Recombinant escherichia coli strain cryopreservation solution as well as preparation method and application thereof

Abstract

The invention discloses a recombinant escherichia coli strain cryopreservation solution as well as a preparation method and application thereof. The formula of the recombinant escherichia coli strain cryopreservation liquid comprises 200-500 g / L of glycerin, 20-100 g / L of cane sugar, 20-50 g / L of skim milk powder, 1-5 g / L of BSA, 4-8 g / L of sodium chloride and 1-5 g / L of vitamin C. The recombinant Escherichia coli strain cryopreservation liquid is used for preserving the recombinant Escherichia coli strain, the method has the advantages of simplicity and practicability in operation, long preservation time, stable and reliable strain quality and low preservation cost, and the recombinant Escherichia coli strain preserved according to the method disclosed by the invention is relatively high in protein expression quantity through strain recovery detection; therefore, the method has a good application prospect in freezing preservation of recombinant escherichia coli strains.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial engineering, and more specifically to a recombinant Escherichia coli strain freezing solution, a preparation method thereof and an application thereof. Background Art

[0002] Bacterial strain resources are precious resources for mankind. Domestic and foreign research institutions have paid great attention to strain preservation. The significance of strain preservation lies in maintaining the stability of its original shape and vitality as much as possible, ensuring that the strains do not die, mutate, or be contaminated, so as to facilitate research, exchange, and use. Strain preservation is to artificially create conditions to isolate the strains from the outside world. The principle of strain preservation is to reduce the metabolism of microorganisms as much as possible to reduce mutation. Generally, the strains can be kept in a "semi-dormant" or "dormant" state by limiting nutrition, hypoxia, drying, and low temperature, etc., to reduce the mutation and death probability of the strains and maintain good genetic genes.

[0003] Recombinant Escherichia coli is a genetically engineered bacterium that contains exogenous DNA fragments carried by vector plasmids, etc. These fragments are usually genetically unstable and can easily lose their exogenous plasmid replicators during the culture process. During the culture preservation and rejuvenation stage, it is also easy to lose the exogenous plasmids, which directly affects the relevant properties of the genetically engineered strains. Recombinant Escherichia coli strains are usually preserved in glycerol tubes at -80℃ ultra-low temperature or liquid nitrogen. The survival rate of strains is significantly reduced after a period of ultra-low temperature preservation at -80℃ in glycerol tubes; liquid nitrogen preservation has strict storage conditions. Not only is the initial preservation cost high, but during the preservation process, the temperature rises due to the evaporation of liquid nitrogen, and the state of ice crystals changes, which can easily lead to the death of strains. Therefore, it is necessary to pay close attention to the residual amount of liquid nitrogen and replenish liquid nitrogen regularly. During replenishment, there is also the risk of explosion due to exposure of liquid nitrogen to air, and operators need to have a high level of operational proficiency. Summary of the invention

[0004] The purpose of the present invention is to provide a recombinant Escherichia coli strain freezing solution and a preparation method and application thereof, so as to solve the problems of reduced survival rate, strict storage conditions and high cost in the storage of recombinant Escherichia coli strains in the prior art.

[0005] In order to solve the above technical problems, the technical solutions of the present invention are as follows: According to a first aspect of the present invention, a recombinant Escherichia coli strain freezing solution is provided, wherein the formula of the recombinant Escherichia coli strain freezing solution is: 200-500 g / L glycerol, 20-100 g / L sucrose, 20-50 g / L skimmed milk powder, 1-5 g / L BSA, 4-8 g / L sodium chloride, and 1-5 g / L vitamin C.

[0006] Preferably, the formula of the recombinant Escherichia coli strain freezing solution is: 200-400 g / L glycerol, 50-100 g / L sucrose, 20 g / L skim milk powder, 1-5 g / L BSA, 4.5-7.2 g / L sodium chloride, and 1-2 g / L vitamin C.

[0007] According to a preferred embodiment of the present invention, the formula of the recombinant Escherichia coli strain freezing solution is: 200 g / L glycerol, 50 g / L sucrose, 20 g / L skim milk powder, 5 g / L BSA, 7.2 g / L sodium chloride, and 2 g / L vitamin C. This formula is the most preferred embodiment of the present invention. The recombinant Escherichia coli strain freezing solution using this formula has the best protection effect on the recombinant Escherichia coli strain.

[0008] According to another preferred embodiment of the present invention, the formula of the recombinant Escherichia coli strain freezing solution is: 200 g / L glycerol, 50 g / L sucrose, 20 g / L skimmed milk powder, 2 g / L BSA, 7.2 g / L sodium chloride, and 1 g / L vitamin C.

