CN112961880B - Construction method of algae-bacteria symbiotic system for removing ARGs in water body - Google Patents
Construction method of algae-bacteria symbiotic system for removing ARGs in water body Download PDFInfo
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 title claims abstract description 41
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- 241000194108 Bacillus licheniformis Species 0.000 claims abstract description 40
- 241000195493 Cryptophyta Species 0.000 claims abstract description 40
- 230000003115 biocidal effect Effects 0.000 claims abstract description 39
- 239000010865 sewage Substances 0.000 claims abstract description 33
- 239000001963 growth medium Substances 0.000 claims abstract description 30
- 241000894006 Bacteria Species 0.000 claims abstract description 26
- 239000011259 mixed solution Substances 0.000 claims abstract description 26
- 239000013612 plasmid Substances 0.000 claims abstract description 26
- 240000009108 Chlorella vulgaris Species 0.000 claims abstract description 24
- 235000007089 Chlorella vulgaris Nutrition 0.000 claims abstract description 24
- 238000005286 illumination Methods 0.000 claims abstract description 24
- 238000012258 culturing Methods 0.000 claims abstract description 23
- 238000004520 electroporation Methods 0.000 claims abstract description 14
- 238000000034 method Methods 0.000 claims abstract description 14
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- 101150015970 tetM gene Proteins 0.000 claims abstract description 11
- 101150004433 tetQ gene Proteins 0.000 claims abstract description 11
- 101100424890 Butyrivibrio fibrisolvens tetW gene Proteins 0.000 claims abstract description 10
- 101100223892 Escherichia coli sulI gene Proteins 0.000 claims abstract description 8
- 238000002156 mixing Methods 0.000 claims abstract description 5
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- 239000013049 sediment Substances 0.000 claims description 10
- 239000002609 medium Substances 0.000 claims description 7
- 239000003242 anti bacterial agent Substances 0.000 claims description 6
- 238000007664 blowing Methods 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 4
- 229930006000 Sucrose Natural products 0.000 claims description 4
- 230000001580 bacterial effect Effects 0.000 claims description 4
- 239000005720 sucrose Substances 0.000 claims description 4
- 230000003698 anagen phase Effects 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 230000000694 effects Effects 0.000 abstract description 6
- -1 sul2 Proteins 0.000 abstract description 3
- 239000008239 natural water Substances 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 61
- 241000195649 Chlorella <Chlorellales> Species 0.000 description 19
- 210000001082 somatic cell Anatomy 0.000 description 14
- 229940088710 antibiotic agent Drugs 0.000 description 5
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- 101100367123 Caenorhabditis elegans sul-1 gene Proteins 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 229940124530 sulfonamide Drugs 0.000 description 3
- 241001185310 Symbiotes <prokaryote> Species 0.000 description 2
- 239000004098 Tetracycline Substances 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
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- 235000019364 tetracycline Nutrition 0.000 description 2
- 229940072172 tetracycline antibiotic Drugs 0.000 description 2
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- 229940123317 Sulfonamide antibiotic Drugs 0.000 description 1
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- 241000894007 species Species 0.000 description 1
- FDDDEECHVMSUSB-UHFFFAOYSA-N sulfanilamide Chemical compound NC1=CC=C(S(N)(=O)=O)C=C1 FDDDEECHVMSUSB-UHFFFAOYSA-N 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
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- 229940040944 tetracyclines Drugs 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
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Abstract
The invention discloses a construction method of an algae-bacteria symbiotic system for removing antibiotic resistance genes in a water body, which comprises the following steps: s1: inoculating chlorella vulgaris cells and bacillus licheniformis into the sterilized BG11-LB culture medium; s2: placing the mixed culture medium inoculated with the chlorella vulgaris cells and the bacillus licheniformis cells into a sterile room with constant temperature and constant illumination for culture; s3: preparing artificial sewage containing antibiotic resistance gene plasmid; s4: mixing the mixed culture medium in the step S2 with the artificial sewage in the step S3 to form an algae-bacteria symbiotic system; s5: taking 1-2mL of the mixed solution of the algae bacteria in the step S4, performing cell perforation on the taken mixed solution of the algae bacteria by adopting a cell electroporation method, cooling, and then placing the mixed solution of the algae bacteria in the symbiotic system of the algae bacteria in the step S4 again, and continuously culturing in a sterile room with constant temperature and constant illumination. The algae-bacteria symbiont has good removal effect on ARGs such as sul1, sul2, tetM, tetQ, tetW and the like in natural water bodies.
