A recombinant escherichia coli strain cryopreservation solution, a preparation method and application thereof

Abstract

The application discloses a kind of recombinant escherichia coli strain freezing solution and preparation method and application thereof.The formula of the recombinant escherichia coli strain freezing solution is: glycerol 200~500g / L, sucrose 20~100g / L, skimmed milk powder 20~50g / L, BSA 1~5g / L, sodium chloride 4~8g / L, vitamin C 1~5g / L.The recombinant escherichia coli strain freezing solution is used to preserve recombinant escherichia coli strain, and the method has the advantages of simple operation, long preservation time, stable and reliable strain quality, low preservation cost, and the recombinant escherichia coli strain preserved by the method has high protein expression amount through resuscitation strain detection, so it has good application prospect in the freezing preservation of recombinant escherichia coli strain.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial engineering, more particularly to a recombinant Escherichia coli strain cryopreservation solution, a preparation method and application thereof. BACKGROUND

[0002] Strain resource is a precious resource for human beings, and domestic and foreign research institutions pay great attention to strain preservation. The importance of strain preservation lies in keeping the original shape and activity of the strain as stable as possible, ensuring that the strain is not dead, not mutated, and not contaminated, so as to meet the needs of research, exchange and use. Strain preservation is to artificially create conditions to make the strain isolated from the outside world. The principle of strain preservation is to reduce the metabolism of microorganisms as much as possible to reduce mutation. Generally, by limiting nutrition, oxygen, drying and low temperature, the strain can be in a "semi-dormant" or "dormant" state, reducing the mutation probability and death probability of the strain, and keeping good genetic genes.

[0003] The recombinant Escherichia coli is a genetically engineered bacterium containing exogenous DNA fragments carried by a vector plasmid. These fragments are usually genetically unstable, and the exogenous plasmid replicon is easily lost during subculture. The loss of exogenous plasmid directly affects the related performance of genetically engineered strains during strain preservation and rejuvenation. The strain preservation of recombinant Escherichia coli usually adopts glycerol tube-80℃ ultra-low temperature or liquid nitrogen preservation. The survival rate of the strain is significantly reduced after a period of glycerol tube-80℃ ultra-low temperature preservation. The preservation condition of liquid nitrogen preservation is harsh. Not only is the initial preservation cost high, but also the temperature rises due to the evaporation of liquid nitrogen during the preservation process, which easily leads to the death of the strain. Therefore, the residual amount of liquid nitrogen needs to be closely monitored, and liquid nitrogen needs to be added regularly. When adding liquid nitrogen, there is a risk of explosion due to the exposure of liquid nitrogen in the air, and the operator needs to have high operation proficiency. SUMMARY

[0004] The purpose of the present application is to provide a recombinant Escherichia coli strain cryopreservation solution, a preparation method and application thereof, so as to solve the problems of reduced survival rate, harsh preservation condition and high cost in the prior art.

[0005] In order to solve the above technical problems, the technical scheme of the present application is as follows:

[0006] According to the first aspect of the present application, a recombinant Escherichia coli strain cryopreservation solution is provided, and the formula of the recombinant Escherichia coli strain cryopreservation solution is as follows: glycerol 200-500g / L, sucrose 20-100g / L, skimmed milk powder 20-50g / L, BSA 1-5g / L, sodium chloride 4-8g / L, and vitamin C 1-5g / L.

[0007] Preferably, the formulation of the recombinant E. coli strain cryopreservation solution is: glycerol 200-400 g / L, sucrose 50-100 g / L, skimmed milk powder 20 g / L, BSA 1-5 g / L, sodium chloride 4.5-7.2 g / L, and vitamin C 1-2 g / L.

[0008] According to a preferred embodiment of the present application, the formulation of the recombinant E. coli strain cryopreservation solution is: glycerol 200 g / L, sucrose 50 g / L, skimmed milk powder 20 g / L, BSA 5 g / L, sodium chloride 7.2 g / L, and vitamin C 2 g / L. This formulation is the most preferred embodiment of the present application. The recombinant E. coli strain cryopreservation solution with this formulation has the best protective effect on the recombinant E. coli strain.

[0009] According to another preferred embodiment of the present application, the formulation of the recombinant E. coli strain cryopreservation solution is: glycerol 200 g / L, sucrose 50 g / L, skimmed milk powder 20 g / L, BSA 2 g / L, sodium chloride 7.2 g / L, and vitamin C 1 g / L.