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

[0010] According to a second aspect of the present invention, there is provided a method for preparing a recombinant Escherichia coli strain freezing solution, comprising the following steps: A1: weighing 200-500 g of glycerol, 20-100 g of sucrose, and 4-8 g of sodium chloride respectively, adding them to distilled water in sequence, stirring until basically dissolved, and then making up the volume to 1 L with pure water; A2: sterilizing at 115°C for 15-30 min, cooling to room temperature for use; A3: weighing 20-50 g of skim milk powder, 1-5 g of BSA, and 1-5 g of vitamin C respectively, adding them to the above solutions in sequence, stirring until dissolved, filtering and sterilizing with a 0.22 μm filter membrane, and then storing at 2-8°C for use or storing for a long time below -20°C.

[0011] According to a third aspect of the present invention, a method for freezing recombinant Escherichia coli strains is provided, comprising the following steps: B1: preparing a freezing solution of recombinant Escherichia coli strains as described above; B2: taking 100 ng of the target plasmid DNA and adding it to 50 µL of Escherichia coli (E. coli) BL21 (DE3) competent cells, gently mixing and ice bathing for 20 minutes, placing it in a 42°C water bath for 45 seconds, quickly transferring it to an ice bath for 2 minutes, adding 800 µL of LB liquid culture medium, culturing it at 37°C for 45 minutes, and then inoculating it on an LB solid plate containing Amp, and inverting the plate into a 37°C constant temperature incubator for 16 hours; B3: using LB culture medium with an antibiotic concentration of 100 μg / mL to culture the recombinant Escherichia coli strains, picking a monoclonal colony into the LB culture medium, culturing it at 37°C and 220 rpm for 6 to 8 hours, using Gram staining for microscopic examination, and stopping the culture immediately if contamination occurs; using absorbance to detect the activity of the strain to achieve the required OD 600 Stop culturing when the value is between 0.8 and 2.0; B4: extract the recombinant Escherichia coli culture freezing solution of step B1 and the culture solution of step B2 in a volume ratio of 1:1 and add them to the cryopreservation tube, cover the cryopreservation tube bottle cap tightly, turn it upside down to mix it thoroughly; package the obtained culture, label it and store it at -20℃~-196℃ for long-term storage.

[0012] In order to overcome the problems of reduced survival rate of recombinant Escherichia coli strains, harsh storage conditions and high costs in the prior art, it is necessary to develop a new strain preservation method. The inventors tried to further optimize the frozen strains in glycerol tubes and developed a new type of freezing solution with the aid of a variety of protective agents. However, during the freezing, storage and recovery process of recombinant Escherichia coli strains, there are many factors that induce strain death, loss of exogenous plasmids, and decreased strain survival rate. In addition, many factors such as the concentration of the strain suspension, the age of the strain material, the type of culture medium, and the protective agent will also affect the frozen preservation of the strain. Therefore, it is necessary to select the appropriate type and amount of protective agents according to the characteristics of the strain to ensure the long-term stable storage and production application of the strain. Therefore, a preservation method suitable for the preservation of recombinant Escherichia coli strains is developed, which has a simple operation method, mild conditions, and can ensure the stability of strain performance and achieve long-term preservation. It has great practical significance for the maintenance, replication and continuation of the excellent performance of genetically engineered bacteria, and is more conducive to industrial stable production.

[0013] The cryoprotectant components used in the prior art generally include glycerol and certain polyol compounds such as mannitol, and some cryoprotectants also include dimethyl sulfoxide. The main function of glycerol and polyols is to prevent water from freezing, and dimethyl sulfoxide can lower 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 metabolites. However, dimethyl sulfoxide has a strong toxic effect on cells at room temperature.

[0014] The present invention provides a novel recombinant Escherichia coli strain freezing solution with glycerol as the main component. When selecting the formula, the influence of multiple factors on the recombinant Escherichia coli during the freezing process is taken into consideration, and protective agents with different mechanisms are selected to enhance or synergistically protect the strain.

[0015] Among them, the effect of glycerol is to prolong the freezing process, because glycerol will not enter the ice, so as the freezing progresses, the concentration of glycerol increases, which further lowers the freezing point. Since freezing is relatively slow, the static pressure of the water may be distributed more evenly, and the bacteria may also freeze ahead of the environment, which are all conducive to their survival. Glycerol also protects the cell membrane. The problem with conventional freezing is that the ice crystals formed in the cells may damage the fragile cell membrane structure, so that the cells cannot survive normally after thawing. Glycerol may increase the ductility of the lipid bilayer, thereby protecting the cell membrane structure.

