CN109294917A - A kind of culture presevation agent and the method using the preserving agent superfreeze preservation genetic engineering bacterium - Google Patents
A kind of culture presevation agent and the method using the preserving agent superfreeze preservation genetic engineering bacterium Download PDFInfo
- Publication number
- CN109294917A CN109294917A CN201811313646.XA CN201811313646A CN109294917A CN 109294917 A CN109294917 A CN 109294917A CN 201811313646 A CN201811313646 A CN 201811313646A CN 109294917 A CN109294917 A CN 109294917A
- Authority
- CN
- China
- Prior art keywords
- agent
- culture presevation
- genetic engineering
- preservation
- parts
- 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
- C12N1/04—Preserving or maintaining viable microorganisms
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (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)
Abstract
The invention belongs to cell preservation technique field more particularly to a kind of culture presevation agent and utilize the method for the preserving agent superfreeze preservation genetic engineering bacterium.Culture presevation agent of the present invention, the component containing following mass parts: 50~70 parts of cryoprotective agents and 2~5 parts of antibiotic selective agents.Wherein antibiotic selective agent can maintain the genetic stability of the exogenous dna fragment carried in genetic engineering bacterium body by vector plasmid during superfreeze preservation and rejuvenation of spawn, thallus exogenous dna fragment is unstable when solving genetic engineering bacterium superfreeze preservation, plasmid problem easy to be lost.Superfreeze method for preserving provided by the invention is easy to operate, storage is convenient, the bacteria live phase is long, the thallus high survival rate of -80 DEG C of rejuvenation after preservation 1 year is up to 96%, it is applied to the preservation of genetic engineering bacterium, it is ensured that the genetic engineering bacterium after rejuvenation can efficiently maintain the corresponding function of exogenous dna fragment.
Description
Technical field
The invention belongs to cell preservation technique field more particularly to a kind of culture presevation agent and utilize the preserving agent ultralow temperature
The method of freezing genetic engineering bacterium.
Background technique
Culture presevation is essential in microbe research and practical application, since microorganism has the spy for being easy variation
Property, therefore, in preserving process, it is necessary to so that the metabolism of microorganism is in least active or opposing stationary state, it could be in length
So that it is not morphed in time and is able to maintain viability.Low temperature, drying and isolation air are to enable microbial metabolism
An important factor for power reduces, though the culture collection process is more, is designed according to these three factors.
Superfreeze preservation is that culture presevation is most direct, is most simply also one of most common method, has necessary expense
With it is low, be less likely to occur variation, it is easy to operate, the holding time is long the advantages that.Wherein preserving agent has the survival rate of microorganism
Large effect can protect the integrality of somatic cells film and protein structure and function in preserving process, lower cell
The physiological damage being subject to.
Containing the exogenous dna fragment by carryings such as vector plasmids in engineering strain, the usually heredity of these segments is not
It is stable, its exogenous plasmid replicon is easily lost in succeeding generations, culture presevation and rejuvenation stage be also easy to because
It loses exogenous plasmid and influences the correlation function of engineering strain.
Summary of the invention
Exogenous plasmid replicon is easily lost during ultralow temperature preservation and rejuvenation of spawn to solve genetic engineering bacterium
Problem the present invention provides a kind of culture presevation agent and utilizes the method for the preserving agent superfreeze preservation genetic engineering bacterium.
Technical solution of the present invention:
A kind of culture presevation agent, the component containing following mass parts: 50~70 parts of cryoprotective agents and 2~5 parts of antibiotic
Selective agent.
Further, the preserving agent is also 10 parts of buffer containing mass parts, the buffer by oligosaccharide with it is sweet
Reveal alcohol 1:1 in mass ratio composition.
Further, the skim milk that the preserving agent is also 3~5 parts containing mass parts.
Further, the cryoprotective agent is one of glycerol, DMSO or glycerine.
Further, the antibiotic selective agent is one or more of kanamycins, ampicillin or chloramphenicol
Combination.
Further, the kanamycins, ampicillin or chloramphenicol are the liquid that concentration is 20~25mg/L, and
Preserving agent is added after 0.22 μm of membrane filtration degerming.