[0010] According to still another preferred embodiment of the present application, the formulation of the recombinant E. coli strain cryopreservation solution is: glycerol 200 g / L, sucrose 50 g / L, skimmed milk powder 20 g / L, BSA 1 g / L, sodium chloride 7.2 g / L, and vitamin C 2 g / L.

[0011] According to a second aspect of the present application, a preparation method of a recombinant E. coli strain cryopreservation solution is provided, comprising the following steps: A1: respectively weighing glycerol 200-500 g, sucrose 20-100 g, and sodium chloride 4-8 g, and adding them into distilled water in sequence, stirring until they are basically dissolved, and then adding purified water to make the volume 1 L; A2: high-temperature sterilization at 115 ℃ for 15-30 min, and cooling to room temperature for standby; A3: respectively weighing skimmed milk powder 20-50 g, BSA 1-5 g, and vitamin C 1-5 g, and adding them into the above solution in sequence, stirring until they are dissolved, filtering with a 0.22 μm filter membrane to remove bacteria, and then storing at 2-8 ℃ for standby or storing at-20 ℃ or below for long-term storage.

[0012] According to the third aspect of the present application, a method for cryopreservation of recombinant E. coli strain is provided, comprising the following steps: B1: preparing a recombinant E. coli strain cryopreservation solution as described above; B2: taking 100 ng of target plasmid DNA and adding it into 50 μL of E. coli BL21 (DE3) competent cells, mixing gently, and then placing in an ice bath for 20 min, placing in a 42°C water bath for 45 s, quickly transferring to an ice bath for 2 min, adding 800 μL of LB liquid medium, and then incubating at 37°C for 45 min before inoculating on an LB solid plate containing Amp, and then placing the plate in a 37°C constant temperature incubator for 16 h; B3: using LB medium with an antibiotic concentration of 100 μg / mL to culture the recombinant E. coli strain, picking a single colony into LB medium, and then incubating at 37°C and 220 rpm for 6-8 h, and then performing microscopic examination using Gram staining, and stopping the culture immediately if contamination occurs; using the absorbance method to detect the strain viability, and stopping the culture when the required OD 600 value of 0.8-2.0 is reached; B4: sequentially taking the recombinant E. coli strain cryopreservation solution of step B1 and the bacterial solution of step B2 at a volume ratio of 1:1, adding them into a cryopreservation tube, tightly covering the cryopreservation tube cap, and then turning it up and down to mix thoroughly; packaging the obtained strain and labeling it, and then storing it at -20°C to -196°C for long-term preservation.

[0013] To overcome the problems of reduced survival rate, harsh preservation conditions, and high cost of the recombinant E. coli strain in the prior art, it is necessary to develop a new strain preservation method. The inventors tried to further optimize the glycerol tube cryopreservation strain and developed a new cryopreservation solution with the aid of various protective agents. However, during the freezing, storage, and recovery of the recombinant E. coli strain, there are many factors that induce strain death, loss of exogenous plasmid, and decrease in strain viability. In addition, the concentration of the strain suspension, the age of the strain material, the type of culture medium, and the protective agent all affect the cryopreservation of the strain. Therefore, the appropriate type and amount of protective agent should be selected according to the characteristics of the strain to ensure long-term stable storage and production application of the strain. Therefore, the development of a preservation method suitable for the preservation of recombinant E. coli strain, which is simple in operation, mild in conditions, and can ensure stable performance of the strain and long-term preservation, has great practical significance for maintaining, replicating, and extending the excellent performance of genetically engineered bacteria, and is more conducive to stable industrial production.

[0014] The cryoprotectant ingredients used in the prior art are generally selected from glycerol and some polyhydric alcohol compounds such as mannitol, etc., and some cryoprotectants are selected from dimethyl sulfoxide. The main role of glycerol and polyhydric alcohol is to make water not easy to freeze, dimethyl sulfoxide can reduce the freezing point of cells, reduce the formation of ice crystals, reduce the damage of free radicals to cells, and change the permeability of biological membranes to electrolytes and metabolic products, but dimethyl sulfoxide has strong toxic effect on cells at room temperature.

[0015] The present application provides a new glycerol-based recombinant E. coli strain cryopreservation solution, which considers the influence of multiple factors on the recombinant E. coli during the freezing process, and selects protective agents with different mechanisms to strengthen or synergistically protect the strain.

[0016] Among them, the function of glycerol is to prolong the freezing process, because glycerol will not enter the ice, so the concentration of glycerol is increased with the freezing process, and the freezing point is further reduced. Because the freezing is relatively slow, the hydrostatic pressure of water can be more evenly distributed, and the bacteria can also freeze first relative to the environment, which is beneficial to their survival. Glycerol can also protect the cell membrane. The problem of general cryopreservation is that the ice crystals formed in the cells can damage the fragile cell membrane structure, so that the cells cannot survive normally after thawing. Glycerol can increase the ductility of the lipid bilayer, thereby protecting the membrane structure of the cells.

