CN112980866B - Plasmid transformation method of bacillus polymyxa - Google Patents
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Abstract
The invention discloses a multi-adhesiveMethod for transforming a bacillus by means of a plasmid on an oligotrophic medium + 、Ca 2+ 、Mg 2+ The bacillus polymyxa is stimulated to form competence together, and then plasmid transformation of the strain is completed on a culture medium. In addition, the method can be carried out on a solid culture medium, and compared with liquid transformation, the method has higher efficiency and more convenient operation.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a plasmid transformation method of bacillus polymyxa.
Background
The bacillus polymyxa can prevent and treat various plant diseases caused by pathogenic bacteria, pathogenic fungi and root-knot nematodes, is a biocontrol agent widely applied to agricultural production, and researches show that a bacillus polymyxa fermentation product can help crops to resist diseases and promote the income of crops to a certain extent, and is a typical plant rhizosphere growth promoting bacterium. How the rhizosphere growth-promoting bacteria colonize around the root system and how the rhizosphere growth-promoting bacteria act around the root system as a biocontrol agent all need to carry out molecular biological marking on the bacteria so as to be convenient for tracking, so the rhizosphere growth-promoting bacteria need to be modified on a gene level.
After the bacillus polymyxa is domesticated for a long time under laboratory conditions, a few strains establish a stable plasmid operation system. However, the method for industrially producing the bacillus polymyxa is still in the early stage of isolation, purification, identification and reactor application because the bacillus polymyxa is isolated from nature, and the bacillus polymyxa isolated from nature has various restriction-modification systems, so that few reports are made on the methods for manipulating plasmids of the bacillus polymyxa.
At present, methods for transforming plasmids for bacillus include electric transformation and natural transformation. The electrical transformation method is to make the surface of the cell have small holes by using an electric shock method so as to make DNA enter the cell. The applicant tried to transform by the electric transformation method but failed to achieve the transformation method, because the electric transformation method has higher requirements on the cell density of the bacillus polymyxa, but the industrial bacillus polymyxa KN-03 is easier to autolyze, and autolysis occurs when the surface ions of the cells are washed in the electric transformation process, and finally the electric transformation fails. The natural transformation method is to induce the expression of competence formation related genes in thallus and the expression of uptake genes by adding certain chemical reagents so as to enable exogenous plasmids to enter cells, and the current common chemical method for preparing competent cells is a liquid natural transformation method. The method needs more solutions and is troublesome to prepare, and the method has lower transformation efficiency than an electric transformation method, so the liquid natural transformation method is rarely used for transformation of bacillus plasmids.
According to the principle of the traditional liquid natural transformation method, a set of high-efficiency solid plate natural plasmid transformation method suitable for bacillus polymyxa is developed on the basis of the method. The invention changes the characteristic of low transformation efficiency of the traditional liquid natural transformation method, provides a molecular tool for the operation of the bacterial plasmid and the molecular biological marking, and has very profound significance.
Disclosure of Invention
The invention aims to provide a method for transforming bacillus polymyxa plasmids, and aims to improve the success rate and the transformation efficiency of the bacillus polymyxa plasmids.
A method for transforming a plasmid of Bacillus polymyxa, comprising the steps of:
1) Culturing the bacillus polymyxa strain until the OD600 value is between 0.9 and 1.5, and collecting the strain;
2) Culturing the thalli by using an amplification culture medium, transferring the thalli to an oligotrophic culture medium for primarily stimulating competent cells to form, culturing for 5-10 h, then transferring the thalli to an oligotrophic culture medium for forming final competent cells, and continuously culturing for 1-3 h;
3) Reacting the exogenous plasmid with the competent cell, and transferring the exogenous plasmid into the competent cell.
By K + 、Ca 2+ Under the stimulation of the expression level of the comK gene of the core regulatory gene for controlling the formation of cell competence, the expression of the subsequent related genes of cell fragment adsorption, fragment shearing and uptake is started, and then Mg 2+ The permeability of the cell membrane is enhanced by stimulating the cell membrane, and the fragment is further facilitated to enter the cell.