Description
Technical Field
The invention belongs to the technical field of pollution treatment of antibiotic resistance genes, and particularly relates to a construction method of an algae-bacteria symbiotic system for removing antibiotic resistance genes in a water body.
Background
Contamination with antibiotic resistance genes (Antibiotic resistance genes, ARGs) has become a concern for water contamination in the world today. The large number of uses of antibiotics in the medical, livestock, aquaculture, etc. fields causes accumulation of ARGs pollution in the water body in the environment. ARGs in various water bodies in China are reported to be detected to different degrees, wherein the ARGs of sulfonamides and tetracyclines are ubiquitous and have higher concentration. The water body is an important medium for releasing and diffusing ARGs, and the ARGs can enter a human food chain through water circulation, so that serious threat is formed to the ecological environment of the water body and human health. Therefore, methods for controlling and repairing ARGs pollution in water bodies are actively explored.
Disclosure of Invention
In order to overcome the defects of the existing technology for removing the pollutants of the antibiotic resistance genes in the water body, the invention provides a construction method of an algae-bacteria symbiotic system for removing the antibiotic resistance genes in the water body, and the algae-bacteria symbiont has good removal effect on ARGs such as sulfanilamide antibiotic resistance genes (sul 1 and sul 2), tetracycline antibiotic resistance genes (tetM, tetQ, tetW) and the like in the natural water body.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
a construction method of an algae-bacteria symbiotic system for removing antibiotic resistance genes in a water body comprises the following steps:
s1: inoculating chlorella vulgaris and bacillus licheniformis into sterilized 150-200mL BG11-LB culture medium;
s2: placing the mixed culture medium inoculated with the chlorella vulgaris and the bacillus licheniformis in a sterile room with constant temperature and constant illumination for culture;
s3: preparing artificial sewage containing antibiotic resistance gene plasmid;
s4: mixing the mixed culture medium in the step S2 with the artificial sewage in the step S3 to form an algae-bacteria symbiotic system;
s5: taking 1-2mL of the mixed solution of the algae bacteria in the step S4, performing cell perforation on the taken mixed solution of the algae bacteria by adopting a cell electroporation method, cooling, and then placing the mixed solution of the algae bacteria in the symbiotic system of the algae bacteria in the step S4 again, and continuously culturing in a sterile room with constant temperature and constant illumination.
The artificial sewage preparation method containing the antibiotic resistance gene plasmid in the step S3 comprises the following steps: the plasmid finished product of the constructed target antibiotic resistance gene and the initial antibiotic resistance gene abundance of the artificial sewage are calculated according to N ARGs ＝C Plasmid(s) ×6.05×10 4 /M DNA Calculating plasmid concentration; preparing artificial sewage of the required ARGs type and abundance by using the mixed culture medium in the step S2, and calculating the concentration of the antibiotic resistance gene plasmid;
wherein N in the formula ARGs Initial ARGs abundance is set according to ARGs pollution level, and the unit is copies/mL;
C plasmid(s) Concentration of the antibiotic resistance gene plasmid is given in ng/mL;
M DNA DNA molecular weight of the antibiotic resistance gene.