[0017] Sucrose is a non-permeable sugar that can increase the osmotic pressure of the cryopreservation solution, which helps the cells to maintain water balance, and in addition, sucrose can also inhibit the formation of ice crystals in the cell tissue, protect the integrity of the plasma membrane and protein structure, etc.

[0018] The addition of physiological level of sodium chloride can reduce the hypertonic effect of glycerol, and can better protect the strain.

[0019] The high molecular protective agent skimmed milk powder is a mixture containing various substances such as proteins, salts and vitamins, and the proteins provide a protective outer membrane for the strain cells, and the phosphates and citrates in it can play a buffering role to stabilize the pH value.

[0020] Bovine serum albumin (BSA) provides protection for enzymes, cell membranes and whole cells in a low temperature environment, and also has certain antioxidant activity, thereby better protecting the strain.

[0021] As an antioxidant, vitamin C can avoid the oxidative damage of bacteria during freezing and storage, and improve the survival rate and stability of bacteria. As an antioxidant, vitamin C can eliminate the corresponding cell membrane oxidative damage by forming free radicals with oxygen, and at the same time, vitamin C can prevent oxidative damage to important bacterial components such as proteins and DNA by preventing the carbonylation of alpha-amino or lysine, cysteine or histidine residues, thereby enhancing the survival rate and storage stability of bacteria.

[0022] The application point of the present application mainly lies in providing such a recombinant E. coli strain cryopreservation solution formula. E. coli is mostly cryopreserved by using 50% glycerol cryopreservation method and skimmed milk powder cryopreservation method. Among them, the 50% glycerol cryopreservation method can reduce the plasmid stability of the recombinant E. coli due to the high glycerol concentration, thereby affecting the expression stability of the recombinant bacteria; the skimmed milk powder cryopreservation method has a high cost and is relatively less used in E. coli cryopreservation. The recombinant E. coli strain cryopreservation solution provided by the present application can enhance the plasmid stability of the bacteria by reducing the glycerol concentration on the one hand, and can enhance the cryopreservation protection effect by using sucrose, skimmed milk powder and glycerol on the other hand, and can balance the osmotic pressure by adding sodium chloride, and at the same time, can further reduce the oxidative damage in the cryopreservation and recovery process by adding BSA and vitamin C. The present application provides such a new recombinant E. coli strain cryopreservation solution taking glycerol as the main body, and in the selection of the formula, the influence of multiple factors on the recombinant E. coli in the cryopreservation process is considered, and different mechanism protectants are selected for strengthening or synergistic protection of the bacteria.

[0023] Another application point of the present application also lies in that through continuous optimization of the formula, a plurality of preferred recombinant E. coli strain cryopreservation solution formulas are finally obtained. Among them, the formula of the most preferred recombinant E. coli strain cryopreservation solution is: glycerol 200 g / L, sucrose 50 g / L, skimmed milk powder 20 g / L, BSA 5 g / L, sodium chloride 7.2 g / L, and vitamin C 2 g / L. The recombinant E. coli strain cryopreservation solution using the formula has the best protection effect on the recombinant E. coli strain, which is better than all other cryopreservation solutions.

[0024] The cryopreservation solution composition used in the present application has a good effect for preserving bacteria, the recombinant E. coli cryopreserved by using the cryopreservation solution and the preservation method has stable quality, the cryopreservation method is simple, the cryopreservation range is wide (-20℃~-196℃), the bacteria can be preserved for more than 3 years at -80℃, and the bacteria after preservation have high activity in recovery, the preservation process is simple, and the bacteria are easy to store and find. The method has the advantages of simple and practical operation, long preservation time, stable and reliable bacteria quality, and low preservation cost. The recombinant E. coli bacteria preserved by the method have a high protein expression amount after four generations of culture after recovery of the bacteria.

