CN114574367A - Freeze-drying protective agent for escherichia coli DH5 alpha competent cells and use method and application thereof - Google Patents
Freeze-drying protective agent for escherichia coli DH5 alpha competent cells and use method and application thereof Download PDFInfo
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- 238000004108 freeze drying Methods 0.000 title claims abstract description 28
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 title claims abstract description 24
- 239000003223 protective agent Substances 0.000 title claims abstract description 23
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 28
- 239000013612 plasmid Substances 0.000 claims abstract description 19
- 230000009466 transformation Effects 0.000 claims abstract description 19
- 239000000843 powder Substances 0.000 claims abstract description 18
- 235000020183 skimmed milk Nutrition 0.000 claims abstract description 18
- 230000001580 bacterial effect Effects 0.000 claims abstract description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 10
- 229920001223 polyethylene glycol Polymers 0.000 claims abstract description 7
- 239000002202 Polyethylene glycol Substances 0.000 claims abstract description 5
- 239000000126 substance Substances 0.000 claims abstract description 5
- 229940057838 polyethylene glycol 4000 Drugs 0.000 claims abstract description 3
- 241000588724 Escherichia coli Species 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 9
- 239000002904 solvent Substances 0.000 claims description 8
- 239000001963 growth medium Substances 0.000 claims description 7
- 239000002244 precipitate Substances 0.000 claims description 7
- 241001052560 Thallis Species 0.000 claims description 6
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 5
- 238000007710 freezing Methods 0.000 claims description 5
- 230000008014 freezing Effects 0.000 claims description 5
- 102000019197 Superoxide Dismutase Human genes 0.000 claims description 4
- 108010012715 Superoxide dismutase Proteins 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 239000002953 phosphate buffered saline Substances 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 239000001110 calcium chloride Substances 0.000 claims description 3
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 3
- 230000035939 shock Effects 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 2
- 230000004913 activation Effects 0.000 claims description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 2
- 238000011084 recovery Methods 0.000 claims description 2
- 238000009472 formulation Methods 0.000 claims 2
- 230000000694 effects Effects 0.000 abstract description 12
- 238000010369 molecular cloning Methods 0.000 abstract description 8
- 230000008569 process Effects 0.000 abstract description 5
- 238000005138 cryopreservation Methods 0.000 abstract description 3
- 238000003860 storage Methods 0.000 abstract description 2
- 238000004321 preservation Methods 0.000 description 8
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 description 5
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 238000012258 culturing Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
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- 230000001954 sterilising effect Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 230000032683 aging Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 239000002577 cryoprotective agent Substances 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012827 research and development Methods 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 235000011089 carbon dioxide Nutrition 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
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- 230000009897 systematic effect Effects 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
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Abstract
The invention discloses a freeze-drying protective agent for escherichia coli DH5 alpha competent cells and a using method and application thereof, belonging to the technical field of bacterial strain cryopreservation and plasmid transformation, and comprising polyethylene glycol 4000, skimmed milk powder, dimethyl sulfoxide and water, wherein the content of each substance in each 100ml of freeze-drying protective agent is as follows: 40000.5-4 g of polyethylene glycol, 10-25 g of skimmed milk powder, 2-8 ml of dimethyl sulfoxide and the balance of water. The freeze-drying protective agent for the escherichia coli DH5 alpha competent cells utilizes the freeze-drying strain with higher activity to directly prepare the competent cells, the method greatly shortens the experimental process of molecular cloning, and the obtained freeze-drying active strain has wider temperature adaptability, can be stored in the temperature environment of-20 ℃ to-80 ℃, and effectively reduces the transportation and storage cost of the competent cells.
Description
Technical Field
The invention relates to the technical field of cryopreservation of strains and plasmid transformation, in particular to a freeze-drying protective agent for escherichia coli DH5 alpha competent cells, and a using method and application thereof.