In the step S5, the cell electroporation method is adopted to carry out cell perforation on the extracted algae bacteria mixed solution, and the specific steps are as follows:
s501: placing 1-2mL of the algae bacteria mixed solution in the step S4 into a sterile centrifuge tube, rapidly placing the sterile centrifuge tube on an ice surface, ensuring that the ice surface covers the centrifuge tube, keeping the temperature for 5min, centrifuging at the temperature of 4 ℃, and retaining sediment;
s502: adding 1-2mL cytomix buffer solution into the precipitate in the step S501, centrifuging for 2-5min at 10000-12000g/min and 0-4deg.C, retaining the precipitate, and repeating the step for 3 times;
s503: adding 100-150 mu L of sucrose solution with mass fraction of 15-20% into 5-10 mu L of artificial sewage obtained in step S3 as a cell protection solution, adding the cell protection solution into the sediment obtained in step S502, and blowing by using a pipetting gun;
s504: transferring the solution blown in the step S503 to a pre-cooling electric shock cup for 5-10min for electric shock pulse treatment;
s505: cooling the solution subjected to electric shock in the step S504 for 15-30min, centrifuging for 10-30S at 8000-10000g/min and 0-4 ℃, reserving sediment, placing the sediment in the algae-bacteria symbiotic system in the step S4 again, continuously culturing in a sterile room with constant temperature and constant illumination, and after culturing for 7-10 days, effectively reducing the relative abundance of ARGs in artificial sewage.
The step S1 specifically comprises the following steps:
s101: inoculating Chlorella vulgaris in logarithmic phase into sterilized BG11 culture medium, performing aseptic culture for 3-5 generations to reach algae cell concentration of 1×10 8 cell/mL-9×10 8 The cell/mL is used for culturing the algae-bacteria symbiota;
s102: inoculating Bacillus licheniformis in logarithmic growth phase into sterilized LB medium, culturing in aseptic chamber under illumination for 5-10 generations to obtain bacterial cell concentration of 1×10 6 CFU/mL-9×10 6 CFU/mL is used for culturing the algae-bacteria symbiota;
s103: taking 1-5mL of chlorella vulgaris and 1-5mL of bacillus licheniformis, inoculating the chlorella vulgaris and the bacillus licheniformis into 150-200mL of BG11-LB culture medium in a sterile room, wherein the BG11 culture medium and the LB culture medium are mixed according to the volume ratio of 1:1-1:5.
The constant temperature and the constant illumination temperature in the step S2 and the step S5 are set to 26-30 ℃, and the illumination intensity is set to 120 mu mol/m 2 /s-150μmol/m 2 /s。
The antibiotic resistance gene is selected from any one or any combination of sulfonamide antibiotic resistance genes (sul 1, sul 2) or tetracycline antibiotic resistance genes (tetM, tetQ, tetW).
By adopting the technical scheme, the invention has the following advantages and positive effects compared with the prior art:
(1) Common chlorella is not easy to be infected by exogenous ARGs, bacillus licheniformis is not easy to be induced by antibiotics to generate ARGs, and even if exogenous ARGs enter algal symbiont cells, the replication and expression of genetic materials are difficult to realize. Therefore, the common chlorella-bacillus licheniformis symbiont can safely and efficiently remove ARGs in the water body.
(2) The method for introducing the ARGs into the chlorella cells and the bacillus licheniformis somatic cells by using the cell electroporation method has the advantages of low cost, quick and simple operation, high survival rate of 80% -90% of the chlorella cells and the bacillus licheniformis somatic cells after perforation, normal growth and propagation of the surviving chlorella cells and the bacillus licheniformis somatic cells (but the ARGs entering the chlorella cells and the bacillus licheniformis somatic cells are not expressed in the chlorella cells and the bacillus licheniformis somatic cells).
(3) The chlorella cells and the bacillus licheniformis somatic cells have higher activity before and after perforation, so that an electric field can be applied to the polluted water body of the ARGs to be treated, pulse cell perforation is realized, ARGs are promoted to efficiently enter the algae cells and the somatic cells, biological genetic blocking of the ARGs in the water body by the common chlorella cells and the bacillus licheniformis is realized, and the removal of the ARGs in the water body is realized by combining a physical method and a biological method.
Drawings
FIG. 1 is a schematic diagram of the mechanism of algae-bacteria symbiotic system for removing ARGs in water after cell electroporation according to the invention.
Detailed Description
The construction method of the algae-bacteria symbiotic system for removing the antibiotic resistance genes in the water body is further described in detail below with reference to the accompanying drawings and specific examples. Advantages and features of the invention will become more apparent from the following description and from the claims.