Further, the oligosaccharide is one of trehalose, sucrose or maltose.
A method of utilizing culture presevation agent superfreeze preservation genetic engineering bacterium provided by the invention, the method
Steps are as follows: taking medium centrifugal to handle, use bacterium genetic engineering bacterium to logarithmic growth phase with 37 DEG C of overnight incubations of LB culture medium
Centrifugation is resuspended kind preserving agent, and bacteria suspension is moved into sterile freezing pipe, freezing pipe is closed, centainly to cool down
Rate is cooled to -80 DEG C, and the freezing at -80 DEG C from 4 DEG C.
Further, it is described take medium centrifugal processing be take 10ml culture solution with 3000rpm be centrifuged 5~10min, it is described
It is to be vibrated centrifugation by magnetic force with 3~4ml culture presevation agent to be resuspended that centrifugation, which is resuspended, with culture presevation agent, makes bacterium
Kind is dispersed in culture presevation agent.
Further, the rate of temperature fall is -1~-10 DEG C/min.
Beneficial effects of the present invention:
One, the antibiotic selective agent containing low concentration in culture presevation agent provided by the invention, provides and is conducive to carry
The growth that the genetic engineering bacterium cell colony of plasmid remains stable selects pressure, can help to maintain plasmid replication and chromosome replication
Coordination, guarantee genetic engineering bacterium the exogenous DNA carried by vector plasmid is maintained during ultralow temperature preservation and rejuvenation of spawn
The genetic stability of segment can effectively prevent the loss of plasmid.
Two, the hydrogen and ionic bond that culture presevation agent provided by the invention is generated by cryoprotective agent produce water and cell
Raw affinity stablizes the configuration of genetic engineering bacterium cell component, to prevent the damage constantly to distil to cell by freezing or moisture
Evil.Oligosaccharide and skim milk etc. then can provide energy for cell, prevent its decline, and cell is allow to sustain life for a long time work
Power extends preservation term.
Three, method for preserving provided by the invention is easy to operate, and storage is convenient, and the bacteria live phase is long, and -80 DEG C after preservation 1 year
Up to 96%, the genetic engineering bacterium after rejuvenation still is able to possess exogenous dna fragment corresponding function the thallus high survival rate of rejuvenation.One
Plant height produce pyruvic acid Recombinant organism application culture presevation agent of the present invention and method for preserving -80 DEG C preservation 1 year
Afterwards, the yield of the strain fermentation production pyruvic acid of rejuvenation is compared with only having dropped 5% before preservation.
Specific embodiment
Below with reference to embodiment, the following further describes the technical solution of the present invention, and however, it is not limited to this, all right
Technical solution of the present invention is modified or replaced equivalently, and without departing from the spirit and scope of the technical solution of the present invention, should all be contained
Lid is within the protection scope of the present invention.
Embodiment 1
A kind of culture presevation agent, the component containing following mass parts: 50~70 parts of cryoprotective agents and 2~5 parts of antibiotic
Selective agent.
Embodiment 2
A kind of culture presevation agent, the component containing following mass parts: 50~70 parts of cryoprotective agents, 2~5 parts of antibiotic choosings
Select agent, 5 parts of oligosaccharide and 5 portions of mannitol.
Embodiment 3
A kind of culture presevation agent, the component containing following mass parts: 50~70 parts of cryoprotective agents, 2~5 parts of antibiotic choosings
Select agent, 5 parts of oligosaccharide, 5 portions of mannitol and 3~5 parts of skim milk.
Embodiment 4
A kind of culture presevation agent, the component containing following mass parts: that is mould for 55 parts of glycerol, the card that 3 parts of concentration are 20mg/L
Element, ampicillin or chloramphenicol, 5 parts of trehalose, 5 portions of mannitol and 3 parts of skim milk.
Embodiment 5
A kind of culture presevation agent, the component containing following mass parts: that is mould for 60 parts of DMSO, the card that 4 parts of concentration are 23mg/L
Element, ampicillin or chloramphenicol, 5 parts of sucrose, 5 portions of mannitol and 4 parts of skim milk.