[0025] In summary, the application provides a recombinant E. coli strain cryopreservation solution which can better protect the recombinant E. coli strain and has a long preservation time, and further provides a recombinant E. coli strain preservation method which is simple and practical to operate, has a long preservation time, stable and reliable strain quality, and low preservation cost. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The influence of the extracellular protective agent, i.e., sucrose and skimmed milk powder, on the cryopreservation effect of the strain in Example 1 is shown; wherein 1-8 are the cryopreservation effects of experimental groups 1-8 in sequence;

[0027] Figure 2 The influence of the intracellular protective agent, i.e., glycerol and sodium chloride, on the cryopreservation effect of the strain in Example 2 is shown; wherein 9-15 are the cryopreservation effects of experimental groups 9-15 in sequence;

[0028] Figure 3 The influence of the ratio of the extracellular protective agent and the intracellular protective agent on the cryopreservation effect of the strain in Example 3 is shown; wherein 16-22 are the cryopreservation effects of experimental groups 16-22 in sequence;

[0029] Figure 4 The influence of the category and ratio of the antioxidant on the cryopreservation effect of the strain in Example 4 is shown; wherein 23-30 are the cryopreservation effects of experimental groups 23-30 in sequence;

[0030] Figure 5 The protein expression amount after the recovery of the strain cryopreserved according to the most preferred recombinant E. coli strain cryopreservation solution provided by the application in Example 5 is shown, wherein the first to fourth bands from left to right are Marker, induction 0h, induction 2h, and induction 4h. DETAILED DESCRIPTION

[0031] The application will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the application but not to limit the scope of the application. Unless otherwise specified, the technical means used in the examples is the conventional operation in the art, or the experimental method suggested by the manufacturers of the kits and instrument equipment. The reagents and materials used in the examples can be obtained from commercial channels unless otherwise specified.

[0032] Example 1: Influence of the category and ratio of the extracellular protective agent on the cryopreservation of the strain

[0033] The extracellular protective agent is a non-permeable protective agent, which is mostly a macromolecular substance and has certain viscosity and cannot enter the cell. The main role is to maintain the stability of the cell membrane, and at the same time, it can combine with water molecules in the solution to reduce the content of free water outside the cell, reduce the freezing point, and reduce the generation of ice crystals. The extracellular protective agent is sucrose and skimmed milk powder, etc.

[0034] I. Preparation of cell cryopreservation solution (the following percentages are by mass):

[0035] Experimental group 1: 50g sucrose, 20g skim milk powder, 500g glycerol, 4.5g sodium chloride.

[0036] Experimental group 2: 50g sucrose, 50g skim milk powder, 500g glycerin, 4.5g sodium chloride.

[0037] Experimental group 3: 50g sucrose, 100g skim milk powder, 500g glycerin, 4.5g sodium chloride.

[0038] Experimental group 4: 20g sucrose, 20g skim milk powder, 500g glycerol, 4.5g sodium chloride.

[0039] Experimental group 5: 100g sucrose, 20g skim milk powder, 500g glycerin, 4.5g sodium chloride.

[0040] Experimental group 6: 20g skim milk powder, 500g glycerin, 4.5g sodium chloride.

[0041] Experimental group 7: 50g sucrose, 500g glycerol, 4.5g sodium chloride.

[0042] Experimental group 8: 500g glycerol, 4.5g sodium chloride (equivalent to a 1:1 mixture of glycerol and physiological saline).

[0043] Accurately weigh each of the reagents in experimental groups 1 to 7, dissolve each group in pure water, and filter sterilize using a 0.22μm filter to obtain the cell cryopreservation solution.

[0044] II. Cryopreservation and thawing of bacterial strains and live cell counting:

[0045] Recombinant Escherichia coli (OD) cultured on LB medium were used in experimental groups 1-7 with cryopreserved solutions. 600 =1.5) mixed 1:1, aliquoted, and stored at -80℃. The frozen bacterial culture was rapidly thawed at 37℃, followed by serial dilution with physiological saline. A certain amount of the dilution was spread on LB agar plates, with three replicates for each group. All plates were then incubated at 37℃ for 16 hours. Colonies were counted and the number of viable bacteria after cryopreservation and thawing was calculated for each group. The protective effect of experimental group 8 was defined as 1, and the protective effects of other experimental groups relative to experimental group 8 were calculated.

[0046] Depend on Figure 1 It can be seen that, compared with the traditional glycerol cryopreservation solution (experimental group 8), the addition of a certain proportion of sucrose and skim milk powder to experimental groups 2, 4 and 5 can improve the cryopreservation effect.

[0047] Example 2: Effect of intracellular protectant ratio on strain cryopreservation

[0048] Intracellular protective agents are permeable protective agents, mostly small molecules, which can enter the cell interior, reduce the water content in the cell, and have a certain molar concentration. They reduce the damage caused by changes in electrolyte concentration and the production of ice crystals during the re-freezing process. Intracellular protective agents include glycerol and the like.