[0016] Sucrose is a non-osmotic sugar that can increase the osmotic pressure of the freezing solution and help cells maintain water balance. In addition, sucrose can inhibit the formation of ice crystals in cell tissues, protect the integrity of plasma membranes and protein structures, etc.

[0017] Adding physiological levels of sodium chloride can reduce the hypertonic effect of glycerol and better protect the bacteria.

[0018] The high molecular weight protective agent skimmed milk powder is a mixture that contains a variety of substances such as protein, salts and vitamins. The protein provides a protective outer membrane for the bacterial cells, and the phosphates and citrates in it can act as a buffer to stabilize the pH value.

[0019] Bovine serum albumin (BSA) provides protection for enzymes, cell membranes and entire cells in low temperature environments, and also has certain antioxidant activity, thereby better protecting bacterial strains.

[0020] As an antioxidant, vitamin C can prevent oxidative damage to bacteria during freezing and storage, and improve the survival rate and stability of bacteria. As an antioxidant, vitamin C eliminates the corresponding oxidative damage to cell membranes by forming free radicals with oxygen. At the same time, vitamin C can also prevent oxidative damage to important bacterial components such as proteins and DNA by preventing the carbonylation of α-amino or lysine, cysteine ​​or histidine residues, thereby enhancing the survival rate and storage of bacteria.

[0021] The main point of the invention is to provide such a formula for recombinant Escherichia coli strain freezing solution. When Escherichia coli is frozen, 50% glycerol freezing method and skimmed milk powder freezing method are mostly used for freezing. Among them, the 50% glycerol freezing method will reduce the plasmid stability of recombinant Escherichia coli due to the high glycerol concentration, thereby affecting the expression stability of the recombinant bacteria; the skimmed milk powder freezing method has a high cost and is relatively less used in Escherichia coli freezing. According to such a recombinant Escherichia coli strain freezing solution provided by the present invention, on the one hand, the glycerol concentration is reduced to enhance the plasmid stability of the strain, and on the other hand, sucrose and skimmed milk powder are used to assist glycerol to enhance the freezing protection effect, and sodium chloride is added to balance the osmotic pressure. At the same time, BSA and vitamin C are added to further reduce the oxidative damage during freezing and recovery. The present invention provides such a new recombinant Escherichia coli strain freezing solution with glycerol as the main body. When selecting the formula, the influence of multiple factors on the recombinant Escherichia coli during the freezing process is taken into account, and protective agents with different mechanisms are selected to strengthen or synergistically protect the strain.

[0022] Another inventive point of the present invention is that, through continuous optimization of the formula, a variety of preferred recombinant Escherichia coli strain freezing solution formulas are finally obtained. Among them, the most preferred formula of a recombinant Escherichia coli strain freezing solution is: 200g / L glycerol, 50g / L sucrose, 20g / L skim milk powder, 5g / L BSA, 7.2g / L sodium chloride, and 2g / L vitamin C. The recombinant Escherichia coli strain freezing solution using this formula has the best protection effect on the recombinant Escherichia coli strain, which is better than all other freezing solutions.

[0023] The present invention uses the freezing solution component to preserve strains with good effect. The quality of recombinant Escherichia coli frozen by the freezing solution and preservation method of the present invention is stable, the freezing method is simple, the freezing range is wide (-20°C~-196°C), the strain preservation time is long, and it can be preserved at -80°C for more than 3 years, and the strains after preservation have high activity when revived, the preservation process is simple, and it is easy to store and find. The method has the advantages of simple and practical operation, long preservation time, stable and reliable strain quality, and low preservation cost. The recombinant Escherichia coli strains preserved by the method of the present invention are tested for expression after revived strains and cultured for four generations, and the protein expression level induced for 2 hours is high.

[0024] In summary, the present invention provides a recombinant Escherichia coli strain freezing solution that can better protect the recombinant Escherichia coli strain and has a long preservation time, and also provides a recombinant Escherichia coli strain preservation method that is simple and practical to operate, has a long preservation time, has stable and reliable strain quality, and has a low preservation cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The effect of the extracellular protective agents, namely sucrose and skimmed milk powder, on the cryopreservation protection effect of the strains in Example 1 is shown; wherein 1-8 are the cryopreservation protection effects of experimental groups 1-8 respectively; Figure 2 The effect of the intracellular protective agents, i.e., glycerol and sodium chloride, on the cryopreservation protection of the strains in Example 2 is shown; wherein 9-15 are the cryopreservation protection effects of the experimental groups 9-15, respectively; Figure 3 The effect of the ratio of the extracellular protective agent to the intracellular protective agent on the cryopreservation protection effect of the strains in Example 3 is shown; wherein, 16-22 are the cryopreservation protection effects of the experimental groups 16-22, respectively; Figure 4 The effects of the types and proportions of antioxidants in Example 4 on the cryopreservation protection of the strains are shown; wherein 23-30 are the cryopreservation protection effects of the experimental groups 23-30, respectively; Figure 5 A schematic diagram of the protein expression level after revival of a most preferred recombinant Escherichia coli strain frozen in a freezing solution according to the present invention in Example 5 is shown, wherein the 1st to 4th bands from left to right are: Marker, induction 0h, induction 2h, induction 4h. DETAILED DESCRIPTION