A construction method of an algae-bacteria symbiotic system for removing antibiotic resistance genes in a water body comprises the following steps:
s1: inoculating chlorella vulgaris and bacillus licheniformis into sterilized 150-200mL BG11-LB culture medium;
s2: placing the mixed culture medium inoculated with Chlorella vulgaris and Bacillus licheniformis in a sterile room with constant temperature and constant illumination, culturing at 26-30deg.C and 120 μmol/m 2 /s-150μmol/m 2 /s；
S3: preparing artificial sewage containing antibiotic resistance gene plasmid;
s4: mixing the mixed culture medium in the step S2 with the artificial sewage in the step S3 to form an algae-bacteria symbiotic system;
s5: taking 1-2ml of the mixed solution of algae in the step S4, performing cell electroporation on the taken mixed solution of algae by adopting a cell electroporation method, cooling, then placing the mixed solution of algae in the symbiotic system of algae in the step S4 again, continuously culturing in a sterile room with constant temperature and constant illumination, setting the temperature to 26-30 ℃ and the illumination intensity to 120 mu mol/m 2 /s-150μmol/m 2 /s。
The artificial sewage preparation method containing the antibiotic resistance gene plasmid in the step S3 comprises the following steps: the plasmid finished product of the constructed target antibiotic resistance gene and the initial antibiotic resistance gene abundance in artificial sewage are processed according to N ARGs ＝C Plasmid(s) ×6.05×10 4 /M DNA Calculating plasmid concentration, wherein the antibiotic resistance gene species is selected from any one or any combination of sul1, sul2 and tetM, tetQ, tetW;
wherein N in the formula ARGs For the initial antibiotic resistance gene abundance of artificial sewage, the initial ARGs abundance is 2.09 multiplied by 10 5 -1.26×10 9 copies/mL；
C Plasmid(s) Concentration of the antibiotic resistance gene plasmid is given in ng/mL;
M DNA DNA molecular weight of the antibiotic resistance gene.
In the step S5, the cell electroporation method is adopted to carry out cell perforation on the extracted algae bacteria mixed solution, and the specific steps are as follows:
s501: placing 1-2mL of the algae bacteria mixed solution in the step S4 into a sterile centrifuge tube, rapidly placing the sterile centrifuge tube on an ice surface, ensuring that the ice surface covers the centrifuge tube, keeping the temperature for 5min, centrifuging at the temperature of 4 ℃, and retaining sediment;
s502: adding 1-2mL cytomix buffer solution into the precipitate in the step S501, centrifuging for 2-5min at 10000-12000g/min and 0-4deg.C, retaining the precipitate, and repeating the step for 3 times;
s503: adding 100-150 mu L of sucrose solution with mass fraction of 15-20% into 5-10 mu L of artificial sewage obtained in step S3 as a cell protection solution, adding the cell protection solution into the sediment obtained in step S502, and blowing by using a pipetting gun;
s504: transferring the solution blown in the step S503 to a pre-cooling electric shock cup for 5-10min for electric shock pulse treatment;
s505: cooling the solution subjected to electric shock in the step S504 for 15-30min, centrifuging for 10-30S at 8000-10000g/min and 0-4 ℃, reserving sediment, placing the sediment in the algae-bacteria symbiotic system in the step S4 again, continuously culturing in a sterile room with constant temperature and constant illumination, and after culturing for 7-10 days, effectively reducing the relative abundance of ARGs in artificial sewage.
The step S1 specifically comprises the following steps:
s101: inoculating Chlorella vulgaris in logarithmic phase into sterilized BG11 culture medium, performing aseptic culture for 3-5 generations to reach algae cell concentration of 1×10 8 cell/mL-9×10 8 The cell/mL is used for culturing the algae-bacteria symbiota;
s102: inoculating Bacillus licheniformis in logarithmic growth phase into sterilized LB medium, culturing in aseptic chamber under illumination for 5-10 generations to obtain bacterial cell concentration of 1×10 6 CFU/mL-9×10 6 CFU/mL is used for culturing the algae-bacteria symbiota;
s103: taking 1-5mL of chlorella vulgaris and 1-5mL of bacillus licheniformis, inoculating the chlorella vulgaris and the bacillus licheniformis into 150-200mL of BG11-LB culture medium in a sterile room, wherein the BG11 culture medium and the LB culture medium are mixed according to the volume ratio of 1:1-1:5.