[0049] I. Preparation of cell freezing solution (the percentage content is by mass):

[0050] Experiment group 9: 50 g of sucrose, 20 g of skimmed milk powder, 600 g of glycerol, and 3.6 g of sodium chloride.

[0051] Experiment group 10: 50 g of sucrose, 20 g of skimmed milk powder, 500 g of glycerol, and 4.5 g of sodium chloride.

[0052] Experiment group 11: 50 g of sucrose, 20 g of skimmed milk powder, 400 g of glycerol, and 5.4 g of sodium chloride.

[0053] Experiment group 12: 50 g of sucrose, 20 g of skimmed milk powder, 300 g of glycerol, and 6.3 g of sodium chloride.

[0054] Experiment group 13: 50 g of sucrose, 20 g of skimmed milk powder, 200 g of glycerol, and 7.2 g of sodium chloride.

[0055] Experiment group 14: 50 g of sucrose, 20 g of skimmed milk powder, 100 g of glycerol, and 9.0 g of sodium chloride.

[0056] Experiment group 15: 500 g of glycerol and 4.5 g of sodium chloride (equivalent to a 1:1 mixture of glycerol and physiological saline).

[0057] The reagents in experiment groups 9-15 were accurately weighed, dissolved in pure water, filtered and sterilized using a 0.22 μm filter, and cell freezing solutions were obtained.

[0058] II. Bacterial strain freezing and recovery and viable cell counting:

[0059] The recombinant E. coli (OD 600 =1.5) cultured in LB medium using the freezing solutions in experiment groups 9-15 was mixed at a ratio of 1:1, aliquoted, and stored at -80°C. The frozen bacterial strains were thawed quickly at 37°C, then gradient diluted with physiological saline, and a certain amount of the diluted solution was spread on LB solid plates, with 3 replicates for each group. All the plates were incubated in a 37°C incubator for 16 h. The colonies were counted and statistically analyzed to calculate the viable cell count after freezing and recovery in each group. The viable cell count in experiment group 15 was used as a standard, and the protective effect was defined as 1. The protective effect of the other experimental groups relative to experiment group 15 was calculated.

[0060] From the results shown in Table 1, it can be seen that the protective effect of experiment group 15 is the best, and the protective effect of experiment group 14 is the worst. Figure 2It can be seen that, compared with the traditional glycerol cryopreservation solution (experimental group 15), the ratio of glycerol to sodium chloride can be changed while maintaining a certain ratio of sucrose and skim milk powder in experimental groups 13 and 11 to improve the cryopreservation effect.

[0061] Example 3: Effect of the ratio of extracellular and intracellular cryopreservatives on the cryopreservation of bacterial strains

[0062] Depend on Figure 1 It can be seen that both sucrose and skim milk powder have good protective effects within a certain range. Figure 2 It was found that 200g glycerol and 7.2g sodium chloride (experimental group 13) and 400g glycerol and 5.4g sodium chloride (experimental group 11) had better protective effects. Therefore, different combinations were used to determine the optimal ratio of extracellular and intracellular protectants.

[0063] I. Preparation of cell cryopreservation solution (the following percentages are by mass):

[0064] Experimental group 16: 50g sucrose, 50g skim milk powder, 400g glycerol, 5.4g sodium chloride.

[0065] Experimental group 17: 50g sucrose, 50g skim milk powder, 200g glycerol, 7.2g sodium chloride.

[0066] Experimental group 18: 50g sucrose, 20g skim milk powder, 400g glycerol, 5.4g sodium chloride.

[0067] Experimental group 19: 50g sucrose, 20g skim milk powder, 200g glycerol, 7.2g sodium chloride.

[0068] Experimental group 20: 20g sucrose, 20g skim milk powder, 400g glycerol, 5.4g sodium chloride.

[0069] Experimental group 21: 20g sucrose, 20g skim milk powder, 200g glycerol, 7.2g sodium chloride.

[0070] Experimental group 22: 500g glycerol, 4.5g sodium chloride (equivalent to a 1:1 mixture of glycerol and physiological saline).

[0071] Accurately weigh each reagent from experimental groups 16 to 22, dissolve each reagent in pure water, and filter sterilize using a 0.22 μm filter to obtain the cell cryopreservation solution.

[0072] II. Cryopreservation and thawing of bacterial strains and live cell counting:

[0073] The cryopreserved solutions from experimental groups 16-22 were used to culture recombinant Escherichia coli (OD) on LB medium. 600=1.5) were mixed at 1:1 and stored at -80℃ after dispensing. The frozen bacteria were quickly thawed at 37℃ and then gradient diluted with physiological saline. A certain amount of diluted solution was spread on LB solid plates, and each group was repeated 3 times. All plates were incubated in a 37℃ incubator for 16 h. The number of viable bacteria after freezing and recovery of each group was calculated by counting the colonies. The protective effect of the experimental group 22 was defined as 1 based on the number of viable bacteria, and the protective effect of other experimental groups relative to the experimental group 22 was calculated.

