CN113308427B - Genetically engineered bacterium capable of efficiently adsorbing lead ions and application thereof - Google Patents
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Abstract
The invention discloses a genetic engineering bacterium capable of efficiently adsorbing lead ions and application thereof, belonging to the technical field of genetic engineering and microbial engineering. The invention successfully constructs a recombinant bacterium for expressing functionalized amyloid fibers by taking escherichia coli BL21(DE3) as an expression host. The invention also applies the genetic engineering bacteria to heavy metal ion adsorption, when the initial lead ion concentration is 50mg/L, the lead ion removal rate reaches 98.26 percent after 6 hours of reaction, and the metal ion removal rate is better; meanwhile, the genetic engineering bacteria provided by the invention also have better removal capacity on other heavy metal ion waste liquids except lead ions, wherein the removal rate is 59.7% for copper ion solution with the final concentration of 80 mg/L; the removal rate of 1mg/L mercury ion solution can reach 96.2%, so the strain has good industrial application value in the field of metal ion adsorption treatment in water environment.
Description
Technical Field
The invention relates to a genetic engineering bacterium capable of efficiently adsorbing lead ions and application thereof, belonging to the technical field of genetic engineering and microbial engineering.
Background
With the acceleration of the industrialization process, the discharge amount of heavy metal wastewater is huge, and the heavy metal pollution is increasingly serious. Heavy metal pollution is mainly represented by heavy metal ions such as copper, cadmium, lead, mercury, hexavalent chromium and the like, and generally comes from electroplating, plastic manufacturing, chemical fertilizers, pigments, mining, metallurgical processes and the like. The heavy metal pollution of water bodies becomes one of the main environmental problems at present, the ecological sustainable development is endangered all the time, and the serious threat to the health of people is formed, so that the effective treatment of the heavy metal pollution is the current work to be solved urgently.
Among the methods for treating heavy metal pollution, compared with the traditional chemical method and physical chemical method, the microbial adsorption technology is a novel and efficient method for removing heavy metal researched recently, and has the characteristics of low cost, low energy consumption and no secondary pollution; however, the prior microbial adsorption technology has the defects of low adsorption efficiency and long adsorption period, so that the application of the microbial adsorption technology is limited. For example, Li D et al (Adsorption of gravity metal ligation to Pb)2+and Cd2+in wastewater[J]Screening of heavy metals Pb from the soil obtained from the heavy metal waste accumulation zone, Environmental Science and Pollution Research,2018,25(32):32156-32162)2+Brevibacterium with high tolerance, preparation of immobilized microbial adsorbent from the strain, and use of the immobilized microbial adsorbent in the preparation of a solution containing Pb2+When the initial concentration is 50mg/L, the adsorption time is 24h, and the reaction time is 24h2+The adsorption rate of (a) is only 30%; when Pb is in solution2+When the initial concentration is 100mg/L, the adsorption time is 24h, and the reaction time is 24h2+The adsorption rate of (2) is less than 20%.
The CsgA nanofiber is formed by self-assembling CsgA monomeric protein, is rich in a beta-folded structure, is a typical amyloid protein, has extremely strong environmental tolerance (for example, can resist high and low temperature, acid-base solution, organic solvent and certain mechanical friction), and can be considered to be applied to adsorption of heavy metal ions.
Chinese patent application publication No. CN111690583A discloses a method for preparing a heavy metal ion bio-adsorption membrane by adding activated carbon into fermentation liquor of a strain expressing functionalized amyloid fiber, but Shewanella is adopted, certain Shewanella is a potential pathogen of human beings and aquatic animals, Shewanella infection reports are more and more, and the host range is continuously expanded, which shows that the potential pathogenicity of the Shewanella can seriously threaten human health and aquaculture. In addition, the activated carbon mainly plays a role in adsorption, the price of the activated carbon continuously rises, and the solid waste and the hazardous waste are difficult to treat. In addition, the membrane is subjected to concentration polarization and membrane fouling after a certain period of use, which are problems inevitably faced by membrane separation techniques in water treatment.
In summary, the strains disclosed in the prior art have low adsorption capacity for lead ions in solution or long adsorption period, which is not suitable for large-scale industrial application, and therefore, how to construct a strain capable of expressing functional amyloid fibers and efficiently absorbing heavy metal ions becomes an urgent problem to be solved.
