CN109439566A - A kind of cultural method of escherichia coli high-level expression Plasmid DNA - Google Patents
A kind of cultural method of escherichia coli high-level expression Plasmid DNA Download PDFInfo
- Publication number
- CN109439566A CN109439566A CN201811175545.0A CN201811175545A CN109439566A CN 109439566 A CN109439566 A CN 109439566A CN 201811175545 A CN201811175545 A CN 201811175545A CN 109439566 A CN109439566 A CN 109439566A
- Authority
- CN
- China
- Prior art keywords
- parts
- escherichia coli
- plasmid dna
- level expression
- expression plasmid
- 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.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses the cultural method of escherichia coli high-level expression Plasmid DNA, include the following steps: the preparation of (1) culture medium；(2) it is inoculated with；(3) thallus culture.The present invention provides a kind of cultural methods of escherichia coli high-level expression Plasmid DNA, on the basis of keeping higher unit thallus plasmid content, the purity of Escherichia coli can be significantly improved, the purifying culture of progress thallus is not needed, simplify the incubation of Escherichia coli, the purity and content of Plasmid DNA are improved, there is good marketing application value.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of cultural method of escherichia coli high-level expression Plasmid DNA.
Background technique
Plasmid has autonomous replication capacity, so that it is also able to maintain constant copy number in progeny cell, and express and taken
The hereditary information of band.Bacterial plasmid is common carrier in DNA recombinant technique.Carrier, which refers to, leads to a useful foreign gene
Genetic engineering means are crossed, is sent into recipient cell and removes the tool for being proliferated and being expressed.The recombination of certain target gene segment is arrived
In plasmid, recombination or recombinant are constituted.Then this recombinant is transferred to recipient cell through microbiological transformation technology
In (such as Escherichia coli), the target gene in recombinant is set to be able to breed or express in recipient bacterium, to change host cell
Original character generates new substance.The first step of the culture of bacterium as production of plasmid DNA, in entire production process
It plays a key role.Its emerging stays directly influences the yield of Plasmid DNA, and under the influence of continue the progress of step.Therefore,
The growth conditions of selection and optimization thallus, is to generate largely stable Plasmid DNA necessary condition, the especially composition of culture medium
Ingredient and concentration have large effect to the growth of bacterium and the content of Plasmid DNA.
Although the existing culture medium for applying to Plasmid DNA engineering bacteria High Cell Density And High Expression can achieve higher cell density
With the target of plasmid total amount, Plasmid DNA is more universal than content lower, using existing culture medium prescription, although Plasmid DNA content
There is a degree of raising, but result is still unsatisfactory.And the cultural method of existing Escherichia coli, it is necessary first to right
Bacterium solution carries out purification process, then expands culture, and after the completion of plasmid extracts, needs that a large amount of man power and material is spent to go to carry out
Purifying, causes the significant wastage of resource, and the quality of extracted Plasmid DNA is unsatisfactory.Therefore, urgent need will develop one
Kind is suitable for the cultural method of escherichia coli high-level expression Plasmid DNA.
Summary of the invention
The purpose of the present invention is being directed to existing problem, a kind of culture of escherichia coli high-level expression Plasmid DNA is provided
Method can significantly improve the purity of Escherichia coli, not need to carry out on the basis of keeping higher unit thallus plasmid content
The purifying culture of thallus, simplifies the incubation of Escherichia coli, improves the purity and content of Plasmid DNA, has good
Marketing application value.
The present invention is achieved by the following technical solutions:
A kind of cultural method of escherichia coli high-level expression Plasmid DNA, includes the following steps:
(1) preparation of culture medium:
A. 2.2 ~ 2.8 parts of 10.5 ~ 11.5 parts of 2.5 ~ 3.5 parts of beef extract, peptone, the sodium chloride of corresponding parts by weight are weighed, is modified
3.2 ~ 3.8 parts of lanthanum carbonate, 1.2 ~ 1.8 parts of active carbon, 0.32 ~ 0.38 part of tartaric acid, 0.12 ~ 0.18 part of citric acid, glycerol 12.5 ~
13.5 parts, 820 ~ 880 parts of deionized water it is spare；
It b. will weighed beef extract in operation a, peptone, chlorination, modified lanthanum carbonate, active carbon, tartaric acid, citric acid, sweet
Oil, deionized water are put into agitator tank jointly, are 97 ~ 99 DEG C by the temperature control in agitator tank, are persistently stirred 32 ~ 38min,
X ray irradiation x processing is carried out while stirring；
When being c. down to 42 ~ 48 DEG C until operating the temperature in b in acquired solution, with the hydrochloric acid solution of 1.2 ~ 1.8mol/L or 1.2
The pH for operating solution obtained in b is adjusted to 7.4 ~ 7.6 by the sodium hydroxide solution of ~ 1.8mol/L；
(2) it is inoculated with:
It takes in 100 ~ 120 μ L ampicillins investment 200 ~ 300mL step (1) prepared culture medium, then takes 100 ~ 120 μ L
E.colistraindh5α is inoculated into the culture medium added with ampicillin, then is weighed special composite powder investment and contained bacterium
In the culture medium of strain and ampicillin；
(3) thallus culture:
Culture medium in step (2) is placed on constant-temperature table and carries out strain culturing, carries out acousto-optic wave alternating while culture
Combined Treatment shakes bacterium culture until the OD600 of bacterium solution reaches 0.9 ~ 1.
