CN115786214B - High-density fermentation culture medium and high-density fermentation culture method for competent escherichia coli - Google Patents
High-density fermentation culture medium and high-density fermentation culture method for competent escherichia coli Download PDFInfo
- Publication number
- CN115786214B CN115786214B CN202211674083.3A CN202211674083A CN115786214B CN 115786214 B CN115786214 B CN 115786214B CN 202211674083 A CN202211674083 A CN 202211674083A CN 115786214 B CN115786214 B CN 115786214B
- Authority
- CN
- China
- Prior art keywords
- fermentation
- medium
- density
- seed
- culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a competent escherichia coli high-density fermentation culture product which comprises a slant culture medium, a seed culture medium and a fermentation culture medium. The seed culture medium is rich in carbon source, nitrogen source, trace elements and amino acids, and is used for further germination, growth and mass propagation of strains, and trace elements ensure that competent cell membranes have more stable osmotic pressure, so that the competent cell membranes become active fermentation seeds. The fermentation medium is rich in rich carbon source, nitrogen source and various types of amino acids, and the rich amino acids enable the fermentation seeds to be quickly propagated to the optimal competence for preparing the growing period-logarithmic metaphase, so that the high activity of the fermentation seeds is maintained, the fermentation scale can be enlarged, and the high yield and large-scale production of competent cells are realized.
Description
Technical Field
The invention relates to the technical field of high-density fermentation, in particular to a high-density fermentation medium and a high-density fermentation culture method of competent escherichia coli.
Background
Competent cells refer to physiological state cells that possess an optimal uptake and accommodation of foreign DNA fragments and achieve gene transfer by recipient (host) cells. Coli as a chassis cell for constructing common plasmids, special plasmids, viral vectors and expression of various libraries and heterologous proteins, the competent cells of the E.coli plays an irreplaceable role in genetic engineering, protein engineering and enzyme engineering. The growth state of competent cells is the most direct and critical factor affecting exogenous gene transfer, i.e., the state of competent cells determines the transformation efficiency. The traditional preparation method of competent cells of escherichia coli is a chemical method-CaCl 2 Methods or Inoue methods, which facilitate the transformation of foreign genes by increasing the permeability of the cell membrane surface, creating some cavities that facilitate the entry of foreign genes or vectors into the cell.
Under the condition of artificial fermentation, the growth of the escherichia coli cells follows an S-shaped growth curve, and the growth process is divided into four stages of an adjustment stage, a logarithmic phase, a flat slow phase and a decay phase, wherein the logarithmic phase can be subdivided into a mid-log stage and a mid-log stage. Is generally in mid-log phase (OD 600nm =0.3 to 0.6) are most viable and most suitable for preparing high quality competence, and related reports also indicate that certain gene-deficient escherichia coli strains have an optimal competence preparation growth phase in the late log phase (OD 600nm =0.6 to 0.9). The cell density of the conventional competent cell fermentation method should not exceed 1×10 8 cell/mL (OD) 600nm ＝0.3～0.6)。
Due to limitations of cell density and culture temperature, fermentation of mainstream competent cells is currently mainly low-density fermentation, and related patent applications mainly focus on preparation methods with high transformation efficiency competence, such as CaCl described in CN114657114A 2 And a method for preparing a nonionic silicone surfactant with high conversion efficiency. Although there are a large number of patent applications for methods of high-density fermentation of genetically engineered escherichia coli, these methods are not suitable for fermentation of original escherichia coli strains for preparing competent cells, such as a high-density fermentation medium of genetically engineered escherichia coli and a fermentation process thereof described in CN114045251B, and the patent emphasizes that the method is suitable for high-density fermentation of most genetically engineered escherichia coli, but the invention is not suitable for the conditions required for preparing competent cells in specific situations for realizing high yield of recombinant proteins.
The traditional competent cell fermentation mode is mainly based on low-density and low-temperature fermentation, and the main defects of the method are as follows: (1) Low density fermentation mode (OD 600nm =0.3 to 0.6), the yield of the thalli is low, the production cost is high, and the mass production is not facilitated. (2) The reported Escherichia coli high-density fermentation method is only suitable for recombinant protein production, but not suitable for high-density fermentation of competent cells. (3) Animal-derived nitrogen sources limit the possibilities of use of E.coli competent cell products in the field of biopharmaceuticals. At present, it is generally considered that the cell density (OD 600nm >20 A) will beThe dispersion difficulty of thalli is increased in the preparation process of competent cells, the cell lysis and the decay are accelerated, and finally the transformation efficiency of the competent cells is obviously reduced.
At present, a high-density fermentation culture medium and a high-density fermentation culture method are not used for large-scale production of competent cells of escherichia coli.
Disclosure of Invention
Based on the above, in order to solve the problems faced by the traditional escherichia coli competent cell fermentation method, the invention provides the escherichia coli high-density fermentation medium which is suitable for large-scale production of escherichia coli competent cells.
In a first aspect of the invention, there is provided a competent E.coli high density fermentation culture comprising a seed medium and/or a fermentation medium.
The seed culture medium comprises 10.0 g/L-15.0 g/L glucose, 4.0 g/L-6.0 g/L yeast powder, 3.0 g/L-5.0 g/L yeast peptone, 1.0 g/L-3.0 g/L citric acid, 0.02 mg/L-0.05 mg/L cobalt chloride, 0.1 g/L-0.3 g/L calcium chloride, 1.0 g/L-0.3 g/L magnesium sulfate, 7.5 mg/L-15.0 mg/L ferric sulfate, 7.5 mg/L-15.0 mg/L manganese sulfate, 0.02 mg/L-0.05 mg/L biotin, 0.5 mg/L-1.5 mg/L VB1, 0.5 mg/L-1.5 g/L-L L-methionine, 0.2 g/L-0.7 g-LL-lysine, 2 g-0.7 g-8 g/L and 0.35 g-84-0.0.84 g-1 g threonine;
the fermentation culture medium comprises 5.0g/L to 7.5g/L glucose, 4.0g/L to 6.0g/L yeast powder, 3.0g/L to 5.0g/L yeast peptone, 0.5g/L to 1.0g/L citric acid, 0.02mg/L to 0.05mg/L cobalt chloride, 0.5g/L to 1.0g/L potassium dihydrogen phosphate, 0.1g/L to 0.3g/L calcium chloride, 0.1g/L to 0.3g/L magnesium sulfate, 7.5mg/L to 15.0mg/L ferric sulfate, 7.5mg/L to 15.0mg/L manganese sulfate, 0.02mg/L to 0.05mg/L biotin VB1 at 0.75mg/L to 1.5mg/L, VH at 0.75mg/L to 1.5mg/L, methionine at 0.1g/L to 0.5g/L to L L-, lysine at 0.2g/L to 0.7g/L L L-, arginine at 0.2g/L to 0.7g/L L-, threonine at 0.1g/L to 0.2g/L L-, valine at 0.3g/L to 0.7g/L L-, isoleucine at 0.5g/L to 1.5g/L L-, glutamic acid at 0.05g/L to 0.2g/L, alanine at 0.1g/L to 0.4g/L L-and serine at 0.6g/L to 0.9g/L L-serine.