Background
The operation of transforming exogenous plasmid DNA into strain cells in order to obtain a cloning vector or an expression product is a routine experimental technique means of genetic engineering. The molecular cloning technology has wide application in the fields of basic research, production research and development and the like. However, since the 70 th 19 th century, the method itself has not been greatly improved, and has been used as a conventional molecular biological technique. The basic experimental process comprises the experimental steps of strain competent cell preparation, competent cell preservation, plasmid transformation, identification and the like. Because the technical principle is clear and the experimental operation is mature, a large number of biological reagent companies are promoted to engage in the production work of the competent cells of common strains, and the frozen products of the competent cells which can be directly used for transformation are released to the market. Among these competent cell production enterprises, well-known biological enterprises at home and abroad are, for example: the whole formula Jinkang is century, TAKARA, Thermo Fisher, etc. The ultralow temperature frozen sensitive strains shorten the experimental flow of conventional molecular cloning to a certain extent, and are one of the largest biological reagents in daily consumption of research institutes.
However, the competent cells commercially available on the market at present do not completely solve the pain point in the molecular cloning experiments. On the one hand, the preservation time of the liquid frozen competent cells is short, the preservation time is about 1 month generally, and the transformation efficiency of the liquid frozen competent cells is reduced rapidly after long-term preservation. On the other hand, the storage conditions of liquid frozen competent cells are severe, the competent cells need to be stored in an ultralow-temperature refrigerator at minus 80 ℃, and the competent cells need to be wrapped in dry ice in the whole transportation process, so that the use cost of commercialized competent cells is increased invisibly. Coli DH5 alpha has wide application in molecular cloning field, is suitable for antibiotic screening of conventional resistance marker vectors, can also be used for blue-white spot screening of LacZ marker vectors, and is the most frequently selected host strain of most cloning plasmids. Therefore, the research and development of the preservation and transformation method of the Escherichia coli DH5 alpha competent strain, which can adapt to a wider temperature range, has a long preservation period and high transformation efficiency, is significant for simplifying the molecular cloning experimental process.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide a freeze-drying protective agent capable of efficiently preserving Escherichia coli DH5 alpha cells, and simultaneously provides an experimental method for rapidly and efficiently transforming exogenous plasmids by directly utilizing freeze-dried Escherichia coli DH5 alpha cells.
The technical scheme adopted by the invention for solving the technical problems is as follows:
the cryoprotectant for escherichia coli DH5 alpha competent cells is composed of polyethylene glycol 4000(PEG4000), skimmed milk powder, dimethyl sulfoxide (DMSO) and water, wherein the content of each substance in each 100ml of the cryoprotectant is as follows: 40000.5-4 g of polyethylene glycol, 10-25 g of skimmed milk powder, 2-8 ml of dimethyl sulfoxide and the balance of water.
Specifically, the skim milk powder is prepared by self-preparation, fat of fresh milk is removed, and the fresh milk is dried to obtain the skim milk powder, and the skim milk powder is prepared by irradiation sterilization, and the purity of the skim milk powder is more than or equal to 99.0%.
Furthermore, the content of each substance in each 100ml of the freeze-drying protective agent is as follows: 40002 g of polyethylene glycol, 20g of skimmed milk powder, 4ml of dimethyl sulfoxide and the balance of water.
Specifically, during preparation, the PEG4000 and DMSO components are sterilized at 121 ℃ for 15min, and the skimmed milk powder is heated in a water bath for sterilization for 15 min.
A method of using a lyoprotectant for escherichia coli DH5 α competent cells, comprising the steps of:
s1: selecting a monoclonal colony, inoculating the colony in a superoxide dismutase culture medium, performing strain activation culture, performing first centrifugal separation on the cultured bacterial liquid to obtain a bacterial precipitate, and performing secondary centrifugal separation after gently washing the bacterial to obtain the bacterial precipitate;
s2: using a freeze-drying protective agent of escherichia coli DH5 alpha competent cells to gently suspend and precipitate, subpackaging suspended thalli, freezing, and then putting the thalli into a freeze dryer for freeze drying to obtain freeze-dried escherichia coli DH5 alpha active cells, wherein: volume of freeze-drying protective agent is equal to volume of superoxide dismutase culture medium/100.