Further, the preparation method of modified lanthanum carbonate includes the following steps: in step (1) the operation a or operation b
1) it impregnates in the hydrochloric acid solution for being 2.5 ~ 3.5% by lanthanum carbonate investment mass fraction, keeps in hydrochloric acid solution when immersion
Temperature be 62 ~ 68 DEG C, filter out after 10.5 ~ 11.5min of immersion treatment spare；
2) in the sodium hydroxide solution for being 4.2 ~ 4.8% by treated the lanthanum carbonate investment mass fraction of acid soak in step 1)
It is impregnated, it is 92 ~ 98 DEG C that the temperature in sodium hydroxide solution is kept when immersion, is filtered out after 17 ~ 19min of immersion treatment spare；
It 3) is 1g:32 ~ 38mL according to mass volume ratio by treated the lanthanum carbonate of soaking with sodium hydroxide in step 2 and modification liquid
Be 0.42 ~ 0.48MPa by the pressure control in reaction kettle in common investment reaction kettle, the temperature control in reaction kettle for 220 ~
280 DEG C, filter residue is filtered to obtain after handling 21 ~ 23min；
4) filter residue obtained in step 3) is put into baking oven and is dried, the temperature control in baking oven is 72 ~ 78 DEG C, and drying is extremely
Moisture content is 3 ~ 4%.
Further, each ingredient and corresponding parts by weight in modification liquid in the step 3) are as follows: 21 ~ 25 parts of glycerol, octyl phenol
3.5 ~ 4.5 parts of polyoxyethylene ether, 12.5 ~ 13.5 parts of tetradecyltrimethylammonium bromide, sodium cetanesulfonate 13.5 ~ 14.5
Part, 420 ~ 480 parts of deionized water.
Further, the form of x ray irradiation x is β ray in the step (1) operation b, the dosage of irradiation is 2.2 ~
2.8Gy/min。
Further, the concentration of ampicillin is 16 ~ 20 μ L/mL in the step (2).
Further, the preparation method of composite powder special in the step (2) is: by malic acid and sodium bicarbonate according to
Weight ratio is after 1:2 ~ 2.4 is mixed, to be placed under ultraviolet lamp and carry out sterilization treatment, and the time of sterilization treatment is 30 ~ 40min.
Further, keeping the temperature in constant incubator in the step (3) when strain culturing is 35 ~ 37 DEG C, shaking table
Revolving speed be 200 ~ 220rpm.
Further, in the step (3) acousto-optic wave alternating Combined Treatment program are as follows: first with frequency be 20 ~ 24kHz
14 ~ 18min of ultrasonication, then with wavelength be 430 ~ 440nm light wave handle 8 ~ 12min, reuse frequency be 20 ~
The ultrasonic wave and wavelength of 24kHz is 5 ~ 7min of light wave Combined Treatment of 430 ~ 440nm.
