Method for efficiently preparing agrobacterium competent cells
The preparation of Agrobacterium competent cells by combining magnesium chloride with acetylsyringone was solved, and the problem of low transformation efficiency in the existing technology was achieved, efficient preparation of Agrobacterium competent cells was achieved, and the operation was simplified and the plant genetic transformation process was improved. efficiency.
Patent Information
- Application Number
- CN202411405237.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-10
- Publication Date
- 2024-12-06
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The existing Agrobacterium competent cell preparation methods have extremely low conversion efficiency and are difficult to meet the needs of plant genetic transformation process.
Agrobacterium competent cells were prepared by combining magnesium chloride with acetylsyringone, replacing the traditional CaCl2 method to increase cell permeability, and a preservative solution rich in nutrients was used to improve the cell's anti-freeze and transformation recovery ability.
It significantly improves the transformation efficiency of Agrobacterium competent cells, reduces the number of repeated transformations, simplifies the operation process, and improves the efficiency of the plant genetic transformation process. It is suitable for different types of Agrobacterium strains.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of microorganisms, and in particular to a method for preparing efficient Agrobacterium competent cells. Background Art
[0002] Bacteria in the rapid growth stage are specially treated to make them easy to accept foreign DNA. The host cell is called a "competent cell". The process of allowing foreign DNA molecules to enter the recipient cell and produce new genetic traits through physical or chemical methods is called "transformation".
[0003] Agrobacterium is a Gram-negative bacterium commonly found in the soil. It can transfer DNA into plant cells and mediate the genetic transformation process of plants. Agrobacterium transformation is one of the key steps in the process of plant transgenics. Plasmids transform Agrobacterium competent cells through physical or chemical methods. Agrobacterium carries exogenous plasmid DNA, transfers and integrates the exogenous DNA into plant cells, and completes the process of plant genetic transformation mediated by Agrobacterium. Foreign genes can be expressed transiently and stably in plants. During transient expression, the T-DNA sequence does not need to be integrated into the host genome, and high-level protein expression can be completed within 24-48 hours. Stable genetic transformation integrates T-DNA into the genome of the cell, and it takes several months to produce genetically transformed plants and obtain new genetic materials.
[0004] The methods for preparing competent cells are mainly physical and chemical methods. The physical method mainly uses electroporation to transform plasmids and has high transformation efficiency. However, electroporation requires expensive electric shock equipment, which cannot be met by general laboratories. The chemical methods for preparing competent cells mainly include the CaCl2 method, the RbCl method, the Inoue method, etc. The chemical transformation method has a low operating threshold and is easy to prepare in the laboratory. However, due to the differences in the chromosome background and cell characteristics of Agrobacterium and Escherichia coli, the method for preparing Escherichia coli competent cells is not very suitable for Agrobacterium. At present, the chemical method for preparing Agrobacterium competent cells is mainly the CaCl2 method, and the existing method for preparing Agrobacterium competent cells has extremely low transformation efficiency. Summary of the invention
[0005] In order to solve the above technical problems, the present invention provides a method for preparing efficient Agrobacterium competent cells, which is used to solve the problem of low preparation transformation efficiency in the above background technology.
[0006] A method for preparing efficient Agrobacterium competent cells, the specific technical scheme includes: step 1) proliferation and cultivation of Agrobacterium; step 2) centrifugation to collect bacteria; step 3) resuspending the bacteria with competent preparation solution; step 4) packaging the preservation solution and freezing in liquid nitrogen;
[0007] The invention is a non-traditional method of using CaCl2 to prepare Agrobacterium competent cells. Instead, a method combining magnesium chloride and acetosyringone is used to increase the permeability of Agrobacterium cells, making it easier for plasmid DNA molecules to be transferred into Agrobacterium competent cells.
[0008] The unique Agrobacterium competent cell preservation solution used in the present invention does not contain traditional glycerol, DMSO and other components that are toxic to cells, thereby reducing damage to competent cells. The use of rich nutrients and ions as the preservation solution not only helps the competent cells to resist freezing, but also helps the recovery process of the competent cells after transformation.