[0026] The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional operations in the art, or according to the experimental methods recommended by the kit and instrument manufacturers. The reagents and materials used in the examples can be obtained from commercial sources unless otherwise specified.

[0027] Example 1: Effects of extracellular protective agent types and ratios on the freezing of bacterial strains Extracellular protective agents are non-permeable protective agents, mostly macromolecular substances with a certain viscosity that cannot enter cells. Their main function is to maintain the stability of the cell membrane and to combine with water molecules in the solution to reduce the extracellular free water content, lower the freezing point, and reduce the formation of ice crystals. Extracellular protective agents include sucrose and skimmed milk powder.

[0028] 1. Prepare cell freezing solution (the following percentages are based on mass): Experimental group 1: 50 g sucrose, 20 g skimmed milk powder, 500 g glycerol, 4.5 g sodium chloride.

[0029] Experimental group 2: 50 g sucrose, 50 g skimmed milk powder, 500 g glycerol, 4.5 g sodium chloride.

[0030] Experimental group 3: 50 g sucrose, 100 g skimmed milk powder, 500 g glycerol, 4.5 g sodium chloride.

[0031] Experimental group 4: 20 g sucrose, 20 g skimmed milk powder, 500 g glycerol, 4.5 g sodium chloride.

[0032] Experimental group 5: 100 g sucrose, 20 g skimmed milk powder, 500 g glycerol, 4.5 g sodium chloride.

[0033] Experimental group 6: 20 g skim milk powder, 500 g glycerol, 4.5 g sodium chloride.

[0034] Experimental group 7: 50 g sucrose, 500 g glycerol, 4.5 g sodium chloride.

[0035] Experimental group 8: 500 g glycerol, 4.5 g sodium chloride (equivalent to a 1:1 mixture of glycerol and saline).

[0036] Accurately weigh each reagent of the above experimental groups 1 to 7, stir and dissolve each group with pure water, and filter and sterilize using a 0.22 μm filter to obtain the cell freezing solution.

[0037] 2. Cryopreservation and resuscitation of bacterial strains and count of viable bacteria: The cryopreserved solutions of experimental groups 1 to 7 were mixed with the recombinant Escherichia coli cultured in LB medium (OD 600 =1.5) 1:1 mix, package and store at -80℃. The frozen bacteria were quickly thawed at 37℃, and then gradiently diluted with physiological saline. A certain amount of dilution was spread on LB solid plates. Each group was repeated 3 times, and then all plates were incubated in a 37℃ incubator for 16 hours. The colonies were counted and the number of viable bacteria in each group after cryopreservation and recovery was calculated. Taking the number of viable bacteria in experimental group 8 as the standard, its protection effect was defined as 1, and the protection effect of other experimental groups relative to experimental group 8 was calculated.

[0038] Depend on Figure 1 It can be seen that compared with the traditional glycerol cryopreservation solution (experimental group 8), adding a certain proportion of sucrose and skimmed milk powder in experimental groups 2, 4, and 5 can improve the protective effect of the cryopreservation solution.

[0039] Example 2: Effect of the ratio of intracellular protective agents on the freezing of bacterial strains Intracellular protective agents are permeable protective agents, mostly small molecules, which can enter the cell and reduce the water molecule content in the cell. They also have a certain molar concentration. They can reduce the damage caused by changes in electrolyte concentration and the production of ice crystals during cell refreezing. Intracellular protective agents include glycerol, etc.

[0040] 1. Prepare cell freezing solution (the following percentages are based on mass): Experimental group 9: 50 g sucrose, 20 g skimmed milk powder, 600 g glycerol, 3.6 g sodium chloride.

[0041] Experimental group 10: 50 g sucrose, 20 g skimmed milk powder, 500 g glycerol, 4.5 g sodium chloride.

[0042] Experimental group 11: 50 g sucrose, 20 g skimmed milk powder, 400 g glycerol, 5.4 g sodium chloride.