The present invention provides a kind of cultural methods of escherichia coli high-level expression Plasmid DNA, from culture medium and cultural method
Two aspects improve, and are used culture medium first, the present invention is on the basis of existing Escherichia coli culture medium is formulated
Very big improvement has been done, has been added to modified lanthanum carbonate in formula, lanthanum carbonate is successively passed through to acid solution and dipping by lye processing, it is soft
Change lanthanum carbonate surface, lays the foundation for subsequent modification, processing is then modified under conditions of high temperature and pressure, it is modified
Glycerol in liquid can increase the mobility of modification liquid, improve the activity on lanthanum carbonate surface, cationic tetradecyltrimethylammonium
Ammonium bromide and anionic sodium cetanesulfonate compounding are made, and use it as dressing agent is improved and are modified to lanthanum carbonate surface
Processing, improves the reactivity on lanthanum carbonate surface, and the compatibility between other compositions in culture medium, modified carbon
Sour lanthanum is added in culture medium, facilitate enhance culture medium in other material compositions coagulability, promote engineering bacteria to effectively at
The absorption divided, improves the reproduction speed of bacterium solution, and modified lanthanum carbonate surface has huge molecular network, engineering can be absorbed
The metabolic waste of bacterium effectively prevents bacterium metabolite from making viable bacteria can be with high efficient expression the toxic action of other viable bacterias.Because
During the long-term preservation of thallus, can inevitably generate the dead bacterium in part and toxic metabolites, the active carbon that the present invention adds with change
Property lanthanum carbonate be used cooperatively, further improve culture medium quality, guarantee thallus health quickly breeding.Applicant is a large amount of
Tartaric acid, citric acid etc. are added in discovery in thallus culture medium in experiment can inhibit the growth of gram-positive bacteria, increase training
The selectivity for supporting base ensure that the purity of Escherichia coli, but add certain single acid for the purity of raising Escherichia coli
The only improvement result of very little, applicant is reasonably combined by tartaric acid and citric acid in a large amount of experiment, with the use of for
The purity for improving Escherichia coli has good improvement result, and then improves the purity of Plasmid DNA, reduces the behaviour of plasmid purification
Make cost.In the preparation process of culture medium, the processing of β x ray irradiation x is carried out, the excitation of β ray generates activated atom and activation point
Son increases the anti-microbe ability of culture medium, prevents culture medium from being infected by miscellaneous bacteria, and x ray irradiation x can assist culture medium
Preparation accelerates crosslinking, polymerization in culture medium between effective component, improves the harmony of culture medium nutrition.In inoculation Shi Pei
It supports and adds composite powder in base, composite powder is the mixed-powder of the malic acid and sodium bicarbonate by sterilizing, and the composite powder of addition adds
It is added in culture medium, carbon dioxide can be discharged, Escherichia coli are facultative anaerobic bacteria, carbon dioxide during thallus culture
The environment of anaerobism is provided, the selectivity of thallus is improved, is conducive to the purity for improving plasmid.During thallus culture, carry out
Acousto-optic wave replaces Combined Treatment, and the sound wave of single frequency or the light wave of single wavelength all can have certain rush to the breeding of bacterium solution
Into effect, but its facilitation effect is not significant enough, and does not have impetus for the purity of Plasmid DNA, and applicant is a large amount of
It was found that, using the sound wave of specific frequency, specific wavelength light wave replace Combined Treatment, significantly improve large intestine bar
The growth characteristics of bacterium improve the selectivity of Escherichia coli, have good improvement result to the purity of Plasmid DNA.
The present invention has the advantage that compared with prior art
The present invention provides a kind of cultural methods of escherichia coli high-level expression Plasmid DNA, are keeping higher unit thallus matter
On the basis of grain content, the purity of Escherichia coli can be significantly improved, the purifying culture for carrying out thallus is not needed, simplifies large intestine bar
The incubation of bacterium improves the purity and content of Plasmid DNA, has good marketing application value.
Specific embodiment
Embodiment 1
A kind of cultural method of escherichia coli high-level expression Plasmid DNA, includes the following steps:
(1) preparation of culture medium:
A. weigh 2.2 parts of 10.5 parts of 2.5 parts of beef extract, peptone, the sodium chloride of corresponding parts by weight, modified 3.2 parts of lanthanum carbonate,
1.2 parts of active carbon, 0.32 part of tartaric acid, 0.12 part of citric acid, 12.5 parts of glycerol, 820 parts of deionized water it is spare；
It b. will weighed beef extract in operation a, peptone, chlorination, modified lanthanum carbonate, active carbon, tartaric acid, citric acid, sweet
Oil, deionized water are put into agitator tank jointly, are 97 DEG C by the temperature control in agitator tank, 32min are persistently stirred, in stirring
X ray irradiation x processing is carried out simultaneously；
When being c. down to 42 DEG C until operating the temperature in b in acquired solution, with the hydrochloric acid solution of 1.2mol/L or 1.2mol/L
The pH for operating solution obtained in b is adjusted to 7.4 by sodium hydroxide solution；
(2) it is inoculated with:
It takes in 100 μ L ampicillins investment 200mL step (1) prepared culture medium, then takes 100 μ L bacillus coli DH 5 alphas
Strain inoculated is into the culture medium added with ampicillin, then weighs special composite powder investment and contain bacterial strain and ammonia benzyl mould
In the culture medium of element；
(3) thallus culture:
Culture medium in step (2) is placed on constant-temperature table and carries out strain culturing, carries out acousto-optic wave alternating while culture
Combined Treatment shakes bacterium culture until the OD600 of bacterium solution reaches 0.9.