[0009] The above steps are as follows:
[0010] 1) Streak the Agrobacterium strain on an LB (rifampicin 25 mg / L) medium plate and culture it in a 28°C incubator for 2 days to obtain a single colony;
[0011] 2) Inoculate the colony in step 1) into 5 mL YEB (rifampicin 25 mg / L) medium and culture with shaking at 28°C overnight; the next day, inoculate the culture into 500 mL YEB medium, culture with shaking at 28°C until OD600 is 0.5-0.6, place on ice for 30 min, centrifuge at 4000 rpm and 4°C for 10 min, and discard the supernatant;
[0012] 3) Resuspend the cells in 20 mL of pre-cooled competent preparation solution, incubate at 4°C 50-60 rpm for 10 min, centrifuge at 4000 rpm and 4°C for 10 min, and discard the supernatant;
[0013] 4) Resuspend the cells in 20 mL of pre-cooled storage solution, dispense 100 μL / tube into pre-cooled EP tubes, snap-freeze with liquid nitrogen, and store in a -80°C refrigerator.
[0014] Preferably, the LB solid medium in step 1) is: 5 g yeast powder, 10 g peptone, 10 g sodium chloride, 20 g agar, and water is added to make the volume 1 L. The pH value is adjusted to 7.0, and the medium is sterilized by high pressure at 121° C. for 15 min and poured into a plate.
[0015] Preferably, the YEB liquid culture medium in step 2) is: 1 g yeast extract, 5 g beef extract, 5 g peptone, 5 g sucrose, 0.5 g magnesium sulfate heptahydrate, add water to make the volume 1 L, adjust the pH to 7.0, and sterilize at 121° C. for 15 min.
[0016] Preferably, the preparation method of the competent state preparation solution in step 3) is: magnesium chloride (10-100mM, preferably 50mM), acetosyringone (50-200μM, preferably 100μM), 2-morpholineethanesulfonic acid (10mM), sucrose (1-5%, preferably 5%), adjusting the pH value to 6.8, sterilizing at high temperature and high pressure, and storing at 4°C.
[0017] Preferably, the preparation method of the preservation solution in step 4) is: add 20g of tryptone, 5g of yeast extract, 0.5g of sodium chloride, and 0.186g of potassium chloride in 900mL of pure water in sequence, adjust the pH value to 6.8, sterilize at high temperature and high pressure, add 10mL each of 1M MgCl2 and 1M MgSO4 that have been sterilized in advance, add 20mL of filtered and sterilized 1M glucose, make the volume to 1L, and store at 4°C.
[0018] In a further embodiment, the transformation process of the Agrobacterium competent cells prepared by the present invention is achieved by the following steps:
[0019] a) Take out competent cells from -80℃, thaw at room temperature, place on ice, add 0.01-1μg plasmid DNA, and mix gently.
[0020] b) Place on ice for 3 min, quick freeze in liquid nitrogen for 3 min, heat shock at 37°C for 3 min, and continue to keep on ice for 3 min.
[0021] c) Add 700 μL of LB medium and culture at 28°C with shaking at 200 rpm for 1-2 h.
[0022] d) Centrifuge at 5000 rpm for 1 min, discard the supernatant, add 100 μL of sterile water to resuspend, spread on the corresponding antibiotic plate, and culture inverted at 28°C for 2-3 days.
[0023] Technical effects and advantages of the present invention:
[0024] 1. Improving the transformation efficiency of Agrobacterium competent cells can reduce the number of repeated transformations, help save scientific research time, and accelerate the subsequent molecular biology research process such as plant genetic transformation.
[0025] 2. Compared with the existing Agrobacterium competent cell preparation technology, the Agrobacterium competent cells prepared by the present invention are simple to operate, have good stability, and greatly improve the transformation efficiency, which can reach 2×10 4 cfu / μg, which has high application value. DETAILED DESCRIPTION
[0026] The present invention will be further described in detail below in conjunction with specific embodiments. The embodiments of the present invention are provided for the purpose of illustration and description, and are not intended to be exhaustive or to limit the present invention to the disclosed forms. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiments are selected and described to better illustrate the principles and practical applications of the present invention, and to enable those of ordinary skill in the art to understand the present invention and thereby design various embodiments with various modifications suitable for specific uses.