Disclosure of Invention
In order to solve the problems that the prior art is not high in adsorption capacity of a strain on metal ions in a solution, long in adsorption period, not beneficial to large-scale application in industry and the like, the invention provides a recombinant escherichia coli, wherein the recombinant escherichia coli over-expresses a subunit csgA of amyloid fiber curli from E.coli K12, and the amino acid sequence of the subunit csgA of the amyloid fiber curli is shown as SEQ ID No. 2.
In one embodiment of the invention, the recombinant Escherichia coli takes Escherichia coli BL21(DE3) as an expression host.
In one embodiment of the invention, the recombinant Escherichia coli uses pET-22b as an expression vector.
In one embodiment of the invention, the nucleotide sequence of the subunit csgA of amyloid fiber curli is shown in SEQ ID No. 1.
The invention also provides a method for constructing the recombinant escherichia coli, which comprises the following steps:
(1) chemically synthesizing a subunit csgA gene sequence of amyloid fiber curli with a nucleotide sequence shown as SEQ ID NO. 1;
(2) and (2) connecting the gene sequence obtained in the step (1) with an expression vector, preparing a recombinant vector, and transferring the recombinant vector into escherichia coli to prepare the recombinant escherichia coli.
In one embodiment of the invention, the method comprises the steps of:
(1) chemically synthesizing a subunit csgA gene segment of amyloid fiber curli of escherichia coli;
(2) assembling the gene fragment csgA obtained in the step (1) with an expression vector pET-22b through Gibson to construct a recombinant plasmid pET22 b-csgA;
(3) transforming the recombinant plasmid pET22b-csgA constructed in the step (2) into a cloning host E.coli JM109, and then transforming the cloning host E.coli JM109 into an expression host E.coli BL21(DE3) to construct recombinant Escherichia coli BL21(DE 3).
In a specific embodiment, the nucleotide sequence encoding the gene fragment csgA in step (1) is shown in SEQ ID NO. 1.
In one embodiment of the present invention, the amino acid sequence of csgA in step (1) is shown in SEQ ID NO. 2.
In one embodiment of the invention, the gene segment csgA takes an escherichia coli genome as a template, a primer is designed for PCR amplification, an upstream primer for amplifying the gene segment is shown as SEQ ID No.3, and a downstream primer sequence is shown as SEQ ID No. 4.
In one embodiment of the invention, the expression vector pET-22b is linearized by a PCR method, and the upstream primer and the downstream primer of the amplified expression vector are respectively shown as SEQ ID NO.5 and SEQ ID NO. 6.
The invention also provides a method for removing metal ions in a wastewater environment, which comprises the step of adding the recombinant escherichia coli or the fermentation broth of the recombinant escherichia coli into the wastewater-containing environment for reaction.
In one embodiment of the invention, the metal ions comprise lead ions, copper ions, mercury ions.
In one embodiment of the invention, the recombinant Escherichia coli is in wastewaterAddition amount OD600The value is at least 1.5.
In one embodiment of the invention, the reaction is carried out at 30 ℃ and 220rpm for 0.1-8 h.
The invention also provides the application of the recombinant escherichia coli or the fermentation liquor of the recombinant escherichia coli in preparing chemicals for removing metal ions in wastewater.
Advantageous effects
(1) The recombinant escherichia coli BL21(DE3) obtained by the method can efficiently adsorb lead ions, the adsorption efficiency of the recombinant escherichia coli BL21 on the lead ions is improved by 24% compared with that of a wild type, and when the initial lead ion concentration is 50mg/L, the lead ion removal rate reaches 98.26% after 6 hours of reaction.
(2) The genetic engineering bacteria provided by the invention also have higher removal efficiency on other heavy metal ion waste liquids except lead ions, wherein the removal rate is 59.7% for copper ion solution with the final concentration of 80 mg/L; the removal rate of 1mg/L mercury ion solution can reach 96.2%, and the gene engineering bacteria have good removal capability on different heavy metal ions.
(3) The recombinant escherichia coli BL21(DE3) obtained by the method has the advantages of low cost and high success rate during construction, can be applied to large-scale industrial production, and has important commercial value in the field of heavy metal ion adsorption in water environment.
Drawings
FIG. 1: the structure diagram of the recombinant plasmid pET-22 b-CsgA.
FIG. 2: control E.coli, recombinant E.coli and Congo red binding profiles at different incubation times.
FIG. 3: and detecting the lead ion concentration standard curve by a color development method.
FIG. 4: comparison graph of lead ion adsorption efficiency (initial lead ion concentration of 100mg/L) of control Escherichia coli and recombinant Escherichia coli.