Embodiment 6
A kind of culture presevation agent, the component containing following mass parts: 65 parts of glycerine, the card that 5 parts of concentration are 25mg/L that
Mycin, ampicillin or chloramphenicol, 5 parts of maltose, 5 portions of mannitol and 5 parts of skim milk.
Antibiotic kanamycins, ampicillin or chloramphenicol used in above-described embodiment be concentration be 20~
The liquid of 25mg/L, and preserving agent is added after 0.22 μm of membrane filtration degerming, kanamycins, ampicillin is specifically added
Or the selectivity of any plasmid carried depending on genetic engineering bacterium of chloramphenicol, it can be adjusted according to the actual situation.
Embodiment 7
The Recombinant organism that the culture presevation agent prepared using embodiment 5 produces pyruvic acid to a plant height carries out bacterium
Kind of preservation, used in antibiotic selective agent be concentration be 23mg/L ampicillin and each 2 parts of chloramphenicol, specific method step
It is rapid as follows:
By the Recombinant organism of high yield pyruvic acid 37 DEG C of overnight incubations of LB culture medium, take 10ml culture solution with
3000rpm is centrifuged 5~10min processing, vibrates centrifugation by magnetic force with 3~4ml culture presevation agent and is resuspended, and oscillation mixes
Bacteria suspension is moved into sterile freezing pipe afterwards, closes freezing pipe, is cooled down with the certain rate of temperature fall of -5 DEG C/min from 4 DEG C
To -80 DEG C, and the freezing at -80 DEG C.
Comparative example 1
A kind of culture presevation agent, the component containing following mass parts: 60 parts of DMSO, 5 parts of sucrose, 5 portions of mannitol and 4
The skim milk of part.
Comparative example 2
The difference of this comparative example and embodiment 7 is only that, this comparative example use that comparative example 1 provides without antibiotic
The culture presevation agent of selective agent.
One, rejuvenation survival rate and genetic engineering bacterium functional verification test
The Recombinant organism culture solution for the high yield pyruvic acid that embodiment 7 is cultivated, carries out before culture presevation
Bacterium colony counts, and culture solution is diluted to 10-7When, the average colony number of spread plate is 26；To this Recombinant organism
Normal fermentation culture is carried out, obtaining output of pyruvic acid is 65g/L；Through 7 culture collection process of embodiment -80 DEG C of freezings 1 year
Afterwards, room temperature rejuvenation is carried out, after passing on for 3 generations, with 37 DEG C of overnight incubations of LB culture medium to logarithmic growth phase, bacterium colony is carried out to culture solution
It counts, culture solution is diluted to 10-7When, the average colony number of spread plate is 25, using 7 culture collection process -80 of embodiment
DEG C after preservation 1 year, the Recombinant organism thallus high survival rate of rejuvenation is up to 96%；To this Recombinant organism
Normal fermentation culture is carried out, obtaining output of pyruvic acid is 61g/L, before the yield of the strain fermentation production pyruvic acid of rejuvenation is compared with preservation
Only have dropped 5%.This illustrates that the foreign gene that genetic engineering bacterium carries remains to efficiently play its function, not in culture presevation
It is lost in the process with rejuvenation.
And the high yield pyruvic acid Recombinant organism after using 2 method for preserving ultralow temperature preservation of comparative example 1 year
Rejuvenation survival rate is 82%, and the bacterial strain output of pyruvic acid after rejuvenation is 46g/L, and the output of pyruvic acid compared with bacterial strain before preservation has dropped
30%.It follows that the culture presevation agent provided by the invention containing antibiotic selective agent and method for preserving can be more efficient
Keep the bioactivity of strain, it is often more important that the exogenous DNA piece in genetic engineering bacterium by carryings such as vector plasmids can be prevented
The loss of section, guarantees that the corresponding function of genetic engineering bacterium does not decline because of ultralow temperature culture presevation.