[0027] Example 1
[0028] The Agrobacterium competent cell state prepared by the present invention is prepared, stored and the transformation efficiency is detected.
[0029] Unless otherwise specified, the raw materials, reagents, strains, etc. used in this application are produced or stored by Beijing Coolbo Company.
[0030] The materials and reagents used in this example are as follows:
[0031] Materials: pCambia2301 plasmid; Agrobacterium GV3101
[0032] Antibiotics: Rifampicin, kanamycin
[0033] Culture medium: LB solid culture medium: 5g yeast powder, 10g peptone, 10g sodium chloride, 20g agar, add water to 1L. Adjust the pH to 7.0 and sterilize by autoclave at 121℃ for 15min. YEB liquid culture medium: 1g yeast extract, 5g beef extract, 5g peptone, 5g sucrose, 0.5g magnesium sulfate heptahydrate, add water to 1L, adjust the pH to 7.0 and sterilize by autoclave at 121℃ for 15min.
[0034] The competent state preparation solution is an aqueous solution of magnesium chloride (50 mM), acetosyringone (100 μM), 2-morpholineethanesulfonic acid (10 mM), and sucrose (5%), with the pH value adjusted to 6.8, sterilized by high temperature and high pressure, and stored at 4°C.
[0035] The method for preparing the preservation solution is as follows: add 20 g of tryptone, 5 g of yeast extract, 0.5 g of sodium chloride, and 0.186 g of potassium chloride in 900 mL of pure water in sequence, adjust the pH value to 6.8, sterilize with high temperature and high pressure, add 10 mL each of 1M MgCl2 and 1MMgSO4 that have been sterilized in advance, add 20 mL of filtered and sterilized 1M glucose, make up to 1L, and store at 4°C.
[0036] The method for preparing the Agrobacterium competent cells of the present invention is as follows:
[0037] 1) Streak the Agrobacterium strain GV3101 on an LB plate (rifampicin 25 mg / L) and culture in a 28°C incubator for 2 days to obtain a single colony;
[0038] 2) Inoculate the colony in step 1) into 5 mL YEB (rifampicin 25 mg / L) liquid culture medium, shake culture at 28°C overnight; the next day, inoculate the culture into 500 mL YEB (rifampicin 25 mg / L) culture medium, shake culture at 28°C until OD600 is 0.5-0.6, and ice bath for 30 minutes. Centrifuge at 4000 rpm, 4°C for 10 minutes, and discard the supernatant.
[0039] 3) Resuspend the cells in 20 mL of pre-cooled competent preparation solution, incubate at 4°C, 50-60 rpm for 10 min, and discard the supernatant.
[0040] 4) Resuspend the cells in 20 mL of pre-cooled storage solution, dispense 100 μL / tube into pre-cooled EP tubes, snap-freeze with liquid nitrogen, and store in a -80°C refrigerator.
[0041] Examples 2-12 are the same as Example 1, except for the concentrations of acetosyringone, sucrose and magnesium chloride, as shown in Table 1.
[0042] Table 1 Differences in competent preparation solutions in Examples 1-12
[0043]
[0044]
[0045] Embodiment 13
[0046] This example is the same as Example 1, except that the Agrobacterium is strain EHA105.
[0047] Embodiment 14
[0048] This example is the same as Example 1, except that the Agrobacterium is strain LBA4404.
[0049] Embodiment 15
[0050] This example is the same as Example 1, except that the Agrobacterium is strain AGL1.
[0051] Example 16
[0052] This example is the same as Example 1, except for the different preservation solution. The preservation solution in this example is the competent preparation solution plus 15% glycerol.
[0053] Example 17: Preparation method of common Agrobacterium competent cells and detection of transformation efficiency.