FIG. 5: comparison graph of lead ion adsorption efficiency (initial lead ion concentration is 50mg/L) of control Escherichia coli and recombinant Escherichia coli.
FIG. 6: and (3) TEM image of the recombinant Escherichia coli after adsorbing lead ions.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below.
Coli BL21(DE3) referred to in the examples below is the laboratory-maintained strain; the salt required to prepare the lead ion standard solution of interest in the following examples was lead chloride, available from shanghai shen di.
The media involved in the following examples are as follows:
ampicillin plates: 10g/L peptone (Oxoid, UK), 5g/L yeast powder (Oxoid), 10g/L sodium chloride and 20g/L agar powder, and adding ampicillin to the sterilized mixture at a final concentration of 100. mu.g/mL.
YES-CA liquid medium: 1g/L of yeast powder (Oxoid) and 10g/L of casein hydrolysis amino acid.
LB liquid medium: peptone (Oxoid, UK) 10g/L, yeast powder (Oxoid)5g/L, and sodium chloride 10 g/L.
The masking agent solutions referred to in the following examples were formulated as follows:
respectively preparing a sodium fluoride solution, a thiourea solution and a trisodium citrate solution with the mass fraction of 1%, and uniformly mixing in equal volume.
The standard curves referred to in the following examples were plotted as follows:
(1) taking 50 mu L of lead ion standard solution (the concentration is 10, 15, 20, 25, 30 and 40 mg/L);
(2) sequentially adding 20 mu L of 1.0mol/L sulfuric acid solution, 20 mu L of masking agent solution, 30 mu L of 20% potassium iodide-ascorbic acid solution, 20 mu L of 0.5% polyvinyl alcohol solution and 20 mu L of 0.5% rhodamine B solution, diluting the solution to 300 mu L by using deionized water, and shaking up;
(3) and (3) after the solution obtained in the step (2) is developed for 10min, measuring the absorbance at the wavelength of 600nm by using an enzyme-labeling instrument.
(4) As shown in fig. 3, the measurement result was obtained by plotting a standard curve with the lead ion concentration as the abscissa and the OD600 value as the ordinate, and obtaining a calculation formula of y being 0.0136x +0.1825, R2Is 0.9935, the detection concentration range of lead ions is 0-50 mg/L.
The detection method of the lead ion content referred to in the following examples is as follows:
the method comprises the following specific steps:
(1) taking 50 mu L of lead ion sample to be detected;
(2) sequentially adding 20 mu L of 1.0mol/L sulfuric acid solution, 20 mu L of masking agent solution, 30 mu L of 20% potassium iodide-ascorbic acid solution, 20 mu L of 0.5% polyvinyl alcohol solution and 20 mu L of 0.5% rhodamine B solution, diluting the solution to 300 mu L by using deionized water, and shaking up;
(3) and (3) after the solution obtained in the step (2) is developed for 10min, measuring the absorbance at the wavelength of 600nm by using an enzyme-labeling instrument.
The method for calculating the removal rate of lead ions by the strains involved in the following examples was as follows:
(1) substituting the absorbance value obtained according to the solution to be detected into a standard curve formula y of 0.0136x +0.1825 to calculate the concentration of the lead ions;
(2) the removal rate of lead ions by the strain was calculated using the following formula.
Wherein A is the lead ion removal rate; x1Lead ion concentration corresponding to the experimental group; x2Lead ion concentration is indicated for the blank.
The CsgA protein expression level calculation method involved in the following examples was as follows:
congo Red (CR) staining to determine CsgA protein expression: 1mL of wild E.coli culture and recombinant E.coli culture incubated for different periods (48h, 72h) were centrifuged (5000rpm, 10min), the supernatant was discarded, the centrifuged pellet was resuspended in 15. mu.g/L CR (dissolved in physiological saline), placed in a 30 ℃ incubator for 10min and centrifuged (5000rpm, 10min), the supernatant was collected, 15. mu.g/L CR was used as a blank, and the remaining amount of CR in the supernatant was measured by a microplate reader at 490nm wavelength.