[0043] Experimental group 12: 50 g sucrose, 20 g skimmed milk powder, 300 g glycerol, 6.3 g sodium chloride.

[0044] Experimental group 13: 50 g sucrose, 20 g skimmed milk powder, 200 g glycerol, 7.2 g sodium chloride.

[0045] Experimental group 14: 50 g sucrose, 20 g skimmed milk powder, 100 g glycerol, 9.0 g sodium chloride.

[0046] Experimental group 15: 500 g glycerol, 4.5 g sodium chloride (equivalent to a 1:1 mixture of glycerol and saline).

[0047] Accurately weigh each reagent in the above experimental groups 9 to 15, dissolve each reagent in pure water, and filter and sterilize using a 0.22 μm filter to obtain the cell freezing solution.

[0048] 2. Cryopreservation and resuscitation of bacterial strains and count of viable bacteria: The cryopreserved liquids of experimental groups 9 to 15 were used to culture the recombinant Escherichia coli (OD 600 =1.5) 1:1 mix, package and store at -80℃. The frozen bacteria were quickly thawed at 37℃, and then gradiently diluted with physiological saline. A certain amount of dilution was spread on LB solid plates. Each group was repeated 3 times, and then all plates were incubated in a 37℃ incubator for 16 h. The colonies were counted and the number of viable bacteria in each group after cryopreservation and recovery was calculated. Taking the number of viable bacteria in experimental group 15 as the standard, its protection effect was defined as 1, and the protection effect of other experimental groups relative to experimental group 15 was calculated.

[0049] Depend on Figure 2It can be seen that compared with the traditional glycerol cryopreservation solution (experimental group 15), changing the ratio of glycerol to sodium chloride on the basis of maintaining a certain ratio of sucrose and skimmed milk powder in experimental groups 13 and 11 can improve the protective effect of the cryopreservation solution.

[0050] Example 3: Effect of the ratio of extracellular protective agent to intracellular protective agent on the freezing of bacterial strains Depend on Figure 1 It can be seen that sucrose and skimmed milk powder have good protective effects within a certain range. Figure 2 It can be seen that 200g glycerol, 7.2g sodium chloride (experimental group 13) and 400g glycerol, 5.4g sodium chloride (experimental group 11) have better protective effects. Therefore, different combinations are used to determine the optimal ratio of extracellular protective agents to intracellular protective agents.

[0051] 1. Prepare cell freezing solution (the following percentages are based on mass): Experimental group 16: 50 g sucrose, 50 g skimmed milk powder, 400 g glycerol, 5.4 g sodium chloride.

[0052] Experimental group 17: 50 g sucrose, 50 g skimmed milk powder, 200 g glycerol, 7.2 g sodium chloride.

[0053] Experimental group 18: 50 g sucrose, 20 g skimmed milk powder, 400 g glycerol, 5.4 g sodium chloride.

[0054] Experimental group 19: 50 g sucrose, 20 g skimmed milk powder, 200 g glycerol, 7.2 g sodium chloride.

[0055] Experimental group 20: 20 g sucrose, 20 g skimmed milk powder, 400 g glycerol, 5.4 g sodium chloride.

[0056] Experimental group 21: 20 g sucrose, 20 g skimmed milk powder, 200 g glycerol, 7.2 g sodium chloride.

[0057] Experimental group 22: 500 g glycerol, 4.5 g sodium chloride (equivalent to a 1:1 mixture of glycerol and saline).

[0058] Accurately weigh each reagent of the above experimental groups 16 to 22, stir and dissolve each group with pure water, and filter and sterilize using a 0.22 μm filter to obtain the cell freezing solution.

[0059] 2. Cryopreservation and resuscitation of bacterial strains and count of viable bacteria: The cryopreserved liquids of experimental groups 16 to 22 were used to culture the recombinant Escherichia coli (OD 600=1.5) 1:1 mix, package and store at -80℃. The frozen bacteria were quickly thawed at 37℃, and then gradiently diluted with physiological saline. A certain amount of dilution was spread on LB solid plates. Each group was repeated 3 times, and then all plates were incubated in a 37℃ incubator for 16 h. The colonies were counted and the number of viable bacteria in each group after cryopreservation and recovery was calculated. Taking the number of viable bacteria in experimental group 22 as the standard, its protection effect was defined as 1, and the protection effect of other experimental groups relative to experimental group 22 was calculated.

[0060] Depend on Figure 3 It can be seen that compared with traditional glycerol cryopreservation solution and other experimental groups, the cryopreservation solution of experimental group 19 has the best protection effect.