Further, adding a pre-cooled re-solvent into a freeze-dried escherichia coli DH5 alpha active cell, carrying out ice bath for 10min to obtain an escherichia coli DH5 alpha competent cell, adding 10-100 ng of plasmid, placing the plasmid into ice for 20min, gently mixing the plasmid and the ice once every 5min, carrying out heat shock at 42 ℃ for 50-55s, carrying out ice bath for 5min, and finally adding an SOB culture medium to carry out recovery culture at 37 ℃ at 150r/min for 30min, wherein the volume of the re-solvent is equal to the volume of the freeze-drying protective agent.
Further, in step S1, the culture conditions were 25 ℃, 150rpm/min, and the culture was stopped when OD was 0.6; the first centrifugation is carried out at 5000rpm for 10min at 4 ℃; centrifuging at 5000rpm for 10min for the second time;
in the step S2, the freezing is carried out at-80 ℃ for 10-12 h, and the drying is carried out in a freeze drier for 4-6 h.
Further, the washing was performed using phosphate buffered saline (PBS buffered saline).
Further, the double solvent contains 0.05mol/L calcium chloride and 0.05mol/L magnesium chloride.
Further, the plasmid was PUC 18.
The application of a lyoprotectant for Escherichia coli DH5 alpha competent cells in plasmid transformation is provided.
Compared with the prior art, the invention has the beneficial effects that:
the invention obtains a freeze-drying protective agent capable of efficiently preserving escherichia coli through systematic experimental optimization, on the basis, the freeze-drying strain with higher activity is used for directly preparing the competent cells, the method greatly shortens the experimental process of molecular cloning, and the obtained freeze-drying active strain has wider temperature adaptability and can be preserved in the temperature environment of-20 ℃ to-80 ℃, thereby effectively reducing the transportation and preservation costs of the competent cells.
Drawings
Other features, objects and advantages of the present application will become more apparent upon reading of the following detailed description of non-limiting embodiments thereof, made with reference to the accompanying drawings in which:
FIG. 1 is a bar graph of plate colony counts;
FIG. 2 is a monoclonal photograph showing the transformation effect of a plate;
FIG. 3 shows the PCR results of monoclonal colonies on the plate;
FIG. 4 is a histogram comparing the rate of aging of E.coli DH5 alpha competent cells.
Detailed Description
The present application will be described in further detail with reference to the drawings and examples. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not to be construed as limiting the invention. It should be noted that, for convenience of description, only the portions related to the present invention are shown in the drawings.
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present application will be described in detail below with reference to the embodiments with reference to the attached drawings.
Example one
Freeze-drying protective agent formula suitable for escherichia coli DH5 alpha high-activity strain and use method thereof
1. Preparation of Escherichia coli DH5 alpha freeze-drying protective agent
The freeze-drying protective agent adopted by the invention comprises PEG4000, DMSO and skimmed milk powder. PEG4000 has high purity which is more than or equal to 99.0 percent; the skimmed milk powder is prepared by removing fat from fresh milk, drying, and sterilizing by irradiation, and has purity of 99.0% or more. In order to optimize the influence of different component ratios on the cryopreservation effect of the strains, an optimal protective agent formula is found by adopting an orthogonal test. Wherein, before the preparation of the freeze-drying protective agent, the components are required to be sterilized, the PEG4000 and DMSO components are sterilized for 15min at 121 ℃, and the skimmed milk powder component can be only heated and sterilized for 15min in a water bath. The orthogonal test groups and the component ratios per 100ml of lyoprotectant are shown in Table 1.