[0054] The materials and reagents used in this example are as follows:
[0055] Materials: pCambia2301 plasmid; Agrobacterium GV3101
[0056] Antibiotics: Rifampicin, kanamycin
[0057] Reagents: LB solid medium: 5g yeast powder, 10g peptone, 10g sodium chloride, 20g agar, add water to 1L. Adjust pH to 7.0, autoclave at 121℃ for 15min. 0.02M CaCl2 solution: 2.22g calcium chloride, add water to 1L. CaCl2 / 15% glycerol mixture: 2.22g calcium chloride, 150mL glycerol, add water to 1L.
[0058] The preparation method of common Agrobacterium competent cells is as follows:
[0059] 1) Streak the Agrobacterium strain GV3101 on an LB plate (rifampicin 25 mg / L) and culture in a 28°C incubator for 2 days to obtain a single colony;
[0060] 2) Inoculate the colony in step 1) into 5 mL LB (25 mg / L rifampicin) medium, and culture at 28°C overnight with shaking; the next day, inoculate the culture into 500 mL LB (25 mg / L rifampicin) medium, and culture at 28°C with shaking until OD600 is 0.5-0.6, and ice bath for 30 minutes. Centrifuge at 4000 rpm, 4°C for 10 minutes, and discard the supernatant.
[0061] 3) Resuspend with 20 mL of pre-cooled 0.02 M CaCl2 solution, place on ice for 20 min, centrifuge at 4000 rpm at 4°C for 10 min, and discard the supernatant.
[0062] 4) Resuspend with 20 mL of pre-cooled CaCl2 / 15% glycerol mixture, dispense 100 μL / tube into pre-cooled EP tubes, snap-freeze in liquid nitrogen, and store in a -80°C refrigerator.
[0063] Competent cell transformation efficiency detection:
[0064] 1) Take out the competent cells from -80℃, thaw at room temperature and place on ice, add 20ng of pCambia2301 plasmid and mix gently.
[0065] 2) Place on ice for 30 min, quick freeze with liquid nitrogen for 5 min, heat shock at 37°C for 5 min, and continue to keep on ice for 2 min.
[0066] 3) Add 700 μL of LB medium and culture at 28°C with shaking at 200 rpm for 1-2 h.
[0067] 4) Centrifuge at 5000 rpm for 1 min, discard the supernatant, add 100 μL of sterile water to resuspend, and spread on LB plates containing 50 mg / L kanamycin and 25 mg / L rifampicin. Incubate at 28°C for 2-3 days, count the colonies grown on the plates, and calculate the transformation efficiency.
[0068] The transformation efficiency of Agrobacterium competent cells in Examples 1-17 is shown in Table 2.
[0069] Table 2 Transformation efficiency of Agrobacterium competent cells in Examples 1-17
[0070]
[0071]
[0072] From the transformation efficiency of Agrobacterium competent cells in the above different embodiments, it can be seen that the transformation efficiency of the Agrobacterium competent cell preparation method of the present invention is high, which is about 100 times that of the conventional method.
[0073] It can be concluded from the transformation efficiency of Agrobacterium competent cells in Example 1 and Examples 13-15 that the efficient Agrobacterium competent cell preparation method of the present application is universal for different Agrobacterium.
[0074] From the comparison of the transformation efficiency of Agrobacterium competent cells in Example 1 and Example 16, it can be concluded that the preservation solution in the present application can greatly improve the transformation efficiency of competent cells compared with the traditional preservation solution.
[0075] Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field and related fields without creative work should fall within the scope of protection of the present invention. The structures, devices and operating methods not specifically described and explained in the present invention are implemented according to the conventional means in the field unless otherwise specified and limited.
Claims
1. A method for preparing efficient Agrobacterium competent cells, It is characterized in that include: Step 1) Proliferation and cultivation of Agrobacterium; Step 2) Collecting bacteria by centrifugation; Step 3) Resuspending the bacteria with competent preparation solution; Step 4) Packaging the preservation solution and freezing in liquid nitrogen.