In one embodiment, the culture medium further comprises a slant culture medium, wherein the slant culture medium comprises 1.0 g/L-5.0 g/L glucose, 5.0 g/L-10.0 g/L yeast powder, 15.0 g/L-20.0 g/L yeast peptone, 5.0 g/L-7.5 g/L NaCl, 1.0 g/L-3.0 g/L monopotassium phosphate, 0.3 g/L-0.5 g/L magnesium sulfate and 15 g/L-20 g/L agar powder.
In one embodiment, the medium is a kit comprising the seed medium, fermentation medium, and slant medium.
In a second aspect of the present invention, there is provided a high-density fermentation method of competent E.coli, comprising the step of subjecting competent E.coli to high-density fermentation using the culture product.
In one embodiment, the step of high density fermentation comprises:
activating competent escherichia coli strains by using the slant culture medium to prepare an activated seed liquid;
inoculating the activated seed liquid to the seed culture medium, and carrying out seed fermentation treatment to prepare primary fermentation liquid;
inoculating the primary fermentation broth to the fermentation medium for fermentation treatment to prepare the escherichia coli high-density fermentation broth.
In one embodiment, the parameters of the seed fermentation process include one or more of the following:
(1) The temperature is 35-37 ℃;
(2) The time is 4.0 to 6.5 hours;
(3) The pressure is 0.03MPa to 0.05MPa;
(4) The pH value is 6.9-7.1;
(5) The dissolved oxygen amount is 30% -75%;
(6) The rotating speed is 250 rpm-510 rpm; and, a step of, in the first embodiment,
(7) The wind speed is 6.5L/min-10.0L/min.
In one embodiment, the parameters of the fermentation process include one or more of the following:
(1) The temperature is 35-37 ℃;
(2) The time is 0.5 h-2.0 h;
(3) The pressure is 0.03MPa to 0.0.5 MPa;
(4) The pH value is 7.0-7.1;
(5) The dissolved oxygen amount is 30% -70%;
(6) The rotating speed is 300 rpm-490 rpm; and, a step of, in the first embodiment,
(7) The wind speed is 15.0L/min-30.0L/min.
In one embodiment, the OD of the primary fermentation broth 600nm 10.0 to 15.0.
In one embodiment, the OD of the E.coli high-density fermentation broth 600nm 4.0 to 6.0.
In one embodiment, the number of times the competent E.coli strain is activated is two; the parameters of the two activations each independently included: the temperature is 35-37 ℃ and the culture time is 14-16 h.
Compared with the traditional technology, the competent escherichia coli high-density fermentation culture product provided by the invention comprises a slant culture medium, a seed culture medium and a fermentation culture medium. The beneficial effects are as follows:
(1) The seed culture medium is rich in carbon source, nitrogen source, trace elements and amino acids, and is used for further germination, growth and mass propagation of strains, and trace elements ensure that competent cell membranes have more stable osmotic pressure, so that the competent cell membranes become active fermentation seeds.
(2) The fermentation medium is rich in rich carbon source, nitrogen source and various amino acids, and the rich amino acids can make the fermentation seed propagate fast to optimal competence to prepare the early growth phase-logarithmic phase, so that the high activity of the fermentation seed is maintained, and the fermentation medium can be used for enlarging the fermentation scale.
(3) The nitrogen source in the culture medium is free of animal source components, so that the competent product prepared at the later stage can be used in the field of biopharmaceuticals.
(4) The seed fermentation medium and the fermentation medium are used for combined culture, the feed supplement is not needed, the fermentation period is obviously shortened (8.5 h), the time and the labor are saved, and the production habit is more met.
(5) Compared with the traditional escherichia coli low-density fermentation medium, the cost of the medium is reduced, and single fermentation bacteria are adoptedThe bulk density is improved by 10 to 15 times (OD) 600nm From 0.3 to 0.6 to 4.0 to 6.0), the productivity is improved by 10 to 15 times, the competent conversion efficiency is improved by 3 to 5 times, and the method can be used for large-scale and standardized production.
Drawings
In order to more clearly illustrate the technical solution in the embodiments of the present invention and to more fully understand the present invention and its advantageous effects, the following brief description will be given with reference to the accompanying drawings, which are required to be used in the description of the embodiments. It is evident that the figures in the following description are only some embodiments of the invention, from which other figures can be obtained without inventive effort for a person skilled in the art.
FIG. 1 is a plot of Dh5α competent cell transformation titers for high density fermentation in example 1;
FIG. 2 is a chart of BL21 competent cell transformation titers for high density fermentation in example 2.
Detailed Description
In order that the invention may be readily understood, a more particular description of the invention will be rendered by reference to specific embodiments that are illustrated in the appended drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
Terminology
In the present invention, a selection range in reference to "and/or", "and/or" includes any one of two or more of the items listed in relation to each other, as well as any and all combinations of the items listed in relation to each other, including any two of the items listed in relation to each other, any more of the items listed in relation to each other, or all combinations of the items listed in relation to each other. It should be noted that when at least three items are connected by a combination of at least two conjunctions selected from the group consisting of "and/or", "and/or", it is to be understood that the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly also includes technical solutions that are all connected by "logical or".
In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.
In the present invention, a numerical range (i.e., a numerical range) is referred to, and optional numerical distributions are considered to be continuous within the numerical range and include two numerical endpoints (i.e., a minimum value and a maximum value) of the numerical range and each numerical value between the two numerical endpoints unless otherwise specified. Where a numerical range merely refers to integers within the numerical range, including both end integers of the numerical range, and each integer between the two ends, unless otherwise indicated, each integer is recited herein as directly, such as where t is an integer selected from 1 to 10, and where t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Further, when a plurality of range description features or characteristics are provided, these ranges may be combined. In other words, unless otherwise indicated, the ranges disclosed herein are to be understood to include any and all subranges subsumed therein.
The temperature parameter in the present invention is not particularly limited, and may be a constant temperature treatment or may vary within a predetermined temperature range. It should be appreciated that the constant temperature process described allows the temperature to fluctuate within the accuracy of the instrument control. Allows for fluctuations within a range such as + -5 ℃, + -4 ℃, + -3 ℃, + -2 ℃, + -1 ℃.