Preferably, the oligotrophic medium used to preliminarily stimulate the formation of competent cells is a K-Ca oligotrophic medium containing potassium and calcium salts; the oligotrophic medium used to form the final competent cells is a K-Mg oligotrophic medium containing potassium and magnesium salts.
Further preferably, the total concentration of potassium and calcium in the K-Ca oligotrophic medium is 0.6-2M, and the molar ratio of potassium to calcium is 6-10: 1. The following examples of the present invention (ii) a; the total concentration of potassium and magnesium in the K-Mg oligotrophic culture medium is 0.8-2M, the molar ratio of potassium to magnesium is 15-20: 1.
further preferably, the total concentration of potassium and calcium in the K-Ca oligotrophic medium is 0.9M, and the molar ratio of potassium to calcium is 8:1; the total concentration of potassium and magnesium in the K-Mg oligotrophic culture medium is 1M, and the molar ratio of potassium to magnesium is 16.7:1.
further preferably, the K-Ca oligotrophic medium and the K-Mg oligotrophic medium contain 0.05-0.2M glucose and 0.001-0.006M ammonium salt.
Preferably, the expansion medium is LB medium.
Preference is given to the ground is a mixture of a plurality of ground, the amplification medium and the oligotrophic medium are both solid media containing agar. Experiments show that competent cell culture under solid conditions has higher plasmid transformation efficiency than culture under liquid conditions.
Preferably, the culturing in step 2) is carried out at 25-35 ℃.
Preferably, the reaction time in step 3) is 1 to 5 hours.
Preferably, the bacillus polymyxa is KN-03, and the preservation number is M2012077.KN-03 is an industrial polymyxa bacterium, and plasmid transformation is more difficult than ordinary polymyxa bacteria such as laboratory bacteria.
The ammonium salt in the present invention includes, but is not limited to, ammonium chloride, ammonium bromide, ammonium iodide, ammonium sulfate, ammonium nitrate, ammonium carbonate and other ionic compounds composed of ammonium ions and acid ions.
The potassium salt, calcium salt and magnesium salt in the invention are composed of K + 、Ca 2+ 、Mg 2+ Salts with acid ions including, but not limited to, chloride, iodide, bromide, carbonate, nitrate, sulfate, oxalate, and the like.
According to an embodiment of the present invention, the best solution is as follows:
1) Inoculating the KN-03 strain to an LB liquid culture medium, culturing until the OD600 value is 1.0-1.2, and collecting a bacterial liquid after the culture is finished.
2) Adding the bacterial liquid to an LB solid culture medium, culturing for 3h at 30 ℃, then transferring to a K-Ca oligotrophic solid culture medium, culturing for 8h to preliminarily stimulate the formation of competent cells, finally transferring to a K-Mg oligotrophic solid culture medium, and continuously culturing for 2h to form final competent cells;
K-Ca oligotrophic solid medium: glucose 0.15M; NH (NH) 4 Cl 0.005M；KCl 0.8M；CaCl 2 0.1M; 15g/L of agar; the balance of water; pH7.4;
K-Mg oligotrophic solid medium: glucose 0.1M; NH 4 Cl 0.003M；KCl 1M；MgCl 2 0.06M; 15g/L of agar; the balance of water; pH7.4.
3) The foreign plasmid was added to the competent cells, and reacted at 30 ℃ for 2 hours, thereby transferring the plasmid into the competent cells.
The invention has the beneficial effects that:
the invention is realized by adding K + and Ca on an oligotrophic culture medium 2+ 、Mg 2+ The bacillus polymyxa is stimulated to form competence together, and then plasmid transformation of the strain is completed on a culture medium.