The formula of the BG11 medium is as follows:
the formulation of LB medium was as follows:
according to the invention, the chlorella vulgaris can resist toxicity induction of antibiotics in water environment and remove antibiotics and ARGs, and particularly has good removal effect on typical dominant ARGs such as sul1, sul2, tetM, tetQ and the like in water. The integration site of the exogenous ARGs entering the chlorella vulgaris cells is located in heterochromatin regions, so that the exogenous ARGs can not be expressed in the cells, and the integration site is one of possible ways for blocking the transmission of the exogenous ARGs by the chlorella vulgaris. The common chlorella is high in safety for removing ARGs in the water body, and is not easy to induce antibiotics in the water body and infect external ARGs. Bacillus licheniformis is widely used as a beneficial bacterium in water, can block the horizontal transfer of ARGs to a certain extent, and is often applied to the removal of ARGs in water. The algae-bacteria symbiote realizes the dual advantages of removing ARGs from algae cells and thalli, and can effectively remove ARGs such as sul1, sul2, tetM, tetQ, tetW and the like in water.
Referring to fig. 1, the method for introducing the ARGs plasmid into the chlorella cells and the bacillus licheniformis somatic cells by using the cell electroporation method provided by the invention has the advantages of low cost, rapid and simple operation, the survival rate of the chlorella cells and the bacillus licheniformis somatic cells is up to 80% -90% after perforation, and the surviving chlorella cells and bacillus licheniformis somatic cells can normally grow and reproduce in a culture medium, but ARGs entering the chlorella cells and the bacillus licheniformis somatic cells are not expressed in the chlorella cells and the bacillus licheniformis somatic cells, the perforated chlorella cells and the bacillus licheniformis somatic cells are placed in artificial sewage containing an algae bacteria mixed solution again, and more ARGs enter the chlorella cells and the bacillus licheniformis somatic cells, so that the biological genetic blocking of the ARGs in a water body by common chlorella cells and bacillus licheniformis is realized, and the removal of the ARGs in the water body is realized.
Therefore, based on the mechanism, an electric field can be applied to the ARGs polluted water body to be treated, so that pulse cell perforation is realized, ARGs are promoted to enter algae cells and thallus cells efficiently, and biological genetic blocking of the ARGs in the water body by common chlorella cells and bacillus licheniformis is realized.
Example 1
The concentration of the algae cells is 1 multiplied by 10 8 cell/mL Chlorella vulgaris (purchased from fresh water algae of China academy of sciences)Seed pool, algal cell number: FACHB-8) 5mL, cell concentration of 1X 10 6 CFU/mL of Bacillus licheniformis (purchased from China academy of sciences strain collection, alga cell number: 1.7461) 5mL; centrifuging (8000 g/min,3 min) respectively, removing supernatant, inoculating the obtained 2 precipitates into 150mL sterilized BG11-LB medium, culturing in a constant temperature and constant illumination sterile room at 28deg.C with illumination intensity of 120 μmol/m 2 And/s, wherein the volume ratio of the BG11 culture medium to the LB culture medium is 1:1;
adding sul1 0.011-65.2 μg, sul2 0.010-58.6 μg, tetM 0.010-58.6 μg, tetQ 0.011-64.2 μg, tetW 0.010-58.6 μg into artificial sewage to obtain ARGs plasmid with concentration of 2.09×10 5 -1.26×10 9 Artificial sewage of cobies/mL;
inoculating the BG11-LB culture medium into 150mL artificial sewage containing ARGs plasmid, and setting the temperature at 28deg.C and illumination intensity at 120 μmol/m in a sterile room with constant temperature and constant illumination 2 And/s, culturing for 5 days to obtain the algae-bacteria symbiote (algae-bacteria mixed solution).