In a first aspect of the invention, there is provided a competent E.coli high density fermentation culture comprising a seed medium and/or a fermentation medium.
The seed culture medium comprises 10.0 g/L-15.0 g/L glucose, 4.0 g/L-6.0 g/L yeast powder, 3.0 g/L-5.0 g/L yeast peptone, 1.0 g/L-3.0 g/L citric acid, 0.02 mg/L-0.05 mg/L cobalt chloride, 0.1 g/L-0.3 g/L calcium chloride, 1.0 g/L-0.3 g/L magnesium sulfate, 7.5 mg/L-15.0 mg/L ferric sulfate, 7.5 mg/L-15.0 mg/L manganese sulfate, 0.02 mg/L-0.05 mg/L biotin, 0.5 mg/L-1.5 mg/L VB1, 0.5 mg/L-1.5 g/L-L L-methionine, 0.2 g/L-0.7 g-LL-lysine, 2 g-0.7 g-8 g/L and 0.35 g-84-0.0.84 g-1 g threonine;
optionally, the seed culture medium further comprises 0.01-0.1 mL/L of bufomide.
The fermentation culture medium comprises 5.0g/L to 7.5g/L glucose, 4.0g/L to 6.0g/L yeast powder, 3.0g/L to 5.0g/L yeast peptone, 0.5g/L to 1.0g/L citric acid, 0.02mg/L to 0.05mg/L cobalt chloride, 0.5g/L to 1.0g/L potassium dihydrogen phosphate, 0.1g/L to 0.3g/L calcium chloride, 0.1g/L to 0.3g/L magnesium sulfate, 7.5mg/L to 15.0mg/L ferric sulfate, 7.5mg/L to 15.0mg/L manganese sulfate, 0.02mg/L to 0.05mg/L biotin VB1 at 0.75mg/L to 1.5mg/L, VH at 0.75mg/L to 1.5mg/L, methionine at 0.1g/L to 0.5g/L to L L-, lysine at 0.2g/L to 0.7g/L L L-, arginine at 0.2g/L to 0.7g/L L-, threonine at 0.1g/L to 0.2g/L L-, valine at 0.3g/L to 0.7g/L L-, isoleucine at 0.5g/L to 1.5g/L L-, glutamic acid at 0.05g/L to 0.2g/L, alanine at 0.1g/L to 0.4g/L L-and serine at 0.6g/L to 0.9g/L L-serine.
Optionally, the fermentation medium further comprises 0.01-0.1 mL/L of bufomide.
Optionally, the culture medium further comprises a slant culture medium, wherein the slant culture medium comprises 1.0 g/L-5.0 g/L glucose, 5.0 g/L-10.0 g/L yeast powder, 15.0 g/L-20.0 g/L yeast peptone, 5.0 g/L-7.5 g/L NaCl, 1.0 g/L-3.0 g/L monopotassium phosphate, 0.3 g/L-0.5 g/L magnesium sulfate and 15 g/L-20 g/L agar powder.
Optionally, the culture medium is a kit product comprising the seed culture medium, the fermentation medium and the slant culture medium.
In a second aspect of the present invention, there is provided a high-density fermentation method of competent E.coli, comprising the step of subjecting competent E.coli to high-density fermentation using the culture product.
Optionally, the step of high density fermentation comprises:
activating competent escherichia coli strains by using the slant culture medium to prepare an activated seed liquid;
inoculating the activated seed liquid to the seed culture medium, and carrying out seed fermentation treatment to prepare primary fermentation liquid;
inoculating the primary fermentation broth to the fermentation medium for fermentation treatment to prepare the escherichia coli high-density fermentation broth.
Optionally, the parameters of the seed fermentation treatment include one or more of the following:
(1) The temperature is 35-37 ℃;
(2) The time is 4.0 to 6.5 hours;
(3) The pressure is 0.03MPa to 0.05MPa;
(4) The pH value is 6.9-7.1;
(5) The dissolved oxygen amount is 30% -75%;
(6) The rotating speed is 250 rpm-510 rpm; and, a step of, in the first embodiment,
(7) The wind speed is 6.5L/min-10.0L/min.
Optionally, the parameters of the fermentation process include one or more of the following:
(1) The temperature is 35-37 ℃;
(2) The time is 0.5 h-2.0 h;
(3) The pressure is 0.03MPa to 0.0.5 MPa;
(4) The pH value is 7.0-7.1;
(5) The dissolved oxygen amount is 30% -70%;
(6) The rotating speed is 300 rpm-490 rpm; and, a step of, in the first embodiment,
(7) The wind speed is 15.0L/min-30.0L/min.
Optionally, the OD of the primary fermentation broth 600nm 10.0 to 15.0.
Optionally, the OD of the escherichia coli high-density fermentation broth 600nm 4.0 to 6.0.
Optionally, the number of times the E.coli species is activated and sensed is two; the parameters of the two activations each independently included: the temperature is 35-37 ℃ and the culture time is 14-16 h.
The following examples are further offered to illustrate, but not to limit, the materials used in the examples are commercially available, and the equipment used is commercially available, and the processes involved are routinely selected by those skilled in the art without any specific description.
The following are specific examples.
Example 1
Example 1 provides a high density fermentation mode of E.coli Dh5α competent strains.
1 Medium formulation
The nitrogen sources of the low-density fermentation culture medium for culturing competent cells of escherichia coli comprise animal peptone-tryptone, the nutrient components are single, the safety risk of potential exogenous factor pollution exists, and the application of the competent cells in the biopharmaceutical fields of vaccines, antibodies, interferon and the like is limited. The yeast peptone has no transgenic dispute, no pathogenicity, no allergen, no common taboo and other problems, can be applied to the field of biological pharmacy, such as vaccine, antibody, interferon production and the like, is rich in protein, peptide, amino acid, nucleotide, B vitamins, biotin and the like, and can provide comprehensive and balanced nutrition for thalli. Can promote the growth and metabolism of microorganism and increase the production efficiency when being used together with yeast extract and other culture medium components.
(1) Slant culture medium
Glucose 2.0g/L, yeast powder 5.0g/L, yeast peptone 15.0g/L, naCl 5.0.0 g/L, potassium dihydrogen phosphate 2.0g/L, magnesium sulfate 0.5g/L, and agar powder 20g/L.