2. The method for preparing a highly efficient Agrobacterium competent cell according to claim 1, It is characterized in that The specific steps are as follows: 1) Streak the Agrobacterium strain on an LB medium plate and culture it in a 28°C incubator for 2 days to obtain a single colony; 2) Inoculate the colony in step 1) into 5 mL YEB medium and culture at 28°C overnight with shaking; the next day, inoculate the culture into 500 mL YEB medium, culture at 28°C with shaking until OD600 is 0.5-0.6, place on ice for 30 min, centrifuge at 4000 rpm and 4°C for 10 min, and discard the supernatant; 3) Resuspend the cells in 20 mL of pre-cooled competent preparation solution, incubate at 4°C 50-60 rpm for 10 min, centrifuge at 4000 rpm and 4°C for 10 min, and discard the supernatant; 4) Resuspend the cells in 20 mL of pre-cooled storage solution, dispense 100 μL / tube into pre-cooled EP tubes, snap-freeze with liquid nitrogen, and store in a -80°C refrigerator.
3. The method for preparing a highly efficient Agrobacterium competent cell according to claim 1, It is characterized in that The LB medium in step 1) is a solid LB medium, which includes: 5 g yeast powder, 10 g peptone, 10 g sodium chloride, 20 g agar, and water is added to make the volume 1 L; the pH value is adjusted to 7.0, and the medium is sterilized by high pressure at 121° C. for 15 min, and poured into a plate.
4. The method for preparing a highly efficient Agrobacterium competent cell according to claim 3, It is characterized in that The YEB medium in step 2) is a YEB liquid medium, which includes: 1 g yeast extract, 5 g beef extract, 5 g peptone, 5 g sucrose, 0.5 g magnesium sulfate heptahydrate, and water is added to make the volume 1 L; the pH is adjusted to 7.0, and the medium is sterilized at 121° C. for 15 min.
5. The method for preparing a highly efficient Agrobacterium competent cell according to claim 1, It is characterized in that The preparation method of the competent state preparation solution in step 3) is: 10-100 mM magnesium chloride, 50-200 μM acetosyringone, 10 mM 2-morpholineethanesulfonic acid, 1-5% sucrose, adjusting the pH value to 6.8, sterilizing at high temperature and high pressure, and storing at 4°C.
6. The method for preparing a highly efficient Agrobacterium competent cell according to claim 1, It is characterized in that The preparation method of the preservation solution in step 4) is as follows: add 20 g of tryptone, 5 g of yeast extract, 0.5 g of sodium chloride, and 0.186 g of potassium chloride in 900 mL of pure water in sequence; adjust the pH value to 6.8, sterilize with high temperature and high pressure, add 10 mL of 1M MgCl2 and 1M MgSO4 sterilized in advance, add 20 mL of filtered and sterilized 1M glucose, adjust the volume to 1 L, and store at 4°C.
7. The method for preparing a highly efficient Agrobacterium competent cell according to claim 5, It is characterized in that The competent state preparation solution includes: 50 mM magnesium chloride, 100 μM acetosyringone, 10 mM 2-morpholineethanesulfonic acid, and 5% sucrose.
8. The method for preparing a highly efficient Agrobacterium competent cell according to claim 4, It is characterized in that The LB medium and the YEB medium both contain 25 mg / L rifampicin.
9. A method for preparing efficient Agrobacterium competent cells according to any one of claims 1 to 8, It is characterized in that The transformation process of the Agrobacterium competent cells prepared in the present invention is achieved by the following steps: (a) Take out the competent cells from -80℃, thaw at room temperature, place on ice, add 0.01-1μg plasmid DNA, and mix gently; (b) Place on ice for 3 min, freeze in liquid nitrogen for 3 min, heat shock at 37°C for 3 min, and continue to stay on ice for 3 min; (c) Add 700 μL of LB medium and culture at 28°C with shaking at 200 rpm for 1-2 h; (d) Centrifuge at 5000 rpm for 1 min, discard the supernatant, add 100 μL of sterile water to resuspend, spread on the corresponding antibiotic plate, and culture inverted at 28°C for 2-3 days.
10. A method for preparing efficient Agrobacterium competent cells according to claims 1-9, It is characterized in that The transformation efficiency of the Agrobacterium competent cells prepared by the present invention reaches 2×10 4 cfu / μg.