[0074] From the above data, it can be seen that the protective effect of the experimental group 19 is the best compared with the traditional glycerol freezing solution and other experimental groups. Figure 3

[0075] Example 4: Effect of antioxidant category and proportion on cell freezing

[0076] Antioxidants eliminate the corresponding oxidative damage to the cell membrane by forming free radicals with oxygen. Bovine serum albumin (BSA) provides protection for enzymes, cell membranes and whole cells in a low temperature environment, and also has certain antioxidant activity. Vitamin C can prevent oxidative damage by preventing the carbonylation of α-amino or lysine, cysteine or histidine residues, thereby enhancing the survival rate and storage of the bacterial strain.

[0077] Experimental group 23: 50 g sucrose, 20 g skim milk powder, 200 g glycerol, 7.2 g NaCl, 1 g BSA, 1 g Vc.

[0078] Experimental group 24: 50 g sucrose, 20 g skim milk powder, 200 g glycerol, 7.2 g NaCl, 2 g BSA, 1 g Vc.

[0079] Experimental group 25: 50 g sucrose, 20 g skim milk powder, 200 g glycerol, 7.2 g NaCl, 1 g BSA, 2 g Vc.

[0080] Experimental group 26: 50 g sucrose, 20 g skim milk powder, 200 g glycerol, 7.2 g NaCl, 2 g BSA, 2 g Vc.

[0081] Experimental group 27: 50 g sucrose, 20 g skim milk powder, 200 g glycerol, 7.2 g NaCl, 2 g BSA, 5 g Vc.

[0082] Experimental group 28: 50 g sucrose, 20 g skim milk powder, 200 g glycerol, 7.2 g NaCl, 5 g BSA, 2 g Vc.

[0083] Experimental group 29: 50 g sucrose, 20 g skim milk powder, glycerol-physiological saline, 5 g BSA, 5 g Vc. ​

[0084] Experimental group 30: 50g sucrose, 20g skim milk powder, 200g glycerol, 7.2g NaCl.

[0085] Accurately weigh each reagent from experimental groups 16 to 22, dissolve each reagent in pure water, and filter sterilize using a 0.22 μm filter to obtain the cell cryopreservation solution.

[0086] II. Cryopreservation and thawing of bacterial strains and live cell counting:

[0087] The experimental group's cryopreserved solution (23-30°C) was used to culture recombinant Escherichia coli (OD) on LB medium. 600 =1.5) mixed 1:1, aliquoted, and stored at -80℃. The frozen bacterial culture was rapidly thawed at 37℃, followed by serial dilution with physiological saline. A certain amount of the dilution was spread on LB agar plates, with three replicates for each group. All plates were then incubated at 37℃ for 16 h. Colonies were counted and the number of viable bacteria after cryopreservation and thawing was calculated for each group. Using the number of viable bacteria in the experimental group 30 as the standard, its protective effect was defined as 1, and the protective effects of other experimental groups relative to the experimental group 30 were calculated.

[0088] Depend on Figure 4 It can be seen that, compared with traditional glycerol cryopreservation solution and other experimental groups, the cryopreservation solution of experimental group 28 has the best protective effect.

[0089] Example 5: Cryopreservation effect of the optimal recombinant Escherichia coli strain cryopreservation solution provided by the present invention.

[0090] Step 1, Preparation of cryopreservation solution:

[0091] According to the present invention, the optimal recombinant Escherichia coli cryopreservation solution, namely Experimental Group 28, is prepared by weighing 200.0g of glycerol, 50.00g of sucrose, and 7.20g of sodium chloride and adding them to distilled water. After stirring until basically dissolved, the solution is brought to a final volume of 1L with pure water. The solution is then sterilized at 115℃ for 15-30 minutes and cooled to room temperature for later use. 20.00g of skim milk powder, 5.00g of BSA, and 2.00g of vitamin C are added to the above solution and stirred until dissolved. The solution is then filtered through a 0.22μm filter membrane for sterilization and subsequently stored at 2-8℃ for later use.