Example 1: construction of recombinant strains
The method comprises the following specific steps:
(1) acquiring a subunit csgA gene sequence of amyloid fiber curli with a nucleotide sequence shown as SEQ ID No.1 from an NCBI database, wherein the nucleotide sequence is derived from E.coli K12, and an amino acid sequence of the subunit csgA gene sequence is shown as SEQ ID No. 2;
(2) respective primers are respectively designed according to a target gene sequence and a vector plasmid sequence, and are shown in table 1;
TABLE 1 primers
(3) PCR amplification was performed using the genome of E.coli BL21(DE3) as a template and csgA primers in Table 1, using high fidelity enzyme Primer Star Max DNA Polymerase (Takara Inc.) under conditions of pre-denaturation 98 ℃ for 3 min; the amplification stage is carried out for 34 cycles according to the conditions of 98 ℃, 10s, 55 ℃, 10s, 72 ℃ and 10 s; extending for 10min at 72 ℃ to obtain a gene segment shown in SEQ ID NO. 1;
(4) PCR amplification was performed using the primers pET-22b in Table 1, and high fidelity enzyme Primer Star Max DNA Polymerase (Takara) was selected for PCR at a pre-denaturation temperature of 98 ℃ for 3 min; the amplification stage is carried out for 34 cycles according to the conditions of 98 ℃, 10s, 55 ℃, 10s, 72 ℃ and 30 s; extending at 72 ℃ for 10min, and linearizing the vector plasmid;
(5) respectively purifying the gene fragment obtained in the step (3) and the vector pET-22b linearization PCR product obtained in the step (4), and then connecting the purified gene fragment and the vector pET-22b linearization PCR product by a Gibson assembly method;
(6) transforming the ligation product into escherichia coli JM109 competence, screening positive clones on an ampicillin plate, verifying the correct fragment size through colony PCR, and then carrying out sequencing identification to finally obtain a recombinant plasmid pET-22b-CsgA (the structure is shown in figure 1) containing a correct sequence;
(7) transforming the recombinant plasmid pET-22b-CsgA prepared in the step (6) into competent escherichia coli BL21 cells, screening positive clones on an ampicillin plate, and finally obtaining a strain E.coli BL21(DE3)/pET-22b-CsgA containing the correct recombinant plasmid after the size of a fragment is verified to be correct through colony PCR;
wherein, the transformation conditions are as follows: 1 μ L plasmid and 100 μ L competent cells were mixed and ice-cooled for 30 min; transferring the mixture to a water bath kettle with the temperature of 42 ℃ for heat shock for 90 s; rapidly transferring the EP pipe to an ice box, and carrying out ice bath for 5 min; adding 600 mu LLB recovery culture solution by using a pipette, and placing the mixture in a shaking table at 37 ℃ for 1 h; taking a proper amount of culture solution and coating the culture solution on an LB solid plate containing ampicillin; culturing in an incubator at 37 ℃ for 12h, and picking transformants for verification.
A control strain E.coli BL21(DE3)/pET-22b was prepared by transferring the empty plasmid pET-22b into E.coli BL21(DE3) as described above.
Example 2: high-efficiency expression of functionalized amyloid fiber CsgA
The method comprises the following specific steps:
(1) the recombinant strain E.coli BL21(DE3)/pET-22b-CsgA and the control strain E.coli BL21(DE3)/pET-22b obtained in example 1 were inoculated into LB liquid medium supplemented with 100. mu.g/mL ampicillin, respectively, and activated, and cultured in a shaker at 37 ℃ at 220rpm for 12-16 hours to prepare a seed solution;
(2) the seed solution was inoculated into 50mL of YES-CA broth containing ampicillin (100. mu.g/mL) in an amount of 1% (v/v), and cultured in a shaker at 30 ℃ at 220rpm until OD600When the concentration is 0.6-0.8, IPTG (0.5 mM of final concentration) is added to induce expression for 48-72 h;
a fermentation broth containing E.coli BL21(DE3)/pET-22b-CsgA and the control strain E.coli BL21(DE3)/pET-22b was prepared.
(3) CsgA protein expression quantity in fermentation liquor is measured by Congo Red (CR) staining, namely, the average CR binding rates per OD after the control escherichia coli and the recombinant escherichia coli are fermented for 48 hours are calculated to be 4.92% and 2.86% respectively.
The results are shown in fig. 2, and it can be seen that the recombinant escherichia coli shows higher CR binding capacity compared with the control strain, indicating that the cell surface of the recombinant escherichia coli synthesizes the amyloid fiber CsgA protein; and the CR binding rate of the recombinant Escherichia coli is higher than 48h after the induction expression is carried out for 72 h.
Example 3: application of recombinant strain in adsorption of lead ions
The method comprises the following specific steps:
1. preparation of bacterial suspension:
the fermentation solutions containing E.coli BL21(DE3)/pET-22b-CsgA and the control strain E.coli BL21(DE3)/pET-22b obtained in example 2 were adjusted to OD concentrations of seed solutions with physiological saline, respectively600The value is 1.5, and bacterial suspensions are respectively obtained.