Further, the preparation method of modified lanthanum carbonate includes the following steps: in step (1) the operation a or operation b
1) it is impregnated in the hydrochloric acid solution for being 2.5% by lanthanum carbonate investment mass fraction, the temperature in hydrochloric acid solution is kept when immersion
Degree is 62 DEG C, is filtered out after immersion treatment 10.5min spare；
2) it is carried out in the sodium hydroxide solution for being 4.2% by treated the lanthanum carbonate investment mass fraction of acid soak in step 1)
It impregnates, it is 92 DEG C that the temperature in sodium hydroxide solution is kept when immersion, is filtered out after immersion treatment 17min spare；
It 3) is that 1g:32mL is common according to mass volume ratio by treated the lanthanum carbonate of soaking with sodium hydroxide in step 2 and modification liquid
It puts into reaction kettle, the pressure in reaction kettle control as 0.42MPa, the temperature control in reaction kettle is 220 DEG C, is handled
Filter residue is filtered to obtain after 21min；
4) filter residue obtained in step 3) is put into baking oven and will be dried, the control of temperature in baking oven is 72 DEG C, dry to containing
Water rate is 3%.
Further, each ingredient and corresponding parts by weight in modification liquid in the step 3) are as follows: 21 parts of glycerol, octyl phenol polyoxy
3.5 parts of vinethene, 12.5 parts of tetradecyltrimethylammonium bromide, 13.5 parts of sodium cetanesulfonate, 420 parts of deionized water.
Further, the form of x ray irradiation x is β ray in step (1) the operation b, and the dosage of irradiation is 2.2Gy/
min。
Further, the concentration of ampicillin is 16 μ L/mL in the step (2).
Further, the preparation method of composite powder special in the step (2) is: by malic acid and sodium bicarbonate according to
Weight ratio is after 1:2 is mixed, to be placed under ultraviolet lamp and carry out sterilization treatment, and the time of sterilization treatment is 30min.
Further, keeping the temperature in constant incubator in the step (3) when strain culturing is 35 DEG C, and shaking table turns
Speed is 200rpm.
Further, in the step (3) acousto-optic wave alternating Combined Treatment program are as follows: be first 20kHz's with frequency
Ultrasonication 14min, then handles 8min with the light wave that wavelength is 430nm, and reuse frequency is the ultrasonic wave and wavelength of 20kHz
For the light wave Combined Treatment 5min of 430nm.
Embodiment 2
A kind of cultural method of escherichia coli high-level expression Plasmid DNA, includes the following steps:
(1) preparation of culture medium:
A. 2.5 parts of 11 parts of 3 parts of beef extract, peptone, sodium chloride, modified 3.5 parts of the lanthanum carbonate, activity of corresponding parts by weight are weighed
1.5 parts of charcoal, 0.35 part of tartaric acid, 0.15 part of citric acid, 13 parts of glycerol, 850 parts of deionized water it is spare；
It b. will weighed beef extract in operation a, peptone, chlorination, modified lanthanum carbonate, active carbon, tartaric acid, citric acid, sweet
Oil, deionized water are put into agitator tank jointly, are 98 DEG C by the temperature control in agitator tank, 35min are persistently stirred, in stirring
X ray irradiation x processing is carried out simultaneously；
When being c. down to 45 DEG C until operating the temperature in b in acquired solution, with the hydrochloric acid solution of 1.5mol/L or 1.5mol/L
The pH for operating solution obtained in b is adjusted to 7.5 by sodium hydroxide solution；
(2) it is inoculated with:
It takes in 110 μ L ampicillins investment 250mL step (1) prepared culture medium, then takes 110 μ L bacillus coli DH 5 alphas
Strain inoculated is into the culture medium added with ampicillin, then weighs special composite powder investment and contain bacterial strain and ammonia benzyl mould
In the culture medium of element；
(3) thallus culture:
Culture medium in step (2) is placed on constant-temperature table and carries out strain culturing, carries out acousto-optic wave alternating while culture
Combined Treatment shakes bacterium culture until the OD600 of bacterium solution reaches 0.95.
Further, the preparation method of modified lanthanum carbonate includes the following steps: in step (1) the operation a or operation b
1) it is impregnated in the hydrochloric acid solution for being 3% by lanthanum carbonate investment mass fraction, the temperature in hydrochloric acid solution is kept when immersion
It is 65 DEG C, is filtered out after immersion treatment 11min spare；
2) it is carried out in the sodium hydroxide solution for being 4.5% by treated the lanthanum carbonate investment mass fraction of acid soak in step 1)
It impregnates, it is 95 DEG C that the temperature in sodium hydroxide solution is kept when immersion, is filtered out after immersion treatment 18min spare；
It 3) is that 1g:35mL is common according to mass volume ratio by treated the lanthanum carbonate of soaking with sodium hydroxide in step 2 and modification liquid
It puts into reaction kettle, the pressure in reaction kettle control as 0.45MPa, the temperature control in reaction kettle is 250 DEG C, is handled
Filter residue is filtered to obtain after 22min；
4) filter residue obtained in step 3) is put into baking oven and will be dried, the control of temperature in baking oven is 75 DEG C, dry to containing
Water rate is 3.5%.