Two, stability verification test
By using the 1 two kinds of culture presevation agent of embodiment 5 and comparative example at -80 DEG C freezing 1 year high yield pyruvic acid
Recombinant organism carry out rejuvenation, after passing on for 10 generations respectively, with 37 DEG C of overnight incubations of LB culture medium, to culture solution into
Row bacterium colony counts, and culture solution is diluted to 10-7When, the average colony number of spread plate is respectively 23 and 22, thallus survival rate
For 88% and 84%.The Recombinant organism for reaching for 10 generations after both rejuvenation carries out normal fermentation culture, using reality
The strain fermentation for applying 5 preserving agent preservation of example and rejuvenation obtains output of pyruvic acid for 60g/L, before the bacterial strain pyruvic acid of rejuvenation is compared with preservation
Only have dropped 7%；The strain fermentation of 1 preserving agent preservation of comparative example and rejuvenation is used to obtain output of pyruvic acid as 47g/L, rejuvenation
Bacterial strain pyruvic acid is compared with only having dropped 27% before preservation, thus comparison is it is found that have the strain of antibiotic selective agent using the present invention
Preserving agent, which carries out ultralow temperature preservation, can preferably maintain the stability of exogenous DNA in genetic engineering bacterium, repeatedly be passed on still not
It can lose.
Claims (10)
1. a kind of culture presevation agent, which is characterized in that the preserving agent contains the component of following mass parts: 50~70 parts of low temperature are protected
Protect agent and 2~5 parts of antibiotic selective agents.
2. a kind of culture presevation agent according to claim 1, which is characterized in that it is 10 parts that the preserving agent, which also contains mass parts,
Buffer, the buffer is made of oligosaccharide and mannitol 1:1 in mass ratio.
3. a kind of culture presevation agent according to claim 1 or claim 2, which is characterized in that it is 3 that the preserving agent, which also contains mass parts,
~5 parts of skim milk.
4. a kind of culture presevation agent according to claim 3, which is characterized in that the cryoprotective agent be glycerol, DMSO or
One of glycerine.
5. a kind of culture presevation agent according to claim 4, which is characterized in that the antibiotic selective agent be kanamycins,
The combination of one or more of ampicillin or chloramphenicol.
6. a kind of culture presevation agent according to claim 5, which is characterized in that the kanamycins, ampicillin or chlorine
Mycin is the liquid that concentration is 20~25mg/L, and adds preserving agent after 0.22 μm of membrane filtration degerming.
7. a kind of culture presevation agent according to claim 2, which is characterized in that the oligosaccharide is trehalose, sucrose or wheat
One of bud sugar.
8. a kind of method using any culture presevation agent superfreeze preservation genetic engineering bacterium of claim 1-7,
It is characterized in that, the method comprises the following steps: genetic engineering bacterium is taken training to logarithmic growth phase with 37 DEG C of overnight incubations of LB culture medium
Centrifugation is resuspended with culture presevation agent for nutrient solution centrifugal treating, and bacteria suspension is moved into sterile freezing pipe, closing freezing
Preservation pipe is cooled to -80 DEG C, and the freezing at -80 DEG C from 4 DEG C with certain rate of temperature fall.
9. a kind of method equal using culture presevation agent superfreeze preservation genetic engineering according to claim 8, special
Sign is, described to take medium centrifugal processing be that 10ml culture solution is taken to be centrifuged 5~10min with 3000rpm, described to use culture presevation
Centrifugation resuspension is to be vibrated centrifugation by magnetic force with 3~4ml culture presevation agent to be resuspended by agent, keeps strain evenly dispersed
In culture presevation agent.