In previous studies, the applicant also found that the two-step transformation method is only suitable for Bacillus polymyxa and not for other Bacillus such as Bacillus subtilis, and the test found that the one-step transformation efficiency of Bacillus subtilis on a solid plate is 1e 3 Whereas the two-step conversion efficiency on solid plates was only 1e 1 -1e 2 。
The plasmid transformation of the invention can be carried out on a solid culture medium, compared with liquid transformation, the solid transformation plasmid is more fully contacted with competent cells, so that the plasmid can be well adsorbed on the surface of the cells and enter the cells.
The invention not only solves the problems of complex preparation process of competent cells, low transformation efficiency and easy autolysis in the electric transformation process in the industrial natural plasmid transformation process of the bacillus polymyxa liquid, which troubles the field for a long time, but also provides reference for the transformation of other bacillus polymyxa microorganisms and provides a favorable tool for the researches of molecular biology, genetic engineering transformation and the like of the bacillus polymyxa.
Drawings
FIG. 1 shows the identification of plasmid extracted from the transformed strain by gel electrophoresis according to the present invention.
FIG. 2 shows PCR gel electrophoresis identification of kanamycin resistance gene of pBE123 plasmid transformed strain according to the present invention.
Detailed Description
The present invention is further illustrated in detail by the following specific examples, which are provided to illustrate the present invention but are not intended to limit the scope thereof. The experimental procedures used in the following examples are, unless otherwise specified, conventional and may be carried out in accordance with the procedures specified in molecular cloning, a laboratory manual (third edition) J. SammBruke, or in accordance with kits and product instructions. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The test strains are Bacillus polymyxa (BaciIllus polymyxa) KN-03 and BNCC194976, wherein the KN-03 strain has the accession number CCTCC NO: M2012077, which is disclosed in the patent literature CN 104611254A on 5/13/2015, and BNCC194976 is another industrial Bacillus polymyxa, commercially available from the Beijing Beinan union Biotechnology research institute, but the embodiments of the present invention are not limited to KN-03 and BNCC194976. The plasmid pBE123 selected for the experiments was a commercial plasmid, which was purchased from Henan biosciences.
Example 1
A method for transforming a plasmid of bacillus polymyxa comprises the following specific steps:
1) Preparation of bacterial liquid
Inoculating the KN-03 strain to an LB liquid culture medium, culturing for 16h, determining the OD600 value to be 1.1, and collecting a bacterial liquid after the culture is finished, wherein the growth state of the strain is in the early and middle stages of exponential growth.
LB liquid medium: tryptone 10g/L; 5g/L of yeast extract; 10g/L of sodium chloride; pH7.4.
2) Two-step method for preparing competent cells on solid plate
Adding 20 microliters of bacterial liquid onto a filter membrane with the aperture of 0.22 micrometer on the surface of an LB solid culture medium, culturing for 3 hours at the temperature of a culture plate being 30 ℃ to enable the thalli to propagate rapidly, then transferring the filter membrane with the lawn on the surface, which is stuck on the LB solid culture medium, to a K-Ca oligotrophic solid culture medium for culturing for 8 hours to preliminarily stimulate the formation of competent cells, and finally transferring the filter membrane with the lawn on the surface, which is stuck on the K-Ca oligotrophic solid culture medium, to the K-Mg oligotrophic solid culture medium for culturing for 2 hours to form the final competent cells.
LB solid medium: tryptone 10g/L; 5g/L of yeast extract; 10g/L of sodium chloride; 15g/L of agar; pH7.4.
K-Ca oligotrophic solid medium: glucose 0.15M; NH (NH) 4 Cl 0.005M；KCl 0.8M；CaCl 2 0.1M; 15g/L of agar; the balance of water; pH7.4;
K-Mg oligotrophic solid medium: glucose 0.1M; NH 4 Cl 0.003M；KCl 1M；MgCl 2 0.06M; 15g/L of agar; the balance of water; pH7.4.
3) Plasmid extraction and transformation
Inoculating Escherichia coli containing plasmid pBE123 into LB liquid culture medium, culturing for 12 hr, extracting plasmid with Tiangen plasmid extraction kit.
2 micrograms of plasmid pBE123 was directly added to the surface of competent cell lawn on the filter, and reacted at 30 ℃ for 2 hours.