(2) Seed culture medium
10.0g/L of glucose, 5.0g/L of yeast powder, 5.0g/L of yeast peptone, 2.0g/L of citric acid, 2.0g/L of monopotassium phosphate, 0.3g/L of calcium chloride, 0.2g/L of magnesium sulfate, 10mg/L of ferric sulfate, 10mg/L of manganese sulfate, 0.03mg/L of cobalt chloride, 0.03mg/L, VB 1.0mg/L, VH 1.0.0 mg/L, L-methionine 0.3g/L, L-lysine 0.5g/L, L-threonine 0.5g/L, L-glutamic acid 0.7g/L and 0.05mL/L of dichlord.
(3) Fermentation medium
Glucose 5.0g/L, yeast powder 5.0g/L, yeast peptone 5.0g/L, citric acid 1.0g/L, potassium dihydrogen phosphate 1.0g/L, calcium chloride 0.3g/L, magnesium sulfate 0.3g/L, ferric sulfate 10mg/L, manganese sulfate 10mg/L, cobalt chloride 0.03mg/L, biotin 0.03mg/L, VB 1.0mg/L, VH 1.0.0 mg/L, L-methionine 0.2g/L, L-lysine 0.5g/L, L-arginine 0.5g/L, L-valine 0.1g/L, L-threonine 0.4g/L, L-glutamic acid 0.5g/L, L-isoleucine 0.1g/L, L-alanine 0.3g/L, L-serine 0.7g/L, and dichlord 0.1mL/L.
2 preparation of culture medium
(1) Slant culture medium: adding the components according to the formula of the slant culture medium, and sterilizing for 30min at 115 ℃; pouring the sterilized culture medium into a test tube or an eggplant-shaped bottle, and obliquely placing to form a slant culture medium.
(2) Seed culture medium: preparing 30% (w/v) glucose mother solution, and sterilizing at 115 ℃ for 35min; according to the proportion of the culture medium, 10L of ultrapure water is used for dissolving each component, the mixture is fully stirred, and after the complete dissolution, the mixture is transferred into a 20L fermentation tank to reach 15L, and the mixture is sterilized for 30min at 121 ℃; before inoculating into the slant thallus, adding glucose with final concentration of 10.0g/L into the seed fermenter by aseptic operation.
(3) Fermentation medium: according to the proportion of the culture medium, dissolving each component by using 50L of ultrapure water, uniformly stirring, transferring into a fermentation tank after complete dissolution, sterilizing for 30min at 121 ℃ after the volume is fixed to 75L; glucose with a final concentration of 5.0g/L was added to the fermenter under aseptic conditions prior to the inoculation of the seed solution.
3 activation of the seed
Resuscitation and activation: under the aseptic condition, DH5 alpha competent cell strain preserved at-80 ℃ is inoculated in a test tube slant culture medium in a streaking mode, and is cultured for 14h at the constant temperature of 37 ℃.
Second generation activation: a10.0 mu L inoculating loop is used for taking a loop of thalli to be uniformly coated on the slant culture medium of the eggplant-shaped bottle, and the culture is carried out for 12 hours at the constant temperature of 37 ℃.
The original strain of the escherichia coli is subjected to activation culture by a two-generation solid culture medium, so that the activity of the escherichia coli is recovered to an optimal state, the overall activity of the original strain population is ensured to be always at an optimal level, and the problem of unstable activity of the strain is solved.
4 seed fermentation
The tank capacity of the seed culture tank is 20L, the actual liquid loading amount is 15L, 100 sterile water is taken and added into 1 activated eggplant-shaped inclined plane, the whole thallus is scraped up by an inoculating loop, and the bacterial suspension is inoculated into the seed fermentation tank;
the fermentation conditions were as follows: culture temperature: 35 ℃; tank pressure: 0.03-0.05 MPa; pH value: the pH value of the fermentation tank is regulated by 25% ammonia water in the whole process, so that the pH value is maintained between 6.9 and 7.1; dissolved oxygen control: maintaining the dissolved oxygen amount between 30% and 80%, and when the dissolved oxygen amount is reduced to be below 30% (2-3 h of culture), increasing the rotating speed, and increasing the rotating speed by 20-50 rpm each time; a rotational speed; the starting rotating speed is 300rpm, and the rotating speed ranges from 300rpm to 550rpm; wind speed: starting wind speed is 7.0L/min, and the wind speed range is 7.0L/min-15.0L/min; the culture time is 5.1h, and the final cell density OD 600 nm The fermentation was stopped at 11.5 to obtain a primary fermentation broth.
5 fermentation culture
The tank capacity of the fermentation culture tank is 100L, the actual liquid loading amount is 75L, and the cultured seed liquid is pumped into the fermentation culture tank in an inoculation amount of 10 percent.
The fermentation conditions were as follows: the culture temperature is 35 ℃; the tank pressure is 0.03-0.05 MPa; the pH value of the fermentation tank is regulated by 25% ammonia water in the whole process, so that the pH value is maintained between 7.0 and 7.1; maintaining the dissolved oxygen amount between 30% and 70%, and when the dissolved oxygen is reduced to below 30% (2-3 h of culture), increasing the rotating speed by 30-50 rpm each time, starting the rotating speed by 330rpm, wherein the rotating speed range is 330-550 rpm; starting wind speed is 20.0L/min, and the wind speed range is 20.0L/min-30.0L/min; the culture time is 1.1h, and the cell density OD 600nm The fermentation was stopped until 5.7 was reached.
6 lower tank control
Temperature is one of the most critical factors affecting the growth of E.coli, and how to maintain E.coli cell state and cell density substantially unchanged during the tank discharging process is critical to maintaining high viability and quality of competent cells. In the scheme, the cooling effect of the high-efficiency refrigerator and the dissolved oxygen control of the fermentation tank are combined, and the tank is realized within 10-20 minThe temperature of the bacterial cells is reduced to 4-8 ℃, and the cell density OD of the bacterial cells is reduced 600nm The fluctuation is less than 0.5, and the specific operation process is as follows:
cooling the temperature of circulating water in a circulating water tank to 3 ℃ in advance, cooling thalli in the tank by circulating cooling water for 15min, and stabilizing the temperature in the tank to 7 ℃; the dissolved oxygen in the tank is 53% when cooling. The cooling causes the cells to grow slowly, so that the dissolved oxygen in the tank increases with time, and the dissolved oxygen in the tank can be maintained by decreasing the rotation speed. The rotating speed is reduced by 50rpm for each wheel, and the rotating speed range is controlled between 150rpm and 300rpm; the pH was adjusted with 25% ammonia and maintained at 7.0.