2. Respectively adding 2mL of the bacterial suspension obtained in the step (1) into a 24-hole deep-hole plate, adding 2mL of physiological saline into a blank group, respectively adding lead ion solutions with final concentrations of 100mg/L and 50mg/L into A, B rows, carrying out oscillation reaction for 0.1-8 h in a deep-hole shaking table at 30 ℃ and 220r/min, centrifuging for 10min at 4000rpm, taking supernatant, and adopting the method for detecting the lead ion content; developing for 10min, and determining the content of residual lead ions by using an enzyme-labeling instrument; wherein the concentrations of the blank groups in the reaction time of 2-8 h are both 100mg/L and 50 mg/L; the adsorption effect of the control strain and the recombinant strain is shown in the following table.
TABLE 2 residual lead ion content after different reaction times
As shown in FIGS. 4 and 5, the removal rate of the recombinant strain is much higher than that of the control strain under the condition that the initial concentration is 50mg/L or 100mg/L lead ions, the removal rate increases with the increase of time and reaches a maximum value at 6h, and the removal effect of lead ions is slightly reduced at 8h, which is probably because the strain is desorbed for a long time; under the condition that the initial concentration is 50mg/L lead ions, the removal rate of the recombinant strain after 6 hours of adsorption is as high as 98.26 percent, which shows that the recombinant strain has good removal effect on the lead ions.
3. The result of a Transmission Electron Microscope (TEM) is performed on the recombinant escherichia coli after adsorbing the lead ions, and is shown in fig. 6, it can be seen that the recombinant escherichia coli under the TEM expresses amyloid fibers on curli pili and adsorbs heavy metal lead ions, which is consistent with the experimental result.
Example 4: survival rate of recombinant strain in simulated industrial wastewater environment
The recombinant strain can adapt to the environment of industrial wastewater, and the survival rate of the strain in the wastewater environment is determined by taking simulated wastewater samples with different pH values as an example.
The method comprises the following specific steps:
(1) preparing bacterial suspension, and calculating the number of viable bacteria in the bacterial suspension before wastewater treatment:
the fermentation broth containing E.coli BL21(DE3)/pET-22b-CsgA prepared in step (2) of example 2 was adjusted to OD by physiological saline600The value is 4, and bacterial suspension is obtained;
the above-mentioned OD is added600The value of 4 of the bacterial suspension is adjusted to OD by physiological saline600The value was 0.5, and the bacterial suspensions were each diluted to 10-5、10-6、10-7Three gradients, two in parallel, were pipetted into 100. mu.L of ampicillin-coated plates, at which the colony count was 1.87X 108cfu/mL。
(2) Reaction of bacterial suspensions in simulated wastewater environments of different pH:
the above-mentioned OD is added600Value 4 bacterial suspension by final concentration OD600The adding amount is 0.5, and the adding amount is respectively added into 20mL of simulated wastewater liquid with different pH values;
wherein, the simulated wastewater liquid comprises the following components: 230mg/L glucose, 60mg/L peptone, 20mg/L beef extract, 40mg/L anhydrous sodium acetate, 198mg/L sodium bicarbonate, 12mg/L potassium dihydrogen phosphate, 170mg/L ammonium bicarbonate, 2.4mg/L magnesium chloride hexahydrate, 1.2mg/L anhydrous calcium chloride and 1mg/L ferric chloride hexahydrate, and adjusting the pH values to be 4, 6, 8 and 10 respectively to obtain reaction systems;
respectively reacting the reaction systems at 30 ℃ and 220rpm, and respectively diluting the reaction liquid after 24h reaction to 10-4、10-5、10-6Three gradients, two for each gradient, were parallel and 100 μ L of ampicillin coated plates were aspirated separately; and calculating the number of colonies corresponding to the recombinant strains in the simulated wastewater samples with different pH values after 24h reaction, and calculating the survival rate, wherein the results are shown in Table 3.
TABLE 3 survival rates of recombinant strains in wastewater samples of different pH values
As can be seen from Table 3, the survival rate of the recombinant Escherichia coli is not high at pH 4 and 6 after 24h, only about 10%, which indicates that the pH value has a certain influence on the survival of the Escherichia coli, and the low pH value leads to the increase of the lethality rate. In the wastewater treatment, if the recombinant Escherichia coli is suicide after adsorbing lead ions with high efficiency, the pH can be appropriately reduced.