4) Dilution coated sheet after conversion
The cell mixture was eluted from the filter with ddH2O, followed by centrifugation at 12000rpm at 4 ℃ for 10min, the supernatant was discarded, and the remaining bacterial suspension was directly spread on a plate of an antibiotic containing kanamycin, followed by culture in an incubator at 30 ℃ for 12 to 16h, and the colony growth was observed.
5) Screening and identification of positive strains
Picking single colony on antibiotic plate, inoculating part of the colony to liquid culture medium containing kanamycin, taking part of the colony for PCR detection and extracting plasmid for identification. Primers for PCR detection primers were designed based on the resistance gene for kanamycin: an upstream sequence 5'-ATGAGAATAGTGAATGGA-3'; downstream sequence 5'-TCAAAATGGTATGCGTTT-3'.
Example 2
1) Preparation of bacterial liquid
And inoculating the KN-03 strain to an LB liquid culture medium, culturing until the OD600 value is 0.9, and collecting a bacterial liquid after the culture is finished.
2) Two-step method for preparing competent cells on solid plate
Adding 20 microliters of bacterial liquid to a filter membrane with a pore diameter of 0.22 micrometer on the surface of an LB solid culture medium, culturing for 4 hours at the temperature of a culture plate of 28 ℃, transferring the filter membrane with the lawn on the surface, which is stuck on the LB solid culture medium, to a K-Ca oligotrophic solid culture medium for culturing for 5 hours, transferring the filter membrane with the lawn on the surface, which is stuck on the K-Ca oligotrophic solid culture medium, to a K-Mg oligotrophic solid culture medium, and culturing for 3 hours to form final competent cells.
K-Ca oligotrophic solid medium: glucose 0.2M, NH 4 Cl 0.006M、KCl 1.2M、CaCl 2 0.2M agar 15g/L; the balance of water; pH7.4;
K-Mg oligotrophic solid medium: glucose 0.15M, NH 4 Cl 0.005M、KCl 1.8M、MgCl 2 0.1M agar 15g/L; the balance of water; pH7.4.
3) Transformation of plasmids
2. Mu.g of plasmid pBE123 was directly added to the surface of competent cell lawn on the filter and reacted for 2 hours at 28 ℃.
The subsequent steps were the same as in example 1.
Example 3
1) Preparation of bacterial liquid
And inoculating the KN-03 strain to an LB liquid culture medium, culturing until the OD600 value is 1.5, and collecting a bacterial liquid after the culture is finished.
2) Two-step method for preparing competent cells on solid plate
Adding 20 microliters of bacterial liquid to a filter membrane with a pore diameter of 0.22 micrometer on the surface of an LB solid culture medium, culturing for 2 hours at the temperature of a culture plate of 32 ℃, transferring the filter membrane with the lawn on the surface, which is stuck on the LB solid culture medium, to a K-Ca oligotrophic solid culture medium for culturing for 10 hours, transferring the filter membrane with the lawn on the surface, which is stuck on the K-Ca oligotrophic solid culture medium, to a K-Mg oligotrophic solid culture medium, and culturing for 1 hour to form final competent cells.
K-Ca oligotrophic solid medium: glucose 0.08M, NH 4 Cl 0.003M、KCl 0.6M、CaCl 2 0.06M agar 15g/L; the balance of water; pH7.4;
K-Mg oligotrophic solid medium: glucose 0.05M, NH 4 Cl 0.002M、KCl 0.8M、MgCl 2 0.05M agar 15g/L; the balance of water; pH7.4.
3) Transformation of plasmids
2. Mu.g of plasmid pBE123 was directly added to the surface of competent cell lawn on the filter and reacted at 32 ℃ for 2 hours.
The subsequent steps were the same as in example 1.
Example 4
1) Preparation of bacterial liquid
And inoculating the KN-03 strain to an LB liquid culture medium, culturing until the OD600 value is 1.1, and collecting a bacterial liquid after the culture is finished.