Example 2
Example 2 provides a fermentation protocol for E.coli expression competent BL21 (DE 3) strain
1 Medium formulation
(1) Slant culture medium
Glucose 3.0g/L, yeast powder 7.5g/L, yeast peptone 20.0g/L, naCl 5.0.0 g/L, potassium dihydrogen phosphate 2.0g/L, magnesium sulfate 0.5g/L, agar powder 20g/L
(2) Seed culture medium
15.0g/L glucose, 5.0g/L yeast powder, 5.0g/L yeast peptone, 2.0g/L citric acid, 2.0g/L potassium dihydrogen phosphate, 0.2g/L calcium chloride, 0.2g/L magnesium sulfate, 10mg/L ferric sulfate, 10mg/L manganese sulfate, 0.03mg/L cobalt chloride, 0.03mg/L, VB 1.0mg/L, VH 1.0.0 mg/L, L-methionine 0.4g/L, L-lysine 0.5g/L, L-threonine 0.5g/L, L-glutamic acid 0.7g/L.
(3) Fermentation medium
7.5g/L glucose, 5.0g/L yeast powder, 5.0g/L yeast peptone, 1.0g/L citric acid, 1.0g/L potassium dihydrogen phosphate, 0.3g/L calcium chloride, 0.3g/L magnesium sulfate, 10mg/L ferric sulfate, 10mg/L manganese sulfate, 0.03mg/L cobalt chloride, 0.03mg/L, VB 1.0mg/L, VH 1.0.0 mg/L, L-methionine 0.2g/L, L-lysine 0.5g/L, L-arginine 0.5g/L, L-valine 0.1g/L, L-threonine 0.4g/L, L-glutamic acid 0.5g/L, L-isoleucine 0.1g/L, L-alanine 0.3g/L, L-serine 0.7g/L, and enemy 0.1mL/L
2 preparation of culture medium
(1) Slant culture medium: the components are added according to the culture medium formula and sterilized for 30min at 115 ℃. Pouring the sterilized culture medium into a test tube or an eggplant-shaped bottle, and obliquely placing to form a slant culture medium.
(2) Seed culture medium: 30% (w/v) glucose mother liquor was prepared and sterilized at 115℃for 35min. According to the proportion of the culture medium, 7.5L of ultrapure water is used for dissolving each component, the mixture is fully stirred, and the mixture is transferred into a 20L fermentation tank to be subjected to constant volume to 10L, and is sterilized for 30min at 121 ℃. Before inoculating into the slant thallus, adding glucose with final concentration of 15.0g/L into the seed fermenter by aseptic operation.
(3) Fermentation medium: according to the proportion of the culture medium, 45L of ultrapure water is used for dissolving each component, the mixture is stirred uniformly, and the mixture is transferred into a fermentation tank to fix the volume to 65L after being completely dissolved, and the fermentation tank is sterilized for 30min at 121 ℃. Before inoculating the seed liquid, adding glucose with final concentration of 7.5g/L into a fermentation tank under aseptic condition.
3 activation of the seed
Resuscitation and activation: under aseptic condition, BL21 (DE 3) competent cell strain preserved at-80 ℃ is inoculated in a test tube slant culture medium in a streaking mode, and the culture is carried out for 16 hours at a constant temperature of 37 ℃.
Second generation activation: a10.0 mu L inoculating loop is used for taking a loop of thalli to be uniformly coated on the slant culture medium of the eggplant-shaped bottle, and the culture is carried out for 14 hours at the constant temperature of 37 ℃.
The original strain of the escherichia coli is subjected to activation culture by a two-generation solid culture medium, so that the activity of the escherichia coli is recovered to an optimal state, the overall activity of the original strain population is ensured to be always at an optimal level, and the problem of unstable activity of the strain is solved.
4 seed fermentation culture
The tank capacity of the seed culture tank is 20L, the actual liquid loading amount is 10L, 100 sterile water is taken and added into 1 activated eggplant-shaped inclined plane, the whole thallus is scraped up by an inoculating loop, and the bacterial suspension is inoculated into the seed fermentation tank;
the fermentation conditions were as follows: the culture temperature is 37 ℃; the tank pressure is 0.03MPa to 0.05MPa; the pH value of the fermentation tank is regulated by 25% ammonia water in the whole process, so that the pH value is maintained between 6.9 and 7.1; maintaining the dissolved oxygen amount between 30% and 75%, and when the dissolved oxygen amount is reduced to be below 30% (2-3 h of culture), increasing the rotating speed, and increasing the rotating speed by 20-50 rpm each time; starting rotation speed is 250rpm, and the rotation speed range is 250 rpm-510 rpm; starting wind speed is 6.5L/min, and the wind speed range is 6.5L/min-10.0L/min; culturing for 5.9h, wherein the final thallus density is 12.8, and stopping fermentation to obtain primary fermentation liquor;
5 fermentation culture
The tank capacity of the fermentation culture tank is 100L, the actual liquid loading amount is 65L, and the cultured seed liquid is pumped into the fermentation culture tank in an inoculation amount of 12 percent;
the fermentation conditions were as follows: the culture temperature is 37 ℃; the tank pressure is 0.03MPa to 0.05MPa; the pH value of the fermentation tank is regulated by 25% ammonia water in the whole process, so that the pH value is maintained between 7.0 and 7.1; maintaining the dissolved oxygen amount between 30% and 70%, and when the dissolved oxygen is reduced to below 30% (2-3 h of culture), increasing the rotating speed by 35rpm each time; starting rotation speed is 300rpm, and the rotation speed range is 300 rpm-490 rpm; starting wind speed is 15.0L/min, and the wind speed range is 15.0L/min-30.0L/min; the culture time is 1.3h, and the cell density OD 600 nm Stopping fermentation until the fermentation time reaches 5.9;
6 lower tank control
Temperature is one of the most critical factors affecting the growth of E.coli, and how to maintain E.coli cell state and cell density substantially unchanged during the tank discharging process is critical to maintaining high viability and quality of competent cells. In the scheme, the temperature reduction effect of the high-efficiency refrigerator and the dissolved oxygen control of the fermentation tank are combined, and the temperature of bacteria in the tank is reduced to 4-8 ℃ within 10-20 min, so that the cell density OD of the bacteria is realized 600nm The fluctuation is less than 0.5, and the specific operation process is as follows:
cooling the temperature of circulating water in a circulating water tank to 3 ℃ in advance, cooling thalli in the tank by circulating cooling water for 16min, and stabilizing the temperature in the tank to 8 ℃; when cooling, the dissolved oxygen in the tank is 53%, the rotating speed is reduced by 50rpm for each wheel, and the rotating speed is controlled to be 150-300 rpm; the pH is regulated by using 25% ammonia water, and the pH is maintained between 6.9 and 7.1.