[0092] Step 2: Transformation of recombinant bacteria

[0093] Take 100 ng of the target plasmid DNA and add it to 50 µL of E. coli ( E. coli) BL21 (DE3) competent cells, gently mixed and placed in ice bath for 20 min, placed in 42℃ water bath for 45 s, quickly transferred to ice bath for 2 min. Add 800 μL LB liquid medium, incubate at 37℃ for 45 min, then inoculate on LB solid plate containing Amp. Invert the plate into a 37℃ constant temperature incubator and incubate overnight.

[0094] Step 3, strain culture

[0095] The LB medium with an antibiotic concentration of 100 μg / mL was used for the culture of recombinant E. coli strain. Single colonies were picked into LB medium and incubated at 37℃, 220 rpm for 6-8 h. Microscopy was performed by Gram staining method, and if contamination occurred, the culture was immediately stopped. The strain activity was detected by absorbance method, and the culture was stopped when the required OD 600 value was between 0.8 and 2.0;

[0096] Step 4, strain induction

[0097] Detection of bacterial liquid OD 600 value between 0.8 and 2.0, the strain induction was performed. 1 mL of bacterial liquid was taken in a 1.5 mL EP tube and centrifuged at 12000 rpm for 1 min, the supernatant was discarded and stored at -20℃ as 0 h sample and the OD 600 value was recorded.

[0098] The remaining bacterial liquid was added with IPTG to a final concentration of 0.01 mM and incubated for 4 h. The OD 600 value of the bacterial liquid was detected and recorded, then 1 mL of bacterial liquid was taken in a 1.5 mL EP tube and centrifuged at 12000 rpm for 1 min, the supernatant was discarded and stored at -20℃ as 4 h sample.

[0099] Step 5, strain detection

[0100] Gram staining microscopy:

[0101] 1) Smear, flame fixation;

[0102] 2) Ammonium oxalate crystal violet staining for 1 min;

[0103] 3) Distilled water washing;

[0104] 4) Add iodine solution to cover the smear and stain for about 1 min;

[0105] 5) Wash with water, and absorb the water with absorbent paper;

[0106] 6) Add a few drops of 95% alcohol and gently shake to decolorize, wash with water after 20 s, and absorb the water;

[0107] 7) Safranin staining solution for 2 min, then distilled water washing.

[0108] Dry, microscopic examination, red rods, indicating that the strain is a typical gram-negative bacillus. Test once every three months, after the 12th month test once a year.

[0109] Step 6, cryopreservation solution and strain preservation

[0110] The cryopreservation solution of step 1 and the bacterial solution of step 3 are sequentially extracted in a volume ratio of 1:1 and added to the cryopreservation tube. The cryopreservation tube cap is tightly covered, and the tube is turned up and down to mix thoroughly. The obtained bacterial strain is packaged and labeled. It is quickly stored at -80°C.

[0111] Step 7, activity verification

[0112] 7.1 Strain recovery

[0113] When the bacterial strain needs to be used, the cryopreservation tube is placed in a 37°C water bath for rapid thawing until the bacterial strain is completely melted. At this time, the bacterial strain can be taken out for experiments. Do not thaw at room temperature. In addition, attention should be paid to avoid repeated freezing and thawing of the bacterial strain to avoid death.

[0114] The melted bacterial strain is inoculated into LB medium for strain recovery. The glycerol bacteria are taken in a volume ratio of 1:200 and inoculated into 5mL of LB liquid medium containing 100μg / mL of ampicillin solution. The culture is incubated at 37°C, 220rpm for 12h. After the bacterial culture becomes turbid, the bacterial solution is taken for smear, gram staining and microscopic examination. The microscopic examination results should be short rod and long rod-shaped bacteria, and no suspicious foreign bacteria are checked in 10 fields.

[0115] 7.2 Strain passage

[0116] 7.2.1 Second-generation culture

[0117] The recovered bacterial strain is inoculated into LB medium for passage. The first-generation bacterial solution is taken in a volume ratio of 1:500 and inoculated into 5mL of LB liquid medium containing 100μg / mL of ampicillin solution. The culture is incubated at 37°C, 220rpm for 6-8h. After the bacterial culture becomes turbid, the bacterial solution is taken for smear, gram staining and microscopic examination. The microscopic examination results should be short rod and long rod-shaped bacteria, and no suspicious foreign bacteria are checked in 10 fields. If the gram staining microscopic examination is unqualified, the passage is terminated.

[0118] 7.2.2 Third-generation culture

[0119] The second generation of bacteria is inoculated into LB medium for subculture, and the second generation of bacteria is inoculated into 5 mL of LB liquid medium containing 100 μg / mL of ampicillin at a volume ratio of 1:500, and then the mixture is incubated at 37°C and 220 rpm for 12 hours. After the bacteria culture becomes turbid, the bacterial liquid is taken for smear, and then the smear is subjected to Gram staining and microscopic examination. The microscopic examination result should be short rod and long rod bacteria, and no suspicious bacteria are observed in 10 fields of view. If the Gram staining microscopic examination is unqualified, the subculture is terminated.