2) Two-step method for preparing competent cells under liquid condition
Transferring 20 microliters of bacterial liquid into 5mL of LB liquid culture medium, culturing at 30 ℃ for 3 hours to enable the bacteria to be rapidly propagated, then centrifuging the LB bacterial suspension at 12000rpm, discarding the supernatant, re-suspending with 5mL of K-Ca oligotrophic liquid culture medium, culturing at 200rpm for 8 hours to preliminarily stimulate the formation of competent cells, then centrifuging the bacterial suspension at 12000rpm, discarding the supernatant, finally re-suspending with 5mL of K-Mg oligotrophic liquid culture medium, and culturing at 200rpm for 2 hours to form the final competent cells.
K-Ca oligotrophic liquid medium: glucose 0.15M, NH 4 Cl 0.005M、KCl 0.8M、CaCl 2 0.1M and the balance of water; pH7.4;
K-Mg oligotrophic liquid medium: glucose 0.1M, NH 4 Cl 0.003M、KCl 1M、MgCl 2 0.06M and the balance of water; pH7.4.
3) Transformation of plasmids
2 micrograms of plasmid pBE123 was directly mixed with 100 microliters of prepared competent cells, and reacted at 30 ℃ for 2 hours.
The subsequent steps were the same as in example 1.
Example 5
1) Preparation of bacterial liquid
And inoculating the KN-03 strain to an LB liquid culture medium, culturing until the OD600 value is 1.1, and collecting a bacterial liquid after the culture is finished.
2) One-step method for preparing competent cells on solid plate
Adding 20 microliters of bacterial liquid to a filter membrane with the aperture of 0.22 micrometer on the surface of an LB solid culture medium, culturing for 3 hours at the temperature of a culture plate of 30 ℃ to enable thalli to propagate rapidly, and then transferring the filter membrane with lawn on the surface, which is stuck on the LB solid culture medium, to a K-Ca-Mg oligotrophic solid culture medium for culturing for 12 hours to form final competent cells.
K-Ca-Mg oligotrophic solid medium: glucose 0.15M, NH 4 Cl 0.005M、KCl 1M、CaCl 2 0.1M、MgCl 2 0.06M agar and 15g/L agar; the balance of water; pH7.4;
3) Plasmid extraction and transformation
2 microgram of plasmid pBE123 was directly added to the surface of competent cell lawn on the filter, and reacted at 30 ℃ for 2 hours.
The subsequent steps were the same as in example 1.
Example 6
1) Preparation of bacterial liquid
And inoculating the KN-03 strain to an LB liquid culture medium, culturing until the OD600 value is 1.1, and collecting a bacterial liquid after the culture is finished.
2) One-step method for preparing competent cells on solid plate
Adding 20 microliters of bacterial liquid to a filter membrane with the aperture of 0.22 micrometer on the surface of an LB solid culture medium, culturing for 3 hours at the temperature of a culture plate of 30 ℃ to enable thalli to propagate rapidly, and then transferring the filter membrane with lawn on the surface, which is stuck on the LB solid culture medium, to a K-Ca oligotrophic solid culture medium for culturing for 12 hours to form final competent cells.
K-Ca oligotrophic solid medium: glucose 0.15M, NH 4 Cl 0.005M、KCl 1M、CaCl 2 0.1M agar 15g/L; the balance of water; pH7.4;
3) Plasmid extraction and transformation
2 micrograms of plasmid pBE123 was directly added to the surface of competent cell lawn on the filter, and reacted at 30 ℃ for 2 hours.
The subsequent steps were the same as in example 1.
Example 7
The procedure and conditions, parameters were the same as in example 1 except that KCl was replaced with NaCl in the two oligotrophic solid media.
Example 8
1) Preparation of bacterial liquid
Inoculating BNCC194976 strain to LB liquid culture medium, culturing until OD600 value is 1.2, and collecting bacterial liquid after culturing.