10. a kind of method equal using culture presevation agent superfreeze preservation genetic engineering according to claim 8 or claim 9,
It is characterized in that, the rate of temperature fall is -1~-10 DEG C/min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811313646.XA CN109294917A (en) | 2018-11-06 | 2018-11-06 | A kind of culture presevation agent and the method using the preserving agent superfreeze preservation genetic engineering bacterium |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811313646.XA CN109294917A (en) | 2018-11-06 | 2018-11-06 | A kind of culture presevation agent and the method using the preserving agent superfreeze preservation genetic engineering bacterium |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109294917A true CN109294917A (en) | 2019-02-01 |
Family
ID=65145468
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811313646.XA Pending CN109294917A (en) | 2018-11-06 | 2018-11-06 | A kind of culture presevation agent and the method using the preserving agent superfreeze preservation genetic engineering bacterium |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109294917A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114292752A (en) * | 2021-12-31 | 2022-04-08 | 山东新时代药业有限公司 | Freeze-dried composition of recombinant escherichia coli strain and preparation method thereof |
| CN115838635A (en) * | 2022-10-08 | 2023-03-24 | 江西国药有限责任公司 | Long-term preservation method of paecilomyces hepiali Cs-4 strain for fermenting cordyceps sinensis powder |
-
2018
- 2018-11-06 CN CN201811313646.XA patent/CN109294917A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| 何国庆等: "《食品微生物学 第3版》", 30 September 2016 * |
| 北钠创联: "菌种和核酸样品保藏的目的和原理www.biaowu.com/html_news/News_18427.html", 《北京标准物质网》 * |
| 吴祖芳主编: "《现代食品微生物学》", 31 January 2017 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114292752A (en) * | 2021-12-31 | 2022-04-08 | 山东新时代药业有限公司 | Freeze-dried composition of recombinant escherichia coli strain and preparation method thereof |
| CN115838635A (en) * | 2022-10-08 | 2023-03-24 | 江西国药有限责任公司 | Long-term preservation method of paecilomyces hepiali Cs-4 strain for fermenting cordyceps sinensis powder |
| CN115838635B (en) * | 2022-10-08 | 2023-08-22 | 江西国药有限责任公司 | Long-term preservation method of paecilomyces hepiali Cs-4 strain for fermented cordyceps sinensis powder |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101264062A (en) | Production technology for preparing freeze-drying genetic engineering bacterium competence cell and protective agent formula | |
| US6610531B1 (en) | Viable dried bacteria produced by drying in the presence of trehalose and divalent cation | |
| Duilio et al. | Recombinant protein production in Antarctic Gram-negative bacteria | |
| CN109294917A (en) | A kind of culture presevation agent and the method using the preserving agent superfreeze preservation genetic engineering bacterium | |
| Day et al. | Cryo-injury in algae and the implications this has to the conservation of micro-algae | |
| CN110468050B (en) | Freeze-drying method for improving survival rate of lactobacillus plantarum by utilizing polysaccharide | |
| WO2001012779A1 (en) | Preservation of bacterial cells at ambient temperatures | |
| Spadaro et al. | Effect of culture age, protectants, and initial cell concentration on viability of freeze-dried cells of Metschnikowia pulcherrima | |
| Moussa et al. | Cell inactivation and membrane damage after long‐term treatments at sub‐zero temperature in the supercooled and frozen states | |
| Cota et al. | Effect of storage temperature on Streptococcus mutans viability | |
| CN109182129B (en) | Bacterium protective agent and application thereof | |
| CN109294916A (en) | A kind of culture presevation agent and the method using the preserving agent anaerobism freezing genetic engineering bacterium | |
| JPS61231994A (en) | Method for preserving acid producing bacteria and composition produced thereby | |
| Acker et al. | Engineering desiccation tolerance in mammalian cells: tools and techniques | |
| CN107058110B (en) | Preservation and recovery method of haematococcus pluvialis cells | |
| CN109055248A (en) | A kind of bio-control yeast bacterium activated freeze dried powder and preparation method thereof | |
| Sidyakina et al. | Viability and genetic stability of the bacterium Escherichia coli HB101 with the recombinant plasmid during preservation by various methods | |
| de Arruda Moura Pietrowski et al. | Viability of Hanseniaspora uvarum yeast preserved by lyophilization and cryopreservation | |
| US20030044965A1 (en) | Long term preservation and storage of viable dried bacteria | |
| Paredes | Vitrification and ultra-rapid laser warming of yeast Saccharomyces cerevisiae | |
| BG100105A (en) | Viable bacteria | |
| CN106867905A (en) | Preservative fluid and recombination bacillus coli fermentation process in high density for culture presevation | |
| Paul et al. | Transposon Mutagenesis of Listeria monocytogenes | |
| CN107881112A (en) | A kind of store method for turning Swine Hepatitis Virus Gene engineering bacteria | |
| Sleesman | Preservation of Phytopathogenic prokaryotes |
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 |