TABLE 1 orthogonal table of components of E.coli DH5 alpha lyophilized protectant mixture ratio
2. Activated freeze-dried Escherichia coli DH5 alpha strain
The monoclonal colonies were picked and inoculated into SOD medium, and activated for culturing at 25 ℃ and 150rpm/min, and the culture was stopped when OD was 0.6. Subpackaging the cultured bacterial liquid into centrifuge tubes, and centrifuging in a centrifuge at 4 deg.C and 5000rpm for 10 min. The supernatant was discarded, and the cells were gently washed with 1mol/L PBS buffer and washed once more. Centrifuging at 5000rpm for 10min to obtain thallus precipitate.
The pellet was gently suspended with 1mL of DH5 α lyoprotectant. The suspended cells were dispensed into 1.5mL EP tubes (100. mu.L/tube). And (4) placing the subpackaged bacterial liquid in a refrigerator at the temperature of-80 ℃ for freezing for 10-12 h. And finally, putting the sample into a freeze dryer for freeze drying for 4-6 h to obtain the freeze-dried Escherichia coli DH5 alpha active cells.
3. Rapid transformation of foreign plasmids
Adding 100 mu L of precooled double solvent into freeze-dried Escherichia coli DH5 alpha, wherein the double solvent contains 0.05mol/L CaCl2And 0.05mol/L MgCl2And standing on ice for 10min to restore the lyophilized sample to a liquid state. And adding 100ng of PUC18 plasmid into the sample for transformation, placing the sample on ice for 20min, and lightly and uniformly mixing the sample once every 5min by using a gun head to ensure that the plasmid is fully contacted with the thalli, thereby improving the transformation efficiency. Heat shock at 42 deg.C for 50-55s, ice-cooling for 5min, adding 500 μ L SOB medium into EP tube, placing on shaker, and activating and culturing at 37 deg.C and 150rpm/min for 30 min.
4. Culturing and detecting after transformation
And (3) centrifugally collecting the thalli in the step (3) (8000rpm, 1min), suspending and precipitating by using 100 mu L of SOD culture medium, then coating the liquid on an LB plate for ampicillin resistance screening, and putting the LB plate into an incubator for culturing for 8-10 h. Finally, the transformation effect of the foreign plasmid was examined by counting the number of single clones in the plate.
The histogram of plate colony counts is shown in FIG. 1, and the monoclonal photograph of plate transformation effects is shown in FIG. 2. The experimental results show that: in 100ml of the freeze-drying protective agent, the ratio of the components is PEG 40002 g, the skim milk powder is 20g, and the DMSO is 4ml, so that the preservation effect on the activity of the thallus is the best.
The positive rate of the monoclonals is detected by a colony PCR mode. Colony PCR primers are FW: 5'-AGAGCAGATTGTACTGAGAG-3', RV: 5'-TCGTATGTTGTGTGGAATTG-3' are provided.
The results showed that the positive rate of the single clone in the plate was 100%, as shown in FIG. 3.
5. Aging detection of lyophilized Escherichia coli DH5 alpha
Lyophilized E.coli DH 5. alpha. was prepared using the best protectant (PEG 40002 g, skimmed milk powder 20g, DMSO 4ml) as the test group, while E.coli DH 5. alpha. competent cells were prepared using the conventional method as the control group. The competent cells were simultaneously placed at-20 ℃ and sampled every 1 day for 5 consecutive times. The transformation activity of E.coli DH5 alpha cells was tested by PUC18 plasmid transformation experiments. As shown in FIG. 4, the results showed that the lyophilized E.coli DH5 a cells obtained by the present method could maintain relatively high transformation activity even in a temperature environment of-20 ℃ compared to E.coli DH5 a competent cells obtained by the conventional method.
The experimental results show that the protective agent formula can preserve the biological activity of the Escherichia coli DH5 alpha to the maximum extent, can be directly used for preparing competent cells, has higher transformation activity and positive rate, and greatly shortens the experimental flow of molecular cloning. Meanwhile, the freeze-dried active cells obtained by the method can be stored at-20 ℃ temporarily, so that the transportation cost of competent cells is greatly reduced.