Example 5: application of recombinant strain in adsorption of other heavy metal ions
The recombinant strain is applied to other heavy metal ion waste liquid except lead ions, and the removal efficiency of the recombinant strain on other heavy metals is measured.
The method comprises the following specific steps:
1. preparation of bacterial suspension:
the fermentation broth containing E.coli BL21(DE3)/pET-22b-CsgA obtained in example 2 was adjusted to OD by physiological saline600A value of 1.5 gave a bacterial suspension.
2. Adding 5mL of the bacterial suspension obtained in the step (1) into a 50mL centrifuge tube, adding 5mL of physiological saline into a blank control, respectively adding a copper ion solution with a final concentration of 80mg/L and a mercury ion solution with a final concentration of 1mg/L, carrying out shake reaction for 6h in a shaking table at 30 ℃ and 220r/min, centrifuging at 4000rpm for 10min, taking a supernatant, measuring the content of the residual copper ions by using an atomic spectrophotometer AA-240, measuring the content of the residual mercury ions by using an atomic fluorescence spectrometer AFS-930, and calculating the removal rate, wherein the contents are shown in the following table.
TABLE 4 residual heavy metal ion content after 6h reaction
As can be seen from Table 4, the recombinant strain has removal capacity for both lead ions and mercury ions, and the removal capacity for a mercury ion solution of 1mg/L can reach 96.20%, so that the recombinant strain has wide industrial application value.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
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gctcatcagt actaaacata tgaaatacct gctgccgacc 40
Claims (7)
1. A method for removing metal ions in a wastewater environment is characterized in that recombinant Escherichia coli or fermentation liquor of the recombinant Escherichia coli is added into wastewater containing the metal ions for reaction; the recombinant escherichia coli overexpresses a subunit csgA of amyloid fiber curli from E.coli K12, and a nucleotide sequence encoding the subunit csgA of the amyloid fiber curli is shown in SEQ ID No. 1.
2. The method of claim 1, wherein the recombinant E.coli is E.coli BL21(DE3) as an expression host.
3. The method of claim 1 or 2, wherein the recombinant escherichia coli uses pET-22b as an expression vector.
4. The method of claim 3, wherein the method of constructing the recombinant E.coli comprises the steps of:
(1) chemically synthesizing a subunit csgA gene sequence of amyloid fiber curli with a nucleotide sequence shown as SEQ ID NO. 1;
(2) and (2) connecting the gene sequence obtained in the step (1) with an expression vector, preparing a recombinant vector, and transferring the recombinant vector into escherichia coli to prepare the recombinant escherichia coli.
5. The method of claim 4, wherein the metal ions comprise lead ions, copper ions, and/or mercury ions.
6. The method of claim 5, wherein the recombinant Escherichia coli is added in an amount OD in the wastewater600The value is at least 1.5.
7. The method of any one of claims 1 to 6, wherein the reaction is carried out at 30 ℃ and 220rpm for 0.1 to 8 hours.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111690583A (en) * | 2020-05-20 | 2020-09-22 | 江苏大学 | Shewanella for expressing functionalized amyloid fiber and construction method and application thereof |
| CN112608876A (en) * | 2020-12-30 | 2021-04-06 | 江苏大学 | Method for marking biotinylation Curli protein living cells and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111690583A (en) * | 2020-05-20 | 2020-09-22 | 江苏大学 | Shewanella for expressing functionalized amyloid fiber and construction method and application thereof |
| CN112608876A (en) * | 2020-12-30 | 2021-04-06 | 江苏大学 | Method for marking biotinylation Curli protein living cells and application thereof |
Non-Patent Citations (5)
| Title |
|---|
| NCBI Reference Sequence: WP_000771437.1;无;《GENBANK》;20201216;ORIGIN * |
| 抗大肠杆菌curli 菌毛CsgA 蛋白特异性抗体的制备及鉴定;冷静等;《免疫学杂志》;20070731;第23卷(第4期);370-372 * |
| 蜂毒肽在大肠杆菌中的高效融合表达与纯化;周丽仙等;《食品与生物技术学报》;20201231;第39卷(第8期);26-33 * |
| 陆利.Curli细胞分泌途径中重要蛋白.《中国优秀硕士学位论文全文数据库》.2017,(第02期), * |
| 魏氏柠檬酸杆菌淀粉样蛋白编码基因csgA的分子功能;王颖思等;《工业微生物》;20210228;第51卷(第1期);1-7 * |
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