[0061] Example 4: Effects of Antioxidant Type and Ratio on Cell Cryopreservation Antioxidants eliminate the corresponding oxidative damage to cell membranes by forming free radicals with oxygen. Bovine serum albumin (BSA) provides protection for enzymes, cell membranes and the entire cell under low temperature conditions, 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 bacterial strains.

[0062] Experimental group 23: 50 g sucrose, 20 g skimmed milk powder, 200 g glycerol, 7.2 g NaCl, 1 g BSA, 1 g Vc.

[0063] Experimental group 24: 50 g sucrose, 20 g skimmed milk powder, 200 g glycerol, 7.2 g NaCl, 2 g BSA, 1 g Vc.

[0064] Experimental group 25: 50 g sucrose, 20 g skimmed milk powder, 200 g glycerol, 7.2 g NaCl, 1 g BSA, 2 g Vc.

[0065] Experimental group 26: 50 g sucrose, 20 g skimmed milk powder, 200 g glycerol, 7.2 g NaCl, 2 g BSA, 2 g Vc.

[0066] Experimental group 27: 50 g sucrose, 20 g skimmed milk powder, 200 g glycerol, 7.2 g NaCl, 2 g BSA, 5 g Vc.

[0067] Experimental group 28: 50 g sucrose, 20 g skimmed milk powder, 200 g glycerol, 7.2 g NaCl, 5 g BSA, 2 g Vc.

[0068] Experimental group 29: 50 g sucrose, 20 g skim milk powder, glycerol-saline, 5 g BSA, 5 g Vc.

[0069] Experimental group 30: 50 g sucrose, 20 g skimmed milk powder, 200 g glycerol, 7.2 g NaCl.

[0070] Accurately weigh each reagent of the above experimental groups 16 to 22, stir and dissolve each group with pure water, and filter and sterilize using a 0.22 μm filter to obtain the cell freezing solution.

[0071] 2. Cryopreservation and resuscitation of bacterial strains and count of viable bacteria: The cryopreserved liquid from the experimental group 23 to 30 was used to culture the recombinant Escherichia coli (OD 600 =1.5) 1:1 mix, package and store at -80℃. The frozen bacteria were quickly thawed at 37℃, and then gradiently diluted with physiological saline. A certain amount of dilution was spread on LB solid plates. Each group was repeated 3 times, and then all plates were incubated in a 37℃ incubator for 16 h. The colonies were counted and the number of viable bacteria in each group after cryopreservation and recovery was calculated. Taking the number of viable bacteria in the experimental group 30 as the standard, its protection effect was defined as 1, and the protection effect of other experimental groups relative to the experimental group 30 was calculated.

[0072] Depend on Figure 4 It can be seen that compared with traditional glycerol cryopreservation solution and other experimental groups, the protection effect of experimental group 28 cryopreservation solution is the best.

[0073] Example 5: Cryopreservation effect of a most preferred recombinant Escherichia coli strain cryopreservation solution provided by the present invention Step 1: Preparation of freezing solution: According to the most preferred recombinant Escherichia coli strain cryopreservation solution provided by the present invention, i.e., Experimental Group 28, according to the formula of Experimental Group 28, 200.0g of glycerol, 50.00g of sucrose, and 7.20g of sodium chloride were weighed and added to distilled water, stirred until basically dissolved, and then diluted to 1L with pure water. Sterilized at 115°C for 15-30min, cooled to room temperature for use; 20.00g of skim milk powder, 5.00g of BSA, and 2.00g of vitamin C were weighed and added to the above solution, stirred until dissolved, and then sterilized by filtration with a 0.22μm filter membrane, and then stored at 2-8°C for use.

[0074] Step 2: Transformation of recombinant bacteria Take 100ng of target plasmid DNA and add 50µL of E. coli ( E. coli ) BL21 (DE3) competent cells, gently mix and place on ice for 20 minutes, place in a 42℃ water bath for 45 seconds, and quickly transfer to an ice bath for 2 minutes. Add 800µL LB liquid medium, culture at 37℃ for 45 minutes, and then inoculate on LB solid plates containing Amp. Invert the plate and culture it in a 37℃ constant temperature incubator overnight.