Cell electroporation treatment: taking 2mL of the algae bacteria mixed solution, rapidly placing the mixed solution on an ice surface, ensuring that the ice surface covers a centrifuge tube, keeping the temperature for 5min, centrifuging at 4 ℃, discarding the supernatant, and retaining the precipitate; adding 2mL cytomix buffer solution into the algae bacteria mixed solution, continuously blowing to fully and uniformly mix the algae bacteria mixed solution buffer solution, centrifuging for 2min at 0-4 ℃ at 10000g/min after uniformly mixing, discarding the supernatant, and repeating the step for 3 times; adding 5 mu L of artificial sewage, blowing uniformly by using a pipetting gun, and adding 100-150 mu L of 15-20% sucrose solution in mass fraction into the 5 mu L of artificial sewage as a protective solution; transferring the solution obtained in the steps into an electroporation cuvette pre-cooled for 5min in advance for electric shock pulse treatment (output voltage is 10-2000V, square wave discharge time is 10-150ms, pulse repetition is set for 1-3 times, and each time is 3-5s apart); placing the electric shock mixed solution of algae bacteria in ice box, cooling for 15min, centrifuging at 8000g/min and 4deg.C for 30s, discarding supernatant, adding the obtained precipitate into algae bacteria symbiont, and culturing for 5-7 days.
Comparative example
Equal amounts of Chlorella vulgaris and Bacillus licheniformis were inoculated alone according to the procedure in example 1.
And then detecting the relative abundance of ARGs in the artificial sewage by using real-time quantitative PCR, wherein the result is shown in a table 1, and the numbers in the table represent the absolute abundance reduction rate of ARGs in the artificial sewage in a single algae cell system, a single bacterial cell system and an algae symbiotic system.
TABLE 1 ARGs absolute abundance reduction Rate for different systems
As can be seen from Table 1, the reduction of ARGs abundance in water can be achieved by the single system of Chlorella vulgaris, the single system of Bacillus licheniformis and the symbiotic system of Chlorella vulgaris and Bacillus licheniformis. The chlorella vulgaris cells have obvious biological removal effects on sulfonamide ARGs (sul 1, sul 2) and tetracycline ARGs (tetM, tetQ, tetW), and compared with the removal effects of bacillus licheniformis on ARGs in water bodies are relatively weak. But the algae-bacillus symbiota formed by the two can absorb most ARGs in the water body into cells after electric shock, so that biological blocking of the ARGs is realized, and the common chlorella-bacillus licheniformis symbiota can effectively remove the ARGs in the water body.
Example 2
Antibiotic resistance gene in actual river sewage is reduced by adopting algae-bacteria symbiotic system
Collecting 200-500mL of river sewage, and detecting to find that the abundance of ARGs in the river water is sul1: 1.5X10 5 copies/mL、sul2:0.4×10 5 copies/mL、tetM：1.2×10 5 copies/mL、tetQ：2.35×10 5 copies/mL、tetW：0.78×10 5 copies/mL，
150mL of BG11-LB culture medium inoculated with chlorella and bacillus licheniformis is mixed with the collected river water, and the initial adding concentration of the algae-bacteria symbiont is 2.5X10 6 cell/mL, culture temperature of 28+ -2deg.C, light-dark ratio of 12h:12h, the illumination intensity is 120 mu mol/m 2 Culture/sCulturing for 5 days under the condition of culturing to obtain an algae-bacteria symbiont (algae-bacteria mixed solution);
after the cell electroporation treatment, continuously placing the cells in the collected river sewage for culture, wherein the relative abundance of sul1, sul2 and tetM, tetQ, tetW in the water body are respectively as follows: 0.3X10 5 copies/mL、0.03×10 5 copies/mL、0.12×10 5 copies/mL、0.27×10 5 copies/mL、0.021×10 5 cobies/mL did decrease the relative abundance of the antibiotic resistance gene.
The embodiments of the present invention have been described in detail with reference to the drawings, but the present invention is not limited to the above embodiments. Even if various changes are made to the present invention, it is within the scope of the appended claims and their equivalents to fall within the scope of the invention.