Further, each ingredient and corresponding parts by weight in modification liquid in the step 3) are as follows: 23 parts of glycerol, octyl phenol polyoxy
4 parts of vinethene, 13 parts of tetradecyltrimethylammonium bromide, 14 parts of sodium cetanesulfonate, 450 parts of deionized water.
Further, the form of x ray irradiation x is β ray in step (1) the operation b, and the dosage of irradiation is 2.5Gy/
min。
Further, the concentration of ampicillin is 18 μ L/mL in the step (2).
Further, the preparation method of composite powder special in the step (2) is: by malic acid and sodium bicarbonate according to
Weight ratio is after 1:2.2 is mixed, to be placed under ultraviolet lamp and carry out sterilization treatment, and the time of sterilization treatment is 35min.
Further, keeping the temperature in constant incubator in the step (3) when strain culturing is 36 DEG C, and shaking table turns
Speed is 210rpm.
Further, in the step (3) acousto-optic wave alternating Combined Treatment program are as follows: be first 22kHz's with frequency
Ultrasonication 16min, then handles 10min with the light wave that wavelength is 435nm, and reuse frequency is the ultrasonic wave and wave of 22kHz
The light wave Combined Treatment 6min of a length of 435nm.
Embodiment 3
A kind of cultural method of escherichia coli high-level expression Plasmid DNA, includes the following steps:
(1) preparation of culture medium:
A. weigh 2.8 parts of 11.5 parts of 3.5 parts of beef extract, peptone, the sodium chloride of corresponding parts by weight, modified 3.8 parts of lanthanum carbonate,
1.8 parts of active carbon, 0.38 part of tartaric acid, 0.18 part of citric acid, 13.5 parts of glycerol, 880 parts of deionized water it is spare；
It b. will weighed beef extract in operation a, peptone, chlorination, modified lanthanum carbonate, active carbon, tartaric acid, citric acid, sweet
Oil, deionized water are put into agitator tank jointly, are 99 DEG C by the temperature control in agitator tank, 38min are persistently stirred, in stirring
X ray irradiation x processing is carried out simultaneously；
When being c. down to 48 DEG C until operating the temperature in b in acquired solution, with the hydrochloric acid solution of 1.8mol/L or 1.8mol/L
The pH for operating solution obtained in b is adjusted to 7.6 by sodium hydroxide solution；
(2) it is inoculated with:
It takes in 120 μ L ampicillins investment 300mL step (1) prepared culture medium, then takes 120 μ L bacillus coli DH 5 alphas
Strain inoculated is into the culture medium added with ampicillin, then weighs special composite powder investment and contain bacterial strain and ammonia benzyl mould
In the culture medium of element；
(3) thallus culture:
Culture medium in step (2) is placed on constant-temperature table and carries out strain culturing, carries out acousto-optic wave alternating while culture
Combined Treatment shakes bacterium culture until the OD600 of bacterium solution reaches 1.
Further, the preparation method of modified lanthanum carbonate includes the following steps: in step (1) the operation a or operation b
1) it is impregnated in the hydrochloric acid solution for being 3.5% by lanthanum carbonate investment mass fraction, the temperature in hydrochloric acid solution is kept when immersion
Degree is 68 DEG C, is filtered out after immersion treatment 11.5min spare；
2) it is carried out in the sodium hydroxide solution for being 4.8% by treated the lanthanum carbonate investment mass fraction of acid soak in step 1)
It impregnates, it is 98 DEG C that the temperature in sodium hydroxide solution is kept when immersion, is filtered out after immersion treatment 19min spare；
It 3) is that 1g:38mL is common according to mass volume ratio by treated the lanthanum carbonate of soaking with sodium hydroxide in step 2 and modification liquid
It puts into reaction kettle, the pressure in reaction kettle control as 0.48MPa, the temperature control in reaction kettle is 280 DEG C, is handled
Filter residue is filtered to obtain after 23min；
4) filter residue obtained in step 3) is put into baking oven and will be dried, the control of temperature in baking oven is 78 DEG C, dry to containing
Water rate is 4%.