[0075] Step 3: Bacteria culture The recombinant E. coli strains were cultured in LB medium with an antibiotic concentration of 100 μg / mL. Monoclonal colonies were picked and placed in LB medium and cultured at 37°C and 220 rpm for 6-8 hours. Gram staining was used for microscopic examination. If contamination occurred, the culture was stopped immediately. The activity of the strains was tested by absorbance to reach the required OD 600 When the value is between 0.8 and 2.0, the culture is stopped; Step 4: Bacteria induction Detection of bacterial solution OD 600 When the value is between 0.8 and 2.0, the strain can be induced. First, extract 1 mL of bacterial solution and centrifuge it in a 1.5 mL EP tube at 12000 rpm for 1 min, discard the supernatant, and store it at -20℃ as the 0 h sample and record its OD 600 value.

[0076] Add IPTG to the remaining bacterial solution to a final concentration of 0.01 mM and continue culturing for 4 h. Detect the OD of the bacterial solution 600 Then, extract 1 mL of bacterial solution and centrifuge it at 12000 rpm for 1 min in a 1.5 mL EP tube. Discard the supernatant and store it at -20°C, which is the 4-h sample.

[0077] Step 5: Bacteria Detection Gram stain microscopy: 1) Smear, flame fixation; 2) Stain with ammonium oxalate crystal violet for 1 min; 3) Rinse with distilled water; 4) Add iodine solution to cover the surface and dye for about 1 minute; 5) Wash with water and absorb the water with absorbent paper; 6) Add a few drops of 95% alcohol and shake gently to decolorize. After 20 seconds, wash with water and absorb the water. 7) After staining with safranin solution for 2 min, rinse with distilled water.

[0078] Dry and examine under a microscope, and the result is red rods, which proves that the bacteria are typical Gram-negative rods. Test once every three months, and once a year after the 12th month.

[0079] Step 6: Adding freezing solution and preserving strains The freezing solution of step 1 and the bacterial solution of step 3 were extracted in a volume ratio of 1:1 and added to the cryopreservation tubes. The caps of the cryopreservation tubes were tightly closed and turned upside down to mix them thoroughly. The obtained bacterial strains were packaged and labeled. The products were quickly stored at -80°C.

[0080] Step 7: Liveness Verification 7.1 Bacterial recovery When you need to use the strains, place the cryopreserved tube in a 37°C water bath to quickly thaw until the strains are completely thawed. At this time, you can take out the strains for experiments, and do not thaw them at room temperature. In addition, be careful not to freeze and thaw the strains repeatedly to avoid death.

[0081] Inoculate the melted bacteria into LB medium for bacterial recovery. Pipette glycerol bacteria at a volume ratio of 1:200 and inoculate into 5mL LB liquid medium containing 100μg / mL ampicillin solution, and culture at 37℃, 220rpm for 12h. After the bacterial culture becomes turbid, take the bacterial solution for smear and Gram staining. The microscopic examination results should be short rod and long rod-shaped bacteria. Check 10 fields of view for no suspicious bacteria.

[0082] 7.2 Subculture of bacterial strains 7.2.1 Second Generation Culture The revived strains were inoculated into LB medium for subculture. The first generation of bacterial solution was inoculated into 5 mL of LB liquid medium containing 100 μg / mL ampicillin solution at a volume ratio of 1:500, and cultured at 37°C and 220 rpm for 6-8 hours. When the strain culture became turbid, the bacterial solution was taken for smear and Gram staining and microscopic examination. The microscopic examination results should be short-rod and long-rod-shaped bacteria. There were no suspicious bacteria in 10 visual fields. If Gram staining and microscopic examination failed, subculture was terminated.

[0083] 7.2.2 Third generation culture The second-generation cultured strains were inoculated into LB medium for subculture. The second-generation bacterial solution was extracted at a volume ratio of 1:500 and inoculated into 5 mL of LB liquid medium containing 100 μg / mL ampicillin solution. The culture was cultured at 37°C and 220 rpm for 12 hours. When the culture became turbid, the bacterial solution was taken for smear and Gram staining and microscopic examination. The microscopic examination results should be short-rod and long-rod-shaped bacteria. There were no suspicious bacteria in 10 visual fields. If the Gram staining and microscopic examination failed, subculture was terminated.

[0084] 7.2.3 Fourth Generation Culture The third-generation cultured bacteria were inoculated into LB medium for subculture. The third-generation culture solution was inoculated into 50 mL of LB liquid medium containing 100 μg / mL ampicillin solution at a volume ratio of 1:200, and cultured at 37°C and 220 rpm for 2-4 hours. During the culture period, the bacterial growth was observed, and 2 mL of the culture solution was taken for detection by UV spectrophotometer. The OD value of the frozen bacteria was required to be 600 When it reaches between 0.8 and 1.5, and the results of Gram staining microscopy are short-rod and long-rod-shaped bacteria, and 10 fields of view are checked for no suspicious bacteria, the culture is terminated; if any step in the process of strain recovery and subculture fails the microscopic examination, the strain recovery and subculture must be repeated until the microscopic examination passes, and then proceed to the next step.