The high cell density and high seed activity are ensured when the seeds are cultured and put in a pot, so that the escherichia coli in the mid-log growth stage, namely OD, is selected 600nm 10.0 to 15.0. When the fermentation culture is carried out in the lower tank, the cell activity needs to be ensured to the greatest extentHigh, but not too high, so that the log front, i.e. OD, is chosen 600nm 4.0 to 6.0.
When the traditional low-density culture is used for preparing competent cells of the escherichia coli, the culture temperature is low (16-25 ℃), the fermentation time is long (16-20 hours), the strain density needs to be monitored for a long time, the time and the labor are wasted, and the standardized production is not facilitated. The competent cells are cultured at high temperature (35-37 ℃) in the whole process, and are matched with low temperature control and dissolved oxygen control in a thallus collecting stage, so that the temperature of the thallus is quickly reduced to 4-10 ℃ by adopting high-efficiency refrigeration, the competent cells are ensured to be always in a stable and high-activity state, the continuous growth, even massive cracking and decay of the thallus caused by the high-density condition of the thallus are effectively avoided, the expanded culture of the competent cells of the escherichia coli is improved, the yield and the conversion rate of the competent cells of the escherichia coli are improved, and the cost is reduced.
Comparative example 1
Comparative example 1 provides a high-density fermentation medium of E.coli Dh5α strain. This comparative example 1 is a comparative example of example 1, and the compositions and arrangement of the slant medium and the fermentation medium of comparative example 1 are the same as those of example 1. Differences with respect to example 1 include: the yeast peptone is replaced by tryptone in the seed culture medium, and the seed fermentation culture is carried out for the same time.
Comparative example 2
Comparative example 2 provides a high density fermentation medium of E.coli Dh5α strain. This comparative example 2 is a comparative example of example 1, and the compositions and arrangement of the slant medium and the fermentation medium of comparative example 2 are the same as those of example 1. Differences with respect to example 1 include: replacing yeast peptone with tryptone in seed culture medium, fermenting and culturing the seeds until the density of the cells is the same as OD 600nm ＝11.5。
Comparative example 3
Comparative example 3 provides a high density fermentation medium of E.coli Dh5α strain. This comparative example 3 is a comparative example of example 1, and the compositions and arrangement of the slant medium and the fermentation medium of comparative example 3 are the same as those of example 1. Differences with respect to example 1 include: amino acid components are not added into the seed culture medium, and the seed is fermented and cultured for the same time.
Comparative example 4
Comparative example 4 provides a high density fermentation medium of E.coli Dh5α strain. This comparative example 4 is a comparative example of example 1, and the compositions and arrangement of the slant medium and the fermentation medium of comparative example 4 are the same as those of example 1. Differences with respect to example 1 include: the seed culture medium is not added with amino acid components, and the seeds are fermented and cultured to the same cell density OD 600nm ＝11.5。
Comparative example 5
Comparative example 5 provides a high-density fermentation medium of the DL21 (DE 3) strain. This comparative example 5 is a comparative example of example 2, and the compositions and arrangement of the slant medium and the seed medium of comparative example 5 are the same as those of example 2. Differences with respect to example 2 include: replacing yeast peptone with tryptone in fermentation medium, fermenting and culturing to the same thallus density OD 600nm ＝5.9。
Comparative example 6
Comparative example 6 provides a high-density fermentation medium of the DL21 (DE 3) strain. This comparative example 5 is a comparative example of example 2, and the compositions and arrangement of the slant medium and the seed medium of comparative example 6 are the same as those of example 2. Differences with respect to example 2 include: amino acid components are not added into the fermentation culture medium, and the fermentation culture is carried out until the density OD of the bacterial cells is the same 600nm ＝5.9。
The culture cycle, cell density, cost and conversion efficiency of Dh5α competent cells prepared in the high density fermentation media of example 1 and comparative examples 1 to 4 are recorded in Table 1.
TABLE 1
As can be seen from the data in Table 1, the total density of the fermented cells obtained from competent Dh5α cells of E.coli according to the present invention was 427.5 (OD 600 nm =5.7x75l), the yield is improved by 1.4-2.0 times compared with the comparative examples 1-4, and the animal source is not animal sourceThe components of the culture medium reduce the total cost of fermentation by 22.6 percent, the whole process is cultivated at 35 ℃ and the cultivation period is shortened by 1.0 to 2.1 hours, and the transformation efficiency of competent cells obtained by the preparation scheme of the invention reaches 5.5 multiplied by 10 8 ～1.3×10 9 The conversion efficiency of the invention is improved by 4.1-10.3 times. The 75L fermentation liquor can prepare over 100 ten thousand Dh5α competence, so that large-scale production can be realized.
The culture cycle, cell density, cost and conversion efficiency of DL21 (DE 3) competent cells prepared by high density fermentation of example 2 and comparative examples 5 to 6 are recorded in table 2.
TABLE 2
As can be seen from Table 2, the present invention has a total cell density of 383.5 (OD) of E.coli expression competent BL21 (DE 3) 600nm Compared with comparative examples 5-6, the yield of the invention is improved by 1.2-1.8 times, the total cost of fermentation is reduced by 21.4% by high-temperature culture in the whole process, the competent conversion efficiency is improved by 3.7-6.7 times, and more than 70 tens of thousands of BL21 (DE 3) competes can be prepared from 65L fermentation broth, and the yield can be further amplified, so that the large-scale production can be realized.
The high-yield competent cell fermentation process provided by the invention consists of a strain activation process, a second-generation fermentation culture process and a tank discharging control process, wherein the second-generation fermentation culture process is subdivided into seed culture and fermentation culture. The seed culture medium is rich in carbon source, nitrogen source and trace elements for further germination, growth and mass propagation of strain, and trace elements ensure that competent cell membranes have more stable osmotic pressure, so that the competent cell membranes become active fermentation seeds. The fermentation medium is rich in rich carbon source, nitrogen source and various amino acids, and the rich amino acids can make the fermentation seeds quickly reproduce to optimal competence to prepare the anagen-mid-log phase, so that the high activity of the fermentation seeds is maintained, and the fermentation medium can be used for enlarging the fermentation scale.