2) Two-step method for preparing competent cells on solid plate
Adding 20 microliters of bacterial liquid to a filter membrane with a pore diameter of 0.22 micrometer on the surface of an LB solid culture medium, culturing for 2 hours at the temperature of a culture plate of 32 ℃, transferring the filter membrane with the lawn on the surface, which is stuck on the LB solid culture medium, to a K-Ca oligotrophic solid culture medium for culturing for 10 hours, transferring the filter membrane with the lawn on the surface, which is stuck on the K-Ca oligotrophic solid culture medium, to a K-Mg oligotrophic solid culture medium, and culturing for 1 hour to form final competent cells.
K-Ca oligotrophic solid medium: glucose 0.1M, NH 4 Cl 0.005M、KCl 1.5M、CaCl 2 0.25M agar 15g/L; the balance of water; pH7.4;
K-Mg oligotrophic solid medium: glucose 0.06M, NH 4 Cl 0.005M、KCl 1.8M、MgCl 2 0.09M agar and 15g/L agar; the balance of water; pH7.4.
3) Transformation of plasmids
2. Mu.g of plasmid pBE123 was directly added to the surface of competent cell lawn on the filter and reacted at 32 ℃ for 3 hours.
Test examples
FIG. 1 shows the results of gel electrophoresis identification of plasmids extracted from the transformant strains according to examples 1 to 7 of the present invention, in which it can be seen that the size of the improved plasmid is 6.2Kb, which is the same as the size of pBE123 plasmid; FIG. 2 shows the results of PCR gel electrophoresis identification of kanamycin resistance gene of the transformant strains of examples 1 to 7 of the present invention, from which it can be seen that the size of the amplified band is 750bp, which is identical to the size of kanamycin resistance gene on pBE 123.
The above results indicate the successful plasmid transformation of plasmid pBE123 in Bacillus polymyxa KN-03.
To further verify the transformation efficiency of the plasmids of the present invention, the number of transformants growing on the selection plate was counted and then divided by the amount of DNA to obtain the transformation efficiency, and the results are shown in Table 1.
TABLE 1 transformation efficiency of plasmids of the invention
| Transformation efficiency (transformant/. Mu.g DNA) | |
| Example 1 | 6.8e 5 |
| Example 2 | 3.7e 5 |
| Example 3 | 4.6e 5 |
| Example 4 | 3.1e 3 |
| Example 5 | 1.2e 1 |
| Example 6 | 1.0e 1 |
| Example 7 | 6.5e 2 |
As can be seen from the above results, examples 1 to 3 had the highest transformation efficiency of the plasmid, and example 4 times, whereas examples 5 to 7 had lower transformation efficiency. On another industrial bacillus polymyxa BNCC194976, the method of the invention can also obtain high plasmid transformation efficiency.
Claims (1)
1. A method for transforming a plasmid of Bacillus polymyxa, the method comprising the steps of:
1) Inoculating the KN-03 strain to an LB liquid culture medium, culturing until the OD600 value is 1.0-1.2, and collecting a bacterial liquid after the culture is finished;
2) Adding the bacterial liquid to an LB solid culture medium, culturing for 3 hours at 30 ℃, then transferring to a K-Ca oligotrophic solid culture medium, culturing for 8 hours to preliminarily stimulate the formation of competent cells, finally transferring to a K-Mg oligotrophic solid culture medium, and continuing to culture for 2 hours to form final competent cells;
K-Ca oligotrophic solid medium: glucose 0.15M; NH 4 Cl 0.005M；KCl 0.8M；CaCl 2 0.1M; 15g/L of agar; the balance of water; pH7.4;
K-Mg oligotrophic solid medium: glucose 0.1M; NH (NH) 4 Cl 0.003M；KCl 1M；MgCl 2 0.06M; 15g/L of agar; the balance of water; pH7.4;
3) Adding exogenous plasmid into competent cell, reacting at 30 deg.C for 2 hr to transfer the plasmid into competent cell,
the preservation number of the KN-03 strain is CCTCC NO: M2012077.
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