Further, each ingredient and corresponding parts by weight in modification liquid in the step 3) are as follows: 25 parts of glycerol, octyl phenol polyoxy
4.5 parts of vinethene, 13.5 parts of tetradecyltrimethylammonium bromide, 14.5 parts of sodium cetanesulfonate, 480 parts of deionized water.
Further, the form of x ray irradiation x is β ray in step (1) the operation b, and the dosage of irradiation is 2.8Gy/
min。
Further, the concentration of ampicillin is 20 μ L/mL in the step (2).
Further, the preparation method of composite powder special in the step (2) is: by malic acid and sodium bicarbonate according to
Weight ratio is after 1:2.4 is mixed, to be placed under ultraviolet lamp and carry out sterilization treatment, and the time of sterilization treatment is 40min.
Further, keeping the temperature in constant incubator in the step (3) when strain culturing is 37 DEG C, and shaking table turns
Speed is 220rpm.
Further, in the step (3) acousto-optic wave alternating Combined Treatment program are as follows: be first 24kHz's with frequency
Ultrasonication 18min, then handles 12min with the light wave that wavelength is 440nm, and reuse frequency is the ultrasonic wave and wave of 24kHz
The light wave Combined Treatment 7min of a length of 440nm.
Comparative example 1
This comparative example 1 compared with Example 2, saves the whole operation of the preparation of step (1) culture medium, and in equal volume
Culture medium in common LB culture medium step of replacing (2), method and step in addition to this are all the same.
Comparative example 2
This comparative example 2 compared with Example 2, saves the special composite powder in step (2), and method and step in addition to this is equal
It is identical.
Comparative example 3
This comparative example 3 compared with Example 2, saves the acousto-optic wave alternating Combined Treatment in step (3), method in addition to this
Step is all the same.
Control group
Application No. is: 201610877655.6 disclosed culture mediums for colibacillus engineering efficient expression plasmid DNA and
Cultural method.
In order to compare effect of the present invention, e.colistraindh5α is chosen as experimental subjects, uses embodiment 2, right respectively
Cultivate than embodiment 1, comparative example 2, comparative example 3, control group, when bacterium solution OD value reaches 0.9, takes bacterium solution
Thallus is harvested with 10000g centrifugal speed, 4 DEG C, 20min centrifugal condition, carries out coherent detection.
(3) plasmid extraction, parametric measurement:
It is carried out using a small amount of extraction agent boxes of qiagen plasmid by operating instruction.Plasmid content is measured using ultraviolet specrophotometer
The A value of 260nm wavelength determines.The measurement of plasmid purity measures the absorption of 260nm, 280nm wavelength using ultraviolet specrophotometer
Ratio determines (ratio met the quality standard should be between 1.75-1.85).
Specific experiment correlation data is as shown in table 1 below:
Table 1
| Unit volume receives bacterium amount (g/L) | Unit volume plasmid content (mg/L) | Unit bacterium amount plasmid content (mg/g) | A260/A280 | |
| Embodiment 2 | 189.46 | 501 | 2.64 | 1.81 |
| Comparative example 1 | 187.58 | 428 | 2.28 | 1.71 |
| Comparative example 2 | 187.34 | 489 | 2.61 | 1.76 |
| Comparative example 3 | 186.87 | 467 | 2.50 | 1.75 |
| Control group | 189.42 | 451 | 2.38 | 1.82 |
The present invention provides a kind of cultural method of escherichia coli high-level expression Plasmid DNA it can be seen from upper table 1, keeping
On the basis of higher unit thallus plasmid content, the purity of Escherichia coli can be significantly improved, does not need the purifying for carrying out thallus
Culture, simplifies the incubation of Escherichia coli, improves the purity and content of Plasmid DNA, and there is good marketing to answer
With value.