[0085] Step 7.3 Detection Keep a sample from the fourth generation of bacteria in step 7.2, and then perform IPTG induction according to step 4 to detect the protein expression of the bacteria. The results are shown in Figure 5 It can be seen that the recombinant Escherichia coli strains preserved according to the freezing solution and method of the present invention, after the strains are revived and cultured for four generations, the protein expression level after induction for 2 hours is still high.

[0086] The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. The above embodiments of the present invention can also be modified in various ways. All simple, equivalent changes and modifications made according to the claims and the description of the present invention fall within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

Claims

1. A recombinant Escherichia coli strain cryopreservation solution, characterized in that: The formula of the recombinant Escherichia coli strain freezing solution is: 200-500 g / L glycerol, 20-100 g / L sucrose, 20-50 g / L skimmed milk powder, 1-5 g / L BSA, 4-8 g / L sodium chloride, and 1-5 g / L vitamin C.

2. The recombinant Escherichia coli strain freezing solution according to claim 1, characterized in that: The formula of the recombinant Escherichia coli strain freezing solution is: 200-400 g / L glycerol, 50-100 g / L sucrose, 20 g / L skimmed milk powder, 1-5 g / L BSA, 4.5-7.2 g / L sodium chloride, and 1-2 g / L vitamin C.

3. The recombinant Escherichia coli strain freezing solution according to claim 1, characterized in that: The formula of the recombinant Escherichia coli strain freezing solution is: 200 g / L glycerol, 50 g / L sucrose, 20 g / L skimmed milk powder, 5 g / L BSA, 7.2 g / L sodium chloride, and 2 g / L vitamin C.

4. The recombinant Escherichia coli strain freezing solution according to claim 1, characterized in that: The formula of the recombinant Escherichia coli strain freezing solution is: 200 g / L glycerol, 50 g / L sucrose, 20 g / L skimmed milk powder, 2 g / L BSA, 7.2 g / L sodium chloride, and 1 g / L vitamin C.

5. The recombinant Escherichia coli strain freezing solution according to claim 1, characterized in that: The formula of the recombinant Escherichia coli strain freezing solution is: 200 g / L glycerol, 50 g / L sucrose, 20 g / L skimmed milk powder, 1 g / L BSA, 7.2 g / L sodium chloride, and 2 g / L vitamin C.

6. A method for preparing a recombinant Escherichia coli strain freezing solution according to any one of claims 1 to 5, characterized in that: The following steps are involved: A1: Weigh 200-500g of glycerol, 20-100g of sucrose, and 4-8g of sodium chloride respectively, add them into distilled water in sequence, stir until they are basically dissolved, and then dilute to 1L with pure water; A2: Sterilize at 115℃ for 15-30min, cool to room temperature for later use; A3: Weigh 20-50 g skim milk powder, 1-5 g BSA, and 1-5 g vitamin C respectively, add them to the solution obtained in step A2 in sequence, stir until dissolved, filter and sterilize using a 0.22 μm filter membrane, and then store at 2-8°C for later use or store below -20°C for long-term storage.

7. A method for freezing recombinant Escherichia coli strains, characterized in that: The following steps are involved: B1: preparing a recombinant Escherichia coli strain freezing solution according to any one of claims 1 to 5; B2: Take 100 ng of target plasmid DNA and add it to 50 µL of E. coli BL21 competent cells. Mix gently and place in ice bath for 20 min. Place in 42°C water bath for 45 s. Quickly transfer to ice bath for 2 min. Add 800 µL of LB liquid medium. Culture at 37°C for 45 min. Inoculate on LB solid plate containing Amp. Place the plate upside down in a 37°C constant temperature incubator and culture for 16 h. B3: Use LB medium with an antibiotic concentration of 100 μg / mL to culture the recombinant E. coli strain, pick a single clone colony and place it in LB medium, culture it at 37°C, 220rpm for 6-8h, use Gram staining to perform microscopic examination, and stop the culture immediately if contamination occurs; use absorbance to detect the activity of the strain to reach the required OD 600 When the value is between 0.8 and 2.0, the culture is stopped; B4: Take the recombinant Escherichia coli culture freezing solution of step B1 and the culture solution of step B2 in a volume ratio of 1:1 and add them to the cryopreservation tube, cover the cryopreservation tube bottle cap tightly, turn it upside down to mix it thoroughly; then quickly package the obtained culture, label it, and store it at -20℃~-196℃ for long-term storage.

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