[0120] 7.2.3 Subculture of the fourth generation

[0121] The third generation of bacteria is inoculated into LB medium for subculture, and the third generation of bacteria is inoculated into 50 mL of LB liquid medium containing 100 μg / mL of ampicillin at a volume ratio of 1:200, and then the mixture is incubated at 37°C and 220 rpm for 2-4 hours. During the incubation, the bacterial growth is observed, 2 mL of the bacterial liquid is taken for detection by using a UV spectrophotometer, and the bacterial liquid is stored at a low temperature when the OD 600 value is 0.8-1.5, and the Gram staining microscopic examination result is short rod and long rod bacteria, and no suspicious bacteria are observed in 10 fields of view. If the Gram staining microscopic examination is unqualified during the bacterial recovery and subculture, the bacterial recovery and subculture are performed again until the microscopic examination is qualified, and then the next step is continued.

[0122] Step 7.3 Detection

[0123] The fourth generation of bacteria in step 7.2 is sampled, and then IPTG induction is performed according to step 4, and the protein expression of the bacteria is detected. The result is shown in Figure 5 It can be seen that the protein expression amount of the recombinant E. coli bacteria preserved by using the freezing solution and the method according to the present application is still high after the bacteria are recovered and subcultured for four generations and then induced for 2 hours.

[0124] The above description is only a preferred embodiment of the present application, and is not intended to limit the scope of the present application. The above embodiment of the present application can be variously changed. Any simple, equivalent change and modification made according to the content of the present application and the description of the claims are within the scope of the present application. The present application is not described in detail, and is a conventional technical content.

Claims

1. A recombinant Escherichia coli strain stock cryo, characterized in that, The formula of the recombinant E. coli strain cryopreservation solution is: glycerol 200 g / L, sucrose 50 g / L, skimmed milk powder 20 g / L, BSA 5 g / L, sodium chloride 7.2 g / L, vitamin C 2 g / L; the recombinant E. coli strain cryopreservation solution is used for the recombinant E. coli BL21 carrying an exogenous plasmid.

2. A method of preparing a recombinant E. coli strain stock cryopreservation solution according to claim 1, characterized by, The method comprises the following steps: A1: respectively weigh 200 g of glycerol, 50 g of sucrose, and 7.2 g of sodium chloride, and add them into distilled water in sequence, stir until they are basically dissolved, and then use pure water to make up to 1 L; A2: high-temperature sterilization at 115 ℃ for 15-30 min, and then cool to room temperature for standby use; A3: respectively weigh 20 g of skimmed milk powder, 5 g of BSA, and 2 g of vitamin C, and add them into the solution obtained in step A2 in sequence, stir until they are dissolved, and then filter sterilization by using a 0.22 μm filter membrane, and then store at 2-8 ℃ for standby use or store at-20 ℃ or below for long-term storage.

3. A method of cryopreservation of a recombinant E. coli strain, characterized in that, The method comprises the following steps: B1: prepare a recombinant E. coli strain cryopreservation solution according to claim 1; B2: take 100 ng of target plasmid DNA and add it into 50 μL of E. coli BL21 competent cells, mix gently, and then perform ice bath for 20 min, place in a 42 ℃ water bath for 45 s, quickly transfer to an ice bath for 2 min, add 800 μL of LB liquid medium, cultivate at 37 ℃ for 45 min, and then inoculate on an LB solid plate containing Amp, and then place the plate into a 37 ℃ constant-temperature incubator for cultivation for 16 h; B3: Recombinant E. coli strain culture was carried out in LB medium with an antibiotic concentration of 100 μg / mL, single colonies were picked into LB medium, incubated at 37°C, 220 rpm for 6-8 h, and then examined by microscopy using Gram staining method. If contamination occurred, the culture was immediately stopped. Bacterial activity was detected by absorbance method, and the culture was stopped when the required OD 600 value was reached in the range of 0.8-2.

0. B4: according to the volume ratio of 1:1, sequentially take the recombinant E. coli strain cryopreservation solution in step B1 and the bacterial liquid in step B2, and add them into a cryopreservation tube, tightly cover the cryopreservation tube bottle cap, and then turn up and down to mix them thoroughly; immediately, quickly package the obtained bacterial strain, label it, and then store it at-80 ℃ to-196 ℃ for long-term storage.

Images