The culture temperature is one of the key factors affecting the growth, propagation and metabolism of escherichia coli, and too high temperature can lead to too fast production of thalli, increase of secondary metabolites, decrease of cell states and finally decrease of conversion efficiency of competent cells. The competent cells in the invention are cultivated at high temperature (35-37 ℃) in the whole process, and are ensured to be always in a stable and high-activity state by being matched with the low-temperature control and dissolved oxygen control in the lower tank process. In the tank discharging stage, an efficient refrigerator is adopted to quickly reduce the temperature of thalli in the tank to 4-10 ℃, so that thalli continuously grow and even crack and decay in a large amount under the condition of high density of thalli are effectively avoided. The high-density fermentation medium and the culture mode are adopted to culture the escherichia coli, so that the high-yield and large-scale production of the competent cells of the escherichia coli are realized, the production cost is reduced, and the conversion rate of the competent cells of the escherichia coli is improved.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. A competent escherichia coli high-density fermentation medium is characterized by comprising a seed medium and a fermentation medium;
the seed culture medium consists of the following components in percentage by weight: 10.0g/L of glucose, 5.0g/L of yeast powder, 5.0g/L of yeast peptone, 2.0g/L of citric acid, 2.0g/L of monopotassium phosphate, 0.3g/L of calcium chloride, 0.2g/L of magnesium sulfate, 10mg/L of ferric sulfate, 10mg/L of manganese sulfate, 0.03mg/L of cobalt chloride, 1.03mg/L, VB of biotin, 1.0mg/L, L-methionine 0.3g/L, L-lysine 0.5g/L, L-threonine 0.5g/L, L-glutamic acid 0.7g/L, bufomide 0.05mL/L and water, or
The seed culture medium consists of the following components in percentage by weight: 15.0g/L glucose, 5.0g/L yeast powder, 5.0g/L yeast peptone, 2.0g/L citric acid, 2.0g/L potassium dihydrogen phosphate, 0.2g/L calcium chloride, 0.2g/L magnesium sulfate, 10mg/L ferric sulfate, 10mg/L manganese sulfate, 0.03mg/L cobalt chloride, 1.03mg/L, VB 1.0mg/L, L-methionine 0.4g/L, L-lysine 0.5g/L, L-threonine 0.5g/L, L-glutamic acid 0.7g/L and water;
the fermentation medium consists of the following components in percentage by weight: 5.0g/L to 7.5g/L glucose, 4.0g/L to 6.0g/L yeast powder, 3.0g/L to 5.0g/L yeast peptone, 0.5g/L to 1.0g/L citric acid, 0.02mg/L to 0.05mg/L cobalt chloride, 0.5g/L to 1.0g/L monopotassium phosphate, 0.1g/L to 0.3g/L calcium chloride, 0.1g/L to 0.3g/L magnesium sulfate, 7.5mg/L to 15.0mg/L ferric sulfate, 7.5mg/L to 15.0mg/L manganese sulfate, 0.77mg/L to 1.55mg/L biotin, 0.75mg/L to 1.5mg/L VB1, 0.1g/L to 0.5g/L L-methionine, 0.2g to 0.7g/L lysine L L g to 96 g, 0.92.7 g to 92 g/L arginine, 0.35 g to 35 g to 26.35 g to 26 g/L alanine, 0.35 g to 35 g to 0.35 g/L serine, 0.35 g to 0.35 g/L alanine.
2. The high-density fermentation medium of claim 1, further comprising a slant medium comprising 1.0g/L to 5.0g/L glucose, 5.0g/L to 10.0g/L yeast powder, 15.0g/L to 20.0g/L yeast peptone, 5.0g/L to 7.5g/L NaCl, 1.0g/L to 3.0g/L potassium dihydrogen phosphate, 0.3g/L to 0.5g/L magnesium sulfate, and 15g/L to 20g/L agar powder.
3. The high-density fermentation medium of claim 2, wherein the high-density fermentation medium is a kit comprising the seed medium, the fermentation medium, and the slant medium.
4. A method of high-density fermentation of competent escherichia coli, characterized in that the method comprises the step of high-density fermentation of competent escherichia coli using the culture product of any one of claims 1 to 3.
5. The high-density culture method according to claim 4, wherein the step of high-density fermentation comprises:
activating competent escherichia coli strains by using the slant culture medium to prepare an activated seed liquid;
inoculating the activated seed liquid to the seed culture medium, and carrying out seed fermentation treatment to prepare primary fermentation liquid;
inoculating the primary fermentation broth to the fermentation medium for fermentation treatment to prepare the escherichia coli high-density fermentation broth.
6. The high density culture method of claim 5, wherein the parameters of the seed fermentation treatment include one or more of the following:
(1) The temperature is 35-37 ℃;
(2) The time is 4.0 to 6.5 hours;
(3) The pressure is 0.03MPa to 0.05MPa;
(4) The pH value is 6.9-7.1;
(5) The dissolved oxygen amount is 30% -75%;
(6) The rotating speed is 250 rpm-510 rpm; and, a step of, in the first embodiment,
(7) The wind speed is 6.5L/min-10.0L/min.
7. The high-density culture method according to claim 5, wherein the parameters of inoculating the primary fermentation broth to the fermentation medium for fermentation treatment include one or more of the following:
(1) The temperature is 35-37 ℃;
(2) The time is 0.5 h-2.0 h;
(3) The pressure is 0.03MPa to 0.05MPa;
(4) The pH value is 7.0-7.1;
(5) The dissolved oxygen amount is 30% -70%;
(6) The rotating speed is 300 rpm-490 rpm; and, a step of, in the first embodiment,
(7) The wind speed is 15.0L/min-30.0L/min.
8. The high-density culture method according to claim 5, wherein the primary fermentation liquid has an OD 600nm 10.0 to 15.0.
