ttctcaacag cttggccgag cccgcctga 399
<210> 4
<211> 726
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
atgatgccag tgtatttggc tgatttgcaa cgacgtctgg cgcgaggcgc cgccctacaa 60
cccaaaactt gcatgatttc gttggcaggc gacgcgcgac atcgggggcg aaaacggcac 120
ggaatggcgg ctttcatgcg agggagccag gggggcttct ggccgggaag cgaccttcgt 180
cacagcgcaa gtcccggtca tgctctcggt gtacgatgtg cggggagaac cgtccgagcg 240
ggacgagaca ttgtggaatc tcaaagtcca tgggggcagc tatggggttt tcttgccgaa 300
agtttctcat tgcccgtgac gatactctct ggcagctgcc aaccaccaaa ttccagcgga 360
tgttgcggga gccggccaac cactgcttgt ccacttttgc cgggcaacgc gcacgcatgg 420
ccgatgtggt cgtcgaactg gtggccagag agccagtgcg cgtcgttcga acgacgtttt 480
ccattctcac gttcgatgcc gaaggttgcc tcgatccagg ggcattcgaa aagcagcaat 540
ttgcgctcgc ggagtcggtc gttgcccccg tcttcgccgc atccgtggat gagagcaagc 600
aacccgtcgt tgacgcgtcc gcgcgattca ttgcgcaagg gggccaatgg gttccgacac 660
gagctttggc gcgcgcgatc gatgaggcgg cgttggggca acgacggtgc ctacgcctgt 720
aggtag 726
<210> 5
<211> 5369
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60
cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120
ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180
gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240
acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300
ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360
ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420
acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480
tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540
tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600
tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660
actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720
gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780
aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840
agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900
cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960
aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020
tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080
tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140
taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200
ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260
tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320
tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380
cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440
cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500
gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560
gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620
agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680
aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740
agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800
cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860
accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920
aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980
ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040
cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100
gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160
tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220
agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280
tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340
caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400
ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460
gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520
gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580
gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640
aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700
ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760
acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820
ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880
tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940
tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000
cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060
gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120
ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180
catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240
ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300
gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360
gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420
ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480
atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540
cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600
tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660
ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720
aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780
atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840
cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900
gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960
tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020
agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080
gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140
ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200
catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260
tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320
tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380
gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440
ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500
tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560
catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620
cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680
tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740
ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800
ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860
cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920
gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980
aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040
ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac 5100
agcagcggcc tggtgccgcg cggcagccat atggctagca tgactggtgg acagcaaatg 5160
ggtcgcggat ccgaattcga gctccgtcga caagcttgcg gccgcactcg agcaccacca 5220
ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc 5280
caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt 5340
tttgctgaaa ggaggaacta tatccggat 5369
<210> 6
<211> 6287
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60
cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120
ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180
gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240
acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300
ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360
ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420
acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480
tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540
tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600
tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660
actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720
gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780
aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840
agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900
cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960
aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020
tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080
tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140
taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200
ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260
tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320
tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380
cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440
cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500
gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560
gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620
agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680
aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740
agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800
cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860
accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920
aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980
ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040
cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100
gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160
tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220
agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280
tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340
caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400
ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460
gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520
gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580
gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640
aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700
ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760
acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820
ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880
tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940
tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000
cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060
gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120
ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180
catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240
ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300
gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360
gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420
ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480
atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540
cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600
tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660
ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720
aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780
atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840
cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900
gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960
tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020
agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080
gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140
ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200
catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260
tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320
tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380
gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440
ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500
tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560
catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620
cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680
tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740
ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800
ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860
cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920
gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980
aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040
ttttgtttaa ctttaagaag gagatatacc atgggcatga ctctcgacaa ggcgttggtg 5100
ctgcgtacct gtgcaaataa catggccgat cactgcggcc ttatatggcc cgcgtccggc 5160
acggtggaat ccagatactg gcagtcaacc aggcggcatg agaatggtct ggtcggttta 5220
ctgtggggcg ctggaaccag cgcttttcta agcgtgcatg ccgatgctcg atggattgtc 5280
tgtgaagttg ccgttgcaga catcatcagt ctggaagagc cgggaatggt caagtttccg 5340
cgggccgagg tggttcatgt cggcgacagg atcagcgcgt cacacttcat ttcggcacgt 5400
caggccgacc ctgcgtcaac gtcaacgtca acgtcaacgt caacgttaac gccaatgcct 5460
acggccatac ccacgcccat gcctgcggta gcaagtgtca cgttaccggt ggccgaacag 5520
gcccgtcatg aagtgttcga tgtcgcgtcg gtcagcgcgg ctgccgcccc agtaaacacc 5580
ctgccggtga cgacgccgca gaatttgcag accggtggtg gtggttccgg atccatgaat 5640
atccagatcg gcgagcttgc caagcgcacc gcatgcccgg tggtgaccat tcgcttctac 5700
gaacaagaag ggctgttgcc gccgccgggc cgcagccggg ggaattttcg cctgtatggc 5760
gaggagcacg tggagcgctt gcagttcatt cgtcactgcc ggtctctgga tatgccgttg 5820
agcgacgtac ggaccttatt gagttaccgg aagcggcccg accaggattg cggtgaagtc 5880
aatatgctct tggatgagca catccgtcag gtcgaatctc ggatcggagc tttgctcgaa 5940
ctgaagcacc atttggtgga actgcgcgaa gcctgttctg gtgccaggcc cgcccaatcg 6000
tgcgggattc tgcagggact gtcggactgc gtgtgtgata cgcgggggac caccgcccat 6060
ccaagcgacc atcatcacca tcaccactag gaattcgagc tccgtcgaca agcttgcggc 6120
cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga 6180
agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa 6240
acgggtcttg aggggttttt tgctgaaagg aggaactata tccggat 6287
<210> 7
<211> 6248
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60
cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120
ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180
gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240
acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300
ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360
ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420
acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480
tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540
tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600
tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660
actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720
gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780
aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840
agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900
cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960
aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020
tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080
tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140
taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200
ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260
tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320
tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380
cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440
cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500
gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560
gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620
agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680
aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740
agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800
cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860
accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920
aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980
ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040
cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100
gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160
tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220
agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280
tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340
caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400
ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460
gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520
gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580
gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640
aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700
ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760
acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820
ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880
tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940
tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000
cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060
gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120
ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180
catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240
ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300
gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360
gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420
ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480
atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540
cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600
tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660
ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720
aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780
atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840
cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900
gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960
tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020
agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080
gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140
ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200
catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260
tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320
tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380
gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440
ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500
tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560
catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620
cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680
tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740
ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800
ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860
cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920
gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980
aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040
ttttgtttaa ctttaagaag gagatatacc atgggcatga ctctcgacaa ggcgttggtg 5100
ctgcgtacct gtgcaaataa catggccgat cactgcggcc ttatatggcc cgcgtccggc 5160
acggtggaat ccagatactg gcagtcaacc aggcggcatg agaatggtct ggtcggttta 5220
ctgtggggcg ctggaaccag cgcttttcta agcgtgcatg ccgatgctcg atggattgtc 5280
tgtgaagttg ccgttgcaga catcatcagt ctggaagagc cgggaatggt caagtttccg 5340
cgggccgagg tggttcatgt cggcgacagg atcagcgcgt cacacttcat ttcggcacgt 5400
caggccgacc ctgcgtcaac gtcaacgtca acgtcaacgt caacgttaac gccaatgcct 5460
acggccatac ccacgcccat gcctgcggta gcaagtgtca cgttaccggt ggccgaacag 5520
gcccgtcatg aagtgttcga tgtcgcgtcg gtcagcgcgg ctgccgcccc agtaaacacc 5580
ctgccggtga cgacgccgca gaatttgcag accggtggtg gtggttccgg atccatgatg 5640
cggatcggtg aactgggcaa gaaggcagat tgcttggtgc agaccgtgcg cttttacgag 5700
tcagaaggct tgctgcccga gcctgcacgt agcgagggca acttcaggct ctatgacgaa 5760
gtccatttgc agcgcttgct gttcatccgc cgctgccggg cgaaggacat gacgctggat 5820
gagatccgtc aactgctgaa cttacgggat cggccagagt tgggctgcgg cgaggtgaac 5880
gcgctggtcg acgctcatat cgcgcaagtg cggaccaaga tgaaggaatt gcgcgccttg 5940
gagcgcgagt taatggatct gcgacgctcc tgcgatagcg cccgaacctc gcgcgagtgc 6000
ggcattctca acagcttggc cgagcccgcc catcatcacc atcaccactg agaattcgag 6060
ctccgtcgac aagcttgcgg ccgcactcga gcaccaccac caccaccact gagatccggc 6120
tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc 6180
ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat 6240
atccggat 6248
<210> 8
<211> 6575
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60
cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120
ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180
gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240
acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300
ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360
ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420
acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480
tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540
tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600
tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660
actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720
gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780
aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840
agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900
cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960
aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020
tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080
tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140
taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200
ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260
tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320
tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380
cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440
cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500
gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560
gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620
agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680
aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740
agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800
cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860
accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920
aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980
ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040
cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100
gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160
tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220
agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280
tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340
caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400
ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460
gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520
gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580
gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640
aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700
ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760
acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820
ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880
tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940
tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000
cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060
gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120
ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180
catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240
ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300
gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360
gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420
ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480
atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540
cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600
tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660
ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720
aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780
atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840
cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900
gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960
tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020
agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080
gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140
ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200
catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260
tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320
tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380
gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440
ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500
tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560
catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620
cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680
tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740
ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800
ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860
cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920
gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980
aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040
ttttgtttaa ctttaagaag gagatatacc atgggcatga ctctcgacaa ggcgttggtg 5100
ctgcgtacct gtgcaaataa catggccgat cactgcggcc ttatatggcc cgcgtccggc 5160
acggtggaat ccagatactg gcagtcaacc aggcggcatg agaatggtct ggtcggttta 5220
ctgtggggcg ctggaaccag cgcttttcta agcgtgcatg ccgatgctcg atggattgtc 5280
tgtgaagttg ccgttgcaga catcatcagt ctggaagagc cgggaatggt caagtttccg 5340
cgggccgagg tggttcatgt cggcgacagg atcagcgcgt cacacttcat ttcggcacgt 5400
caggccgacc ctgcgtcaac gtcaacgtca acgtcaacgt caacgttaac gccaatgcct 5460
acggccatac ccacgcccat gcctgcggta gcaagtgtca cgttaccggt ggccgaacag 5520
gcccgtcatg aagtgttcga tgtcgcgtcg gtcagcgcgg ctgccgcccc agtaaacacc 5580
ctgccggtga cgacgccgca gaatttgcag accggtggtg gtggttccgg atccatgatg 5640
ccagtgtatt tggctgattt gcaacgacgt ctggcgcgag gcgccgccct acaacccaaa 5700
acttgcatga tttcgttggc aggcgacgcg cgacatcggg ggcgaaaacg gcacggaatg 5760
gcggctttca tgcgagggag ccaggggggc ttctggccgg gaagcgacct tcgtcacagc 5820
gcaagtcccg gtcatgctct cggtgtacga tgtgcgggga gaaccgtccg agcgggacga 5880
gacattgtgg aatctcaaag tccatggggg cagctatggg gttttcttgc cgaaagtttc 5940
tcattgcccg tgacgatact ctctggcagc tgccaaccac caaattccag cggatgttgc 6000
gggagccggc caaccactgc ttgtccactt ttgccgggca acgcgcacgc atggccgatg 6060
tggtcgtcga actggtggcc agagagccag tgcgcgtcgt tcgaacgacg ttttccattc 6120
tcacgttcga tgccgaaggt tgcctcgatc caggggcatt cgaaaagcag caatttgcgc 6180
tcgcggagtc ggtcgttgcc cccgtcttcg ccgcatccgt ggatgagagc aagcaacccg 6240
tcgttgacgc gtccgcgcga ttcattgcgc aagggggcca atgggttccg acacgagctt 6300
tggcgcgcgc gatcgatgag gcggcgttgg ggcaacgacg gtgcctacgc ctgtaggcat 6360
catcaccatc accactagga attcgagctc cgtcgacaag cttgcggccg cactcgagca 6420
ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc 6480
tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag 6540
gggttttttg ctgaaaggag gaactatatc cggat 6575
Claims (3)
1. A construction method of surface display engineering bacteria for specifically adsorbing heavy metal lead is characterized by comprising the following steps:
(1) Amplifying nucleotide sequences encoding the dockerin INPN and a lead binding protein selected from the three non-membrane proteins PbrR, pbrR691 or PbrD of Cupridinium metulid CH34, respectively, by PCR reaction; removing a stop codon at the tail end of the anchoring protein INPN during amplification, introducing flexible connecting peptide FL, wherein the amino acid sequence of the flexible connecting peptide FL is GGGGS, and introducing a hexahistidine Tag His-Tag before the stop codon of the lead binding protein coding sequence; the nucleotide sequences of the lead binding proteins PbrR, pbrR691 and PbrD are respectively shown in SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 4; the nucleotide sequence of the INPN is shown as SEQ ID No. 1;
(2) The method comprises the following steps of fusing an anchored protein INPN with a lead binding protein gene sequence in an enzyme digestion connection mode to construct fusion protein modules which are respectively a PbrR fusion module INPN-FL-PbrR, a PbrR691 fusion module INPN-FL-PbrR691 and a PbrD fusion module INPN-FL-PbrD;
(3) Cloning the fusion protein module onto a pET-28a (+) plasmid vector; and introducing the cloned plasmid vector into a host chassis cell BL21 (DE 3), and screening a positive clone strain with a target screening plasmid through colony PCR (polymerase chain reaction), namely the specific lead binding protein surface display engineered strain.
2. The surface display engineering bacterium constructed by the method of claim 1, which is characterized by comprising PbrR surface display engineering bacterium BL21 (pR-FL), wherein the nucleotide sequence of the contained recombinant plasmid pR-FL is SEQ ID No.6; the surface display engineering bacterium BL21 (pR 691-FL) comprising PbrR691, the nucleotide sequence of the contained recombinant plasmid pR691-FL is SEQ ID No.7; the surface display engineering bacterium BL21 (pD-FL) containing PbrD has the nucleotide sequence of SEQ ID No.8.
3. The use of the surface display engineered bacteria of claim 1 in situ remediation of lead ion contaminated environments.
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| CN111979166B (en) * | 2020-08-14 | 2022-11-18 | 中国环境科学研究院 | Engineering bacterium for specifically removing arsenic as well as construction method and application thereof |
| CN111996207A (en) * | 2020-08-14 | 2020-11-27 | 中国环境科学研究院 | Carrier for specifically removing arsenic as well as construction method and application thereof |
| CN114686411B (en) * | 2020-12-29 | 2023-05-05 | 上海科技大学 | Mixture of engineering bacteria, kit for sensing and adsorbing heavy metal pollutants and treatment method |
| CN113755418A (en) * | 2021-08-04 | 2021-12-07 | 天津大学 | Recombinant engineering bacterium for displaying carbonic anhydrase on surface as well as construction method and application thereof |
| CN113637657B (en) * | 2021-08-05 | 2024-01-30 | 云南师范大学 | Carboxylesterase CarCB2 and whole-cell catalyst and application thereof |
| CN114774452B (en) * | 2022-04-29 | 2023-12-26 | 天津大学 | Construction method and application of engineering escherichia coli for adsorbing mercury ions in solution |
| CN115433687B (en) * | 2022-09-28 | 2024-09-10 | 华中科技大学 | Engineering bacteria for removing heavy metal ions by broad-spectrum high-efficiency adsorption and coupling biomineralization |
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