Claims (8)
1. a kind of cultural method of escherichia coli high-level expression Plasmid DNA, which comprises the steps of:
(1) preparation of culture medium:
A. 2.2 ~ 2.8 parts of 10.5 ~ 11.5 parts of 2.5 ~ 3.5 parts of beef extract, peptone, the sodium chloride of corresponding parts by weight are weighed, is modified
3.2 ~ 3.8 parts of lanthanum carbonate, 1.2 ~ 1.8 parts of active carbon, 0.32 ~ 0.38 part of tartaric acid, 0.12 ~ 0.18 part of citric acid, glycerol 12.5 ~
13.5 parts, 820 ~ 880 parts of deionized water it is spare；
It b. will weighed beef extract in operation a, peptone, chlorination, modified lanthanum carbonate, active carbon, tartaric acid, citric acid, sweet
Oil, deionized water are put into agitator tank jointly, are 97 ~ 99 DEG C by the temperature control in agitator tank, are persistently stirred 32 ~ 38min,
X ray irradiation x processing is carried out while stirring；
When being c. down to 42 ~ 48 DEG C until operating the temperature in b in acquired solution, with the hydrochloric acid solution of 1.2 ~ 1.8mol/L or 1.2
The pH for operating solution obtained in b is adjusted to 7.4 ~ 7.6 by the sodium hydroxide solution of ~ 1.8mol/L；
(2) it is inoculated with:
It takes in 100 ~ 120 μ L ampicillins investment 200 ~ 300mL step (1) prepared culture medium, then takes 100 ~ 120 μ L
E.colistraindh5α is inoculated into the culture medium added with ampicillin, then is weighed special composite powder investment and contained bacterium
In the culture medium of strain and ampicillin；
(3) thallus culture:
Culture medium in step (2) is placed on constant-temperature table and carries out strain culturing, carries out acousto-optic wave alternating while culture
Combined Treatment shakes bacterium culture until the OD600 of bacterium solution reaches 0.9 ~ 1.
2. a kind of cultural method of escherichia coli high-level expression Plasmid DNA according to claim 1, which is characterized in that described
Step (1) operation a or the preparation method for operating modified lanthanum carbonate in b include the following steps:
1) it impregnates in the hydrochloric acid solution for being 2.5 ~ 3.5% by lanthanum carbonate investment mass fraction, keeps in hydrochloric acid solution when immersion
Temperature be 62 ~ 68 DEG C, filter out after 10.5 ~ 11.5min of immersion treatment spare；
2) in the sodium hydroxide solution for being 4.2 ~ 4.8% by treated the lanthanum carbonate investment mass fraction of acid soak in step 1)
It is impregnated, it is 92 ~ 98 DEG C that the temperature in sodium hydroxide solution is kept when immersion, is filtered out after 17 ~ 19min of immersion treatment spare；
It 3) is 1g:32 ~ 38mL according to mass volume ratio by treated the lanthanum carbonate of soaking with sodium hydroxide in step 2 and modification liquid
Be 0.42 ~ 0.48MPa by the pressure control in reaction kettle in common investment reaction kettle, the temperature control in reaction kettle for 220 ~
280 DEG C, filter residue is filtered to obtain after handling 21 ~ 23min；
4) filter residue obtained in step 3) is put into baking oven and is dried, the temperature control in baking oven is 72 ~ 78 DEG C, and drying is extremely
Moisture content is 3 ~ 4%.
3. a kind of cultural method of escherichia coli high-level expression Plasmid DNA according to claim 2, which is characterized in that described
Each ingredient and corresponding parts by weight in modification liquid in step 3) are as follows: 21 ~ 25 parts of glycerol, 3.5 ~ 4.5 parts of octyl phenol polyoxyethylene ether, ten
12.5 ~ 13.5 parts of tetraalkyl trimethylammonium bromide, 13.5 ~ 14.5 parts of sodium cetanesulfonate, 420 ~ 480 parts of deionized water.
4. a kind of cultural method of escherichia coli high-level expression Plasmid DNA according to claim 1, which is characterized in that described
The form that step (1) operates x ray irradiation x in b is β ray, and the dosage of irradiation is 2.2 ~ 2.8Gy/min.
5. a kind of cultural method of escherichia coli high-level expression Plasmid DNA according to claim 1, which is characterized in that described
The concentration of ampicillin is 16 ~ 20 μ L/mL in step (2).
6. a kind of cultural method of escherichia coli high-level expression Plasmid DNA according to claim 1, which is characterized in that described
The preparation method of special composite powder is in step (2): according to weight ratio being that 1:2 ~ 2.4 is mixed by malic acid and sodium bicarbonate
Afterwards, it is placed under ultraviolet lamp and carries out sterilization treatment, the time of sterilization treatment is 30 ~ 40min.
7. a kind of cultural method of escherichia coli high-level expression Plasmid DNA according to claim 1, which is characterized in that described
Keeping the temperature in constant incubator in step (3) when strain culturing is 35 ~ 37 DEG C, and the revolving speed of shaking table is 200 ~ 220rpm.