Claims (2)
1. The construction method of the algae-bacteria symbiotic system for removing the antibiotic resistance genes in the water body is characterized in that the antibiotic resistance genes are selected from any one or any combination of sul1, sul2 and tetM, tetQ, tetW, and the construction method comprises the following steps:
s1: inoculating chlorella vulgaris cells and bacillus licheniformis into sterilized 150-200mL BG11-LB culture medium;
s2: placing the mixed culture medium inoculated with the chlorella vulgaris and the bacillus licheniformis in a sterile room with constant temperature and constant illumination for culture;
s3: preparing artificial sewage containing antibiotic resistance gene plasmid;
s4: mixing the mixed culture medium in the step S2 with the artificial sewage in the step S3 to form an algae-bacteria symbiotic system;
s5: taking 1-2mL of the mixed solution of the algae bacteria in the step S4, performing cell perforation on the taken mixed solution of the algae bacteria by adopting a cell electroporation method, cooling, and then placing the mixed solution of the algae bacteria in the symbiotic system of the algae bacteria in the step S4 again, and continuously culturing in a sterile room with constant temperature and constant illumination;
the step S1 specifically comprises the following steps:
s101: inoculating Chlorella vulgaris in logarithmic phase into sterilized BG11 culture medium, and feedingCulturing in sterile condition for 3-5 generations until the concentration of algae cells reaches 1×10 8 cell/mL-9×10 8 The cell/mL is used for culturing the algae-bacteria symbiota;
s102: inoculating Bacillus licheniformis in logarithmic growth phase into sterilized LB medium, culturing in aseptic chamber under illumination for 5-10 generations to obtain bacterial cell concentration of 1×10 6 CFU/mL-9×10 6 CFU/mL is used for culturing the algae-bacteria symbiota;
s103: inoculating 1-5mL of chlorella vulgaris and 1-5mL of bacillus licheniformis into 150-200mL of BG11-LB culture medium in a sterile room, wherein the BG11 culture medium and the LB culture medium are mixed according to the volume ratio of 1:1-1:5;
the artificial sewage preparation method containing the antibiotic resistance gene plasmid in the step S3 comprises the following steps: according to N ARGs ＝C Plasmid(s) ×6.05×10 4 /M DNA Calculating plasmid concentration, and then preparing artificial sewage containing antibiotic resistance gene plasmid;
wherein N in the formula ARGs The gene abundance is the initial antibiotic resistance gene abundance of artificial sewage, and the unit is copies/mL;
C plasmid(s) Concentration of the antibiotic resistance gene plasmid is given in ng/mL;
M DNA DNA molecular weight which is an antibiotic resistance gene;
in the step S5, the cell electroporation method is adopted to carry out cell perforation on the extracted algae bacteria mixed solution, and the specific steps are as follows:
s501: placing 1-2mL of the algae bacteria mixed solution in the step S4 into a sterile centrifuge tube, placing the centrifuge tube on ice, keeping the ice for 5min, centrifuging at the temperature of 4 ℃, and retaining sediment;
s502: adding 1-2mL cytomix buffer solution into the precipitate in the step S501, centrifuging for 2-5min at 10000-12000g/min and 0-4deg.C, retaining the precipitate, and repeating the step for 3 times;
s503: adding 100-150 mu L of sucrose solution with mass fraction of 15-20% into 5-10 mu L of artificial sewage obtained in step S3 as a cell protection solution, adding the cell protection solution into the sediment obtained in step S502, and blowing by using a pipetting gun;
s504: transferring the solution blown in the step S503 to a pre-cooling electric shock cup for 5-10min for motor pulse treatment;
s505: cooling the solution subjected to electric shock in the step S504 for 15-30min, centrifuging at 8000-10000g/min and 0-4deg.C for 10-30S, retaining precipitate, placing the precipitate in the symbiotic system of algae bacteria in the step S4, and continuously culturing in a sterile room with constant temperature and constant illumination.
2. The method for constructing an symbiotic system of algae for removing antibiotic resistance genes from a water body according to claim 1, wherein the constant temperature and constant illumination temperature in step S2 and step S5 are set to 26-30 ℃, and the illumination intensity is set to 120 μmol/m 2 /s-150μmol/m 2 /s。
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