It will be appreciated by a person skilled in the art that the scope of the invention as referred to in the present application is not limited to the embodiments with a specific combination of the above-mentioned features, but also covers other embodiments with any combination of the above-mentioned features or their equivalents without departing from the inventive concept. For example, the above features may be replaced with (but not limited to) features having similar functions disclosed in the present application.
Claims (10)
1. A freeze-drying protective agent for escherichia coli DH5 alpha competent cells is characterized by comprising polyethylene glycol 4000, skimmed milk powder, dimethyl sulfoxide and water, wherein the content of each substance in each 100ml of freeze-drying protective agent is as follows: 40000.5-4 g of polyethylene glycol, 10-25 g of skimmed milk powder, 2-8 ml of dimethyl sulfoxide and the balance of water.
2. The lyoprotectant according to claim 1, wherein the content of each substance per 100ml of lyoprotectant is: 40002 g of polyethylene glycol, 20g of skimmed milk powder, 4ml of dimethyl sulfoxide and the balance of water.
3. The method of using a lyoprotectant according to any of claims 1-2, comprising the steps of:
s1: selecting a monoclonal colony, inoculating the colony in a superoxide dismutase culture medium, performing strain activation culture, performing first centrifugal separation on the cultured bacterial liquid to obtain a bacterial precipitate, and performing secondary centrifugal separation after gently washing the bacterial to obtain the bacterial precipitate;
s2: using a freeze-drying protective agent of escherichia coli DH5 alpha competent cells to gently suspend and precipitate, subpackaging and freezing suspended thalli, and then putting the thalli into a freeze dryer for freeze drying to obtain freeze-dried escherichia coli DH5 alpha active cells, wherein: volume of freeze-drying protective agent is equal to volume of superoxide dismutase culture medium/100.
4. The use method of the lyoprotectant according to claim 3, wherein the pre-cooled re-solvent is added into lyophilized active cells of Escherichia coli DH5 alpha, ice bath is carried out for 10min to obtain competent cells of Escherichia coli DH5 alpha, 10-100 ng of plasmid is added, the competent cells are placed on ice for 20min, the mixture is gently mixed once every 5min, heat shock is carried out at 42 ℃ for 50-55s, ice bath is carried out for 5min, finally, SOB culture medium is added, and the recovery culture is carried out at 37 ℃ and 150r/min for 30min, wherein the volume of the re-solvent is equal to the volume of the lyoprotectant.
5. The method of using a lyoprotectant according to claim 3, wherein the culture conditions in step S1 are 25 ℃, 150rpm/min, and the culture is stopped when OD is 0.6; the first centrifugation is carried out at 5000rpm for 10min at 4 ℃; the second centrifugation was performed at 5000rpm for 10 min.
6. The method for using the lyoprotectant according to claim 3, wherein in step S2, the freezing is performed at-80 ℃ for 10-12 h, and the drying is performed in a freeze dryer for 4-6 h.
7. The method for using lyoprotectant according to claim 3, wherein said rinsing is performed with phosphate buffered saline.
8. The method of using E.coli DH5 a lyoprotectant formulation according to claim 4, wherein said double solvent comprises 0.05mol/L calcium chloride and 0.05mol/L magnesium chloride.
9. The method of using E.coli DH5 a lyoprotectant formulation of claim 4, wherein said plasmid is PUC 18.
10. Use of a lyoprotectant according to any of claims 1-2 for plasmid transformation.
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| CN106047753A (en) * | 2016-06-06 | 2016-10-26 | 厦门生命互联科技有限公司 | Preparation method of competent cells with high transformation efficiency |
| CN113481229A (en) * | 2021-07-08 | 2021-10-08 | 上海佐润生物科技有限公司 | Preparation method for obtaining escherichia coli super competent cells |
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| CN101264062A (en) * | 2007-03-12 | 2008-09-17 | 袁红杰 | Production technology for preparing freeze-drying genetic engineering bacterium competence cell and protective agent formula |
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