8. a kind of cultural method of escherichia coli high-level expression Plasmid DNA according to claim 1, which is characterized in that described
The program of acousto-optic wave alternating Combined Treatment in step (3) are as follows: the 14 ~ 18min of ultrasonication for being first 20 ~ 24kHz with frequency,
Then 8 ~ 12min is handled with the light wave that wavelength is 430 ~ 440nm, the ultrasonic wave and wavelength that reuse frequency is 20 ~ 24kHz be 430 ~
5 ~ 7min of light wave Combined Treatment of 440nm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811175545.0A CN109439566A (en) | 2018-10-10 | 2018-10-10 | A kind of cultural method of escherichia coli high-level expression Plasmid DNA |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811175545.0A CN109439566A (en) | 2018-10-10 | 2018-10-10 | A kind of cultural method of escherichia coli high-level expression Plasmid DNA |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109439566A true CN109439566A (en) | 2019-03-08 |
Family
ID=65546371
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811175545.0A Pending CN109439566A (en) | 2018-10-10 | 2018-10-10 | A kind of cultural method of escherichia coli high-level expression Plasmid DNA |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109439566A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150132740A1 (en) * | 2012-06-05 | 2015-05-14 | 3M Innovative Properties Company | Lanthanum-based concentration agents for microorganisms |
| CN104651263A (en) * | 2014-12-22 | 2015-05-27 | 广西大学 | Culture medium for increasing bacteria specific content in escherichia coli of plasmid DNA (Deoxyribonucleic Acid) content |
-
2018
- 2018-10-10 CN CN201811175545.0A patent/CN109439566A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150132740A1 (en) * | 2012-06-05 | 2015-05-14 | 3M Innovative Properties Company | Lanthanum-based concentration agents for microorganisms |
| CN104651263A (en) * | 2014-12-22 | 2015-05-27 | 广西大学 | Culture medium for increasing bacteria specific content in escherichia coli of plasmid DNA (Deoxyribonucleic Acid) content |
Non-Patent Citations (2)
| Title |
|---|
| 微生物工程编写组: "《微生物工程》", 30 November 1976 * |
| 郑淑真: "重组大肠杆菌生产基因治疗质粒DNA发酵工艺研究", 《中国优秀硕士学位论文全文数据库工程科技I辑》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Gullo et al. | Succession of selected strains of Acetobacter pasteurianus and other acetic acid bacteria in traditional balsamic vinegar | |
| CN106957807B (en) | Bacillus licheniformis strain TA65 and application thereof in promoting compost maturity | |
| CN106011022A (en) | Streptomyces roseoflavus solid fermentation medium and preparation and fermentation method thereof | |
| CN107828702A (en) | A kind of kasugarnycin fermentation medium and fermentation process | |
| CN112280721A (en) | Paenibacillus polymyxa LCP-01, compound microbial inoculum compounded with bile acid and application thereof | |
| JP3327469B2 (en) | Methods for biological inactivation of nucleic acids | |
| CN109439566A (en) | A kind of cultural method of escherichia coli high-level expression Plasmid DNA | |
| CN115704001A (en) | Caproic acid bacteria powder, its preparation method and application | |
| CN106867939B (en) | One plant of bacillus amyloliquefaciens for reducing biogenic amine and its application | |
| CN104830734B (en) | The method of one plant of propionibacterium freudenreichii bacterial strain and its fermented-producing bacteria element | |
| CN108130252A (en) | Application of one plant of production paste flavor bacterium in bran starter Maotai-flavor liquor type white wine | |
| CN109370964A (en) | A kind of culture medium and preparation method thereof of escherichia coli high-level expression Plasmid DNA | |
| CN109133354B (en) | Algae-based ecological bacteriostasis method for wastewater treatment | |
| CN114891701A (en) | Staphylococcus simulans HZ01, microbial inoculum and application thereof | |
| CN109370963A (en) | A kind of cultural method of colibacillus engineering high efficient expression supercoiled plasmid DNA | |
| CN113136210A (en) | Agricultural plant enzyme for naturally repairing and improving soil and preparation method thereof | |
| CN106259058A (en) | A kind of fish and shrimp common disease Therapeutic Method with good result | |
| CN109234247A (en) | A kind of glucose oxidase and preparation method thereof | |
| CN109295084A (en) | A kind of preparation of the culture medium for colibacillus engineering efficient expression plasmid DNA | |
| Eastoe et al. | The effect of nisin on the growth of cells and spores of Clostridium welchii in gelatine | |
| CN105132494B (en) | A method of during the fermentation hyaluronic acid is prepared using modified wheat stalk | |
| CN104798819B (en) | A kind of method for improving turfgrass anti-seismic design using low temperature resistant microbial bacterial agent is strengthened | |
| CN106148257B (en) | The Klebsiella pneumoniae of transformation and its application for producing gluconic acid | |
| CN109294938A (en) | A kind of sick dead pig static fermentation high temperature degradation microbial inoculum and its application | |
| CN107053392B (en) | A kind of maize peel high-effect anti-mould processing method |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190308 |
|
| RJ01 | Rejection of invention patent application after publication |