9. The method according to claim 5, wherein the E.coli high-density fermentation broth has an OD 600nm 4.0 to 6.0.
10. The high-density fermentation method according to claim 5, wherein the number of times of activation of the competent E.coli strain is twice; the parameters of the two activations each independently included: the temperature is 35-37 ℃ and the culture time is 14-16 h.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211674083.3A CN115786214B (en) | 2022-12-26 | 2022-12-26 | High-density fermentation culture medium and high-density fermentation culture method for competent escherichia coli |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211674083.3A CN115786214B (en) | 2022-12-26 | 2022-12-26 | High-density fermentation culture medium and high-density fermentation culture method for competent escherichia coli |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115786214A CN115786214A (en) | 2023-03-14 |
| CN115786214B true CN115786214B (en) | 2023-08-18 |
Family
ID=85426729
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211674083.3A Active CN115786214B (en) | 2022-12-26 | 2022-12-26 | High-density fermentation culture medium and high-density fermentation culture method for competent escherichia coli |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115786214B (en) |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1991010721A2 (en) * | 1990-01-19 | 1991-07-25 | GESELLSCHAFT FüR BIOTECHNOLOGISCHE FORSCHUNG MBH (GBF) | Process for high cell density fermentation of escherichia coli in an agitated boiler fermenter |
| CN101993830A (en) * | 2009-08-28 | 2011-03-30 | 吉林省农业科学院 | Preparation method of escherichia coli competent cell |
| CN103184172A (en) * | 2011-12-30 | 2013-07-03 | 广州拜迪生物医药有限公司 | Culture medium used in Escherichia coli high-density culturing |
| CN104293668A (en) * | 2013-07-17 | 2015-01-21 | 南京朗恩生物科技有限公司 | Recombinant escherichia coli high-density fermentation method |
| CN105420157A (en) * | 2015-12-20 | 2016-03-23 | 深圳宏康生物科技有限公司 | Preparation of escherichia coli competent cells and transformation method of escherichia coli competent cells |
| CN106434426A (en) * | 2016-08-30 | 2017-02-22 | 内蒙古金源康生物工程有限公司 | High-efficiency expression culture medium for escherichia coli |
| WO2017197887A1 (en) * | 2016-05-17 | 2017-11-23 | 河南巨龙生物工程股份有限公司 | Escherichia coli jltrp strain and application thereof in l-tryptophan synthesis |
| CN112080456A (en) * | 2020-09-15 | 2020-12-15 | 江南大学 | Method for inducing and expressing recombinant protein by adopting escherichia coli system |
| CN112625988A (en) * | 2020-12-22 | 2021-04-09 | 江苏诚信药业有限公司 | Escherichia coli fermentation medium, fermentation culture method and application |
| CN114045251A (en) * | 2021-12-02 | 2022-02-15 | 四川百诺吉科技有限公司 | High-density fermentation medium for genetically engineered escherichia coli and fermentation process thereof |
| CN114657114A (en) * | 2022-04-19 | 2022-06-24 | 天津鸿宇泰生物科技有限公司 | Escherichia coli competent cell and preparation method thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7303906B2 (en) * | 2002-09-06 | 2007-12-04 | Wisconsin Alumni Research Foundation | Competent bacteria |
| JP2013085531A (en) * | 2011-10-20 | 2013-05-13 | Tosoh Corp | Method for producing protein with recombinant e.coli |
| KR102036671B1 (en) * | 2012-03-12 | 2019-11-26 | 한미사이언스 주식회사 | Method of culturing E. coli for high density |
-
2022
- 2022-12-26 CN CN202211674083.3A patent/CN115786214B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1991010721A2 (en) * | 1990-01-19 | 1991-07-25 | GESELLSCHAFT FüR BIOTECHNOLOGISCHE FORSCHUNG MBH (GBF) | Process for high cell density fermentation of escherichia coli in an agitated boiler fermenter |
| CN101993830A (en) * | 2009-08-28 | 2011-03-30 | 吉林省农业科学院 | Preparation method of escherichia coli competent cell |
| CN103184172A (en) * | 2011-12-30 | 2013-07-03 | 广州拜迪生物医药有限公司 | Culture medium used in Escherichia coli high-density culturing |
| CN104293668A (en) * | 2013-07-17 | 2015-01-21 | 南京朗恩生物科技有限公司 | Recombinant escherichia coli high-density fermentation method |
| CN105420157A (en) * | 2015-12-20 | 2016-03-23 | 深圳宏康生物科技有限公司 | Preparation of escherichia coli competent cells and transformation method of escherichia coli competent cells |
| WO2017197887A1 (en) * | 2016-05-17 | 2017-11-23 | 河南巨龙生物工程股份有限公司 | Escherichia coli jltrp strain and application thereof in l-tryptophan synthesis |
| CN106434426A (en) * | 2016-08-30 | 2017-02-22 | 内蒙古金源康生物工程有限公司 | High-efficiency expression culture medium for escherichia coli |
| CN112080456A (en) * | 2020-09-15 | 2020-12-15 | 江南大学 | Method for inducing and expressing recombinant protein by adopting escherichia coli system |
| CN112625988A (en) * | 2020-12-22 | 2021-04-09 | 江苏诚信药业有限公司 | Escherichia coli fermentation medium, fermentation culture method and application |
| CN114045251A (en) * | 2021-12-02 | 2022-02-15 | 四川百诺吉科技有限公司 | High-density fermentation medium for genetically engineered escherichia coli and fermentation process thereof |
| CN114657114A (en) * | 2022-04-19 | 2022-06-24 | 天津鸿宇泰生物科技有限公司 | Escherichia coli competent cell and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 大肠杆菌感受态细胞制备及转化研究现状;郭梦姚等;《河北北方学院学报》;全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115786214A (en) | 2023-03-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6937366B2 (en) | Continuous culture of black aspergillus and citric acid production method using it | |
| CN102154160B (en) | Strain capable of producing L-arginine and method for producing L-arginine by same | |
| CN105368766B (en) | One plant of method for producing the genetic engineering bacterium of pentanediamine and its preparing pentanediamine | |
| CN103397005B (en) | Production method of glucose oxidase | |
| CN112625988A (en) | Escherichia coli fermentation medium, fermentation culture method and application | |
| CN108410788A (en) | The method of high-density cultivation of Escherichia coli cell | |
| CN102604904B (en) | Production method of glucose dehydrogenase | |
| CN114214386A (en) | Method for producing astaxanthin by heterotrophic culture of chlorella | |
| CN115786214B (en) | High-density fermentation culture medium and high-density fermentation culture method for competent escherichia coli | |
| CN116731933B (en) | Corynebacterium glutamicum and application thereof in valine production | |
| WO2012140223A1 (en) | High efficiency fermentation process | |
| CN115895976B (en) | Escherichia coli for producing L-tryptophan and application thereof in producing L-tryptophan | |
| CN115161203B (en) | Method for heterotrophic culture of high-yield protein by chlorella | |
| CN101153297A (en) | Novel single-tank hemicontinuous high-strength ferment high optical purity L- lactic acid technique for rhizopus oryzae bacterium ball | |
| JP7151049B2 (en) | How to grow microorganisms | |
| CN114045251B (en) | High-density fermentation medium for genetically engineered escherichia coli and fermentation process thereof | |
| CN104593306A (en) | High-density culture method of escherichia coli strains HY-05C | |
| CN114164160A (en) | Fermentation medium for efficiently producing plasmid DNA and fermentation production method | |
| CN112029810B (en) | Culture medium for producing insulin glargine and fermentation method | |
| CN109295118B (en) | Circulating fermentation method of propionibacterium | |
| CN105296407A (en) | Method for culturing avibacterium paragallinarum bacterial solution | |
| CN104498542A (en) | Method for preparing L-lactic acid employing continuous method in fermentation manner | |
| CN112111476A (en) | Penicillin G acylase high-yield strain breeding and fermentation method | |
| CN116355814B (en) | Escherichia coli and application thereof in fermentation production of L-arginine | |
| CN112391331B (en) | Recombinant escherichia coli for overexpression of GatA gene and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |