CN113817609A - Freeze-drying preservation method of genetically engineered bacteria - Google Patents
Freeze-drying preservation method of genetically engineered bacteria Download PDFInfo
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- CN113817609A CN113817609A CN202111282307.1A CN202111282307A CN113817609A CN 113817609 A CN113817609 A CN 113817609A CN 202111282307 A CN202111282307 A CN 202111282307A CN 113817609 A CN113817609 A CN 113817609A
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- 241000894006 Bacteria Species 0.000 title claims abstract description 34
- 238000004108 freeze drying Methods 0.000 title claims abstract description 33
- 238000004321 preservation Methods 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 27
- 230000001580 bacterial effect Effects 0.000 claims abstract description 29
- 239000007788 liquid Substances 0.000 claims abstract description 27
- 239000001963 growth medium Substances 0.000 claims abstract description 15
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 14
- 230000031700 light absorption Effects 0.000 claims abstract description 12
- 238000009630 liquid culture Methods 0.000 claims abstract description 11
- 239000000243 solution Substances 0.000 claims abstract description 11
- 238000007710 freezing Methods 0.000 claims abstract description 10
- 230000008014 freezing Effects 0.000 claims abstract description 10
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 9
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 8
- 239000008103 glucose Substances 0.000 claims abstract description 8
- 239000013612 plasmid Substances 0.000 claims abstract description 8
- 238000005086 pumping Methods 0.000 claims abstract description 8
- 241001494508 Arundo donax Species 0.000 claims abstract description 6
- 241000588724 Escherichia coli Species 0.000 claims abstract description 6
- 235000014676 Phragmites communis Nutrition 0.000 claims abstract description 6
- 238000007664 blowing Methods 0.000 claims abstract description 5
- 239000011259 mixed solution Substances 0.000 claims abstract description 5
- 230000003321 amplification Effects 0.000 claims abstract description 4
- 238000005119 centrifugation Methods 0.000 claims abstract description 4
- 239000013604 expression vector Substances 0.000 claims abstract description 4
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 4
- 239000006228 supernatant Substances 0.000 claims abstract description 4
- 241001052560 Thallis Species 0.000 claims abstract description 3
- 239000011248 coating agent Substances 0.000 claims abstract description 3
- 238000000576 coating method Methods 0.000 claims abstract description 3
- 239000013049 sediment Substances 0.000 claims abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 239000008223 sterile water Substances 0.000 claims description 5
- 238000002835 absorbance Methods 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 3
- 239000008188 pellet Substances 0.000 claims description 2
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 8
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 5
- 230000004083 survival effect Effects 0.000 description 4
- 239000012530 fluid Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000003223 protective agent Substances 0.000 description 3
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 2
- 101000794603 Paramacrobiotus richtersi Cytosolic-abundant heat soluble protein 106094 Proteins 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- 241000282452 Ailuropoda melanoleuca Species 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 101000637488 Hypsibius dujardini Secretory-abundant heat soluble protein 33020 Proteins 0.000 description 1
- 208000025174 PANDAS Diseases 0.000 description 1
- 208000021155 Paediatric autoimmune neuropsychiatric disorders associated with streptococcal infection Diseases 0.000 description 1
- 240000004718 Panda Species 0.000 description 1
- 235000016496 Panda oleosa Nutrition 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
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- 239000013078 crystal Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
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- 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
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- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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Abstract
The invention discloses a freeze-drying preservation method of genetic engineering bacteria, which comprises the following steps: constructing a gene sequence of the giant reed TDP protein on a pET-28a expression vector to obtain a recombinant plasmid into which a sequence for coding the giant reed TDP protein is introduced; transferring the recombinant plasmid into an escherichia coli competent cell to obtain a transformed bacterial liquid, and coating the bacterial liquid on an LB culture medium; selecting the monoclonal colony cultured on the previous day, inoculating the monoclonal colony into a liquid culture medium for amplification culture, detecting the light absorption value of a bacterial liquid in the liquid culture medium, and obtaining the bacterial liquid to be operated, wherein the light absorption value at the wavelength of 600nm is 0.4-0.6; centrifuging the to-be-operated bacterial liquid with 10^9 cells per milliliter according to the light absorption value of the to-be-operated bacterial liquid, removing supernatant after centrifugation is finished, and blowing off thalli sediment by using glucose solution to obtain mixed liquid; and transferring the mixed solution into a test tube, putting the test tube into a freeze dryer, and sequentially freezing and vacuum-pumping to finish freeze-drying preservation.
Description
Technical Field
The invention relates to the technical field of strain preservation, in particular to a freeze-drying preservation method of genetically engineered bacteria.
Background
In the prior art, the engineering bacteria are characterized in that the original strains have and have the ability to have the form-specific functions by introducing recombinant plasmids by utilizing a synthetic biology means so as to meet the production and living needs. In the past, most of engineering bacteria need to perform functions in a normal-temperature environment in application scenes, but the most extensive strain preservation scheme cannot meet the requirement, and a means for normal-temperature preservation needs to be explored. The normal temperature preservation needs to change the existence form of the strains, namely the most extensive frozen state formed by the ultra-low temperature refrigeration of the mixed solution of the glycerol and the bacteria is changed into a dry powder form. The preparation of dry powder requires the utilization of freeze-drying technology. However, for engineering bacteria still having application requirements, the environmental conditions of the freeze-drying process are extreme, and the bacteria alone are difficult to survive in freeze-drying, so the survival rate is greatly reduced, and the effect of the engineering bacteria needed to be applied next step is also affected. Therefore, the invention aims to improve the stress resistance of the strain to freeze-drying by means of genetic engineering while realizing normal-temperature preservation by freeze-drying.
At present, the conventional strain preservation method is mainly an ultra-low temperature preservation method. As a method generally selected in each laboratory, the ultra-low temperature preservation method requires glycerin and a-80 ℃ refrigerator, which are not available in the ordinary user's home, and thus the method is not feasible.
According to the technical scheme of application patent publication No. CN101264062A, common skim milk, trehalose and the like are selected as the protective agents in the freeze-drying process, so that the engineering bacteria competent cells are stored at the low temperature of-20-4 ℃, but the room-temperature storage is not realized, and the selected freeze-drying protective agents still have the risk of damaging cell membranes by forming ice crystals in the freezing process.
According to the technical scheme of the application patent publication No. CN109055227A, the survival rate of the escherichia coli BL21(DE3) cells after freeze-drying is maintained to a certain extent, but the selected protective agent is formed by mixing a strain fermentation product, human serum albumin and the like, the production is not suitable for mass production, and the cost is high.
The survival rate of the prior engineering bacteria after freeze-drying is basically maintained at about 10 percent and is at a lower level, which is not beneficial to the practical application of the prior engineering bacteria.
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
Disclosure of Invention
For the above reasons, the applicant proposes a freeze-drying preservation method of genetically engineered bacteria, aiming at solving at least one of the above problems.
In order to meet the requirements, the invention aims to provide a freeze-drying preservation method of genetically engineered bacteria, which comprises the following steps:
constructing a gene sequence of the giant reed TDP protein on a pET-28a expression vector to obtain a recombinant plasmid into which a sequence for coding the giant reed TDP protein is introduced;
transferring the recombinant plasmid into an escherichia coli competent cell to obtain a transformed bacterial liquid, and coating the bacterial liquid on an LB culture medium;
selecting a monoclonal bacterial colony cultured on the previous day, inoculating the monoclonal bacterial colony into a liquid culture medium for amplification culture, detecting the light absorption value of bacterial liquid in the liquid culture medium, and obtaining the bacterial liquid to be operated, wherein the light absorption value at the wavelength of 600nm is 0.4-0.6;
centrifuging the bacteria liquid to be operated with 10^9 cells per milliliter according to the light absorption value of the bacteria liquid to be operated, removing supernatant after centrifugation is finished, and blowing off thalli sediment by using glucose solution to obtain mixed liquid;
and transferring the mixed solution into a test tube, putting the test tube into a freeze dryer, and sequentially freezing and vacuum-pumping to finish freeze-drying preservation.
In some embodiments of the invention, the species of E.coli competent cell is BL21(DE 3).
In some embodiments of the present invention, the step of detecting the absorbance of the bacterial fluid in the liquid culture medium comprises measuring the absorbance of the bacterial fluid in the liquid culture medium a plurality of times at intervals.
In some embodiments of the invention, the step of centrifuging the bacterial fluid to be treated at 10^9 cells per ml comprises centrifuging at 4000rpm/min for 3-6 minutes.
In some embodiments of the invention, the step of aspirating the bacterial pellet with a glucose solution comprises aspirating 100 microliters of a 0.3% glucose solution.
In some embodiments of the invention, the tube is a 15ml tube.
In some embodiments of the invention, the sequential freezing and vacuum drying step comprises freezing at-50 ℃ to-90 ℃ for 1-3 hours.
In some embodiments of the present invention, the step of sequentially freezing and vacuum-pumping further comprises vacuum-pumping under a pressure of 1Pa for 6-12 hours.
In some embodiments of the invention, the method further comprises adding sterile water to the sample that has been subjected to lyophilization preservation to dissolve the bottom solid to complete the resuscitation.
In some embodiments of the invention, the sterile water has a volume of 0.5 to 1.5 ml.
Compared with the prior art, the invention has the beneficial effects that: with the protocol proposed in this application, the use of the inherent disordered proteins (TDPs) of the water bombesia makes it possible to protect different strains of escherichia coli under lyophilisation conditions and to allow the subsequent storage of the bacteria at room temperature.
The invention is further described below with reference to the accompanying drawings and specific embodiments.
Drawings
FIG. 1 is a schematic flow chart of the freeze-drying preservation method of the genetically engineered bacteria of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above should not be understood to necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples described in this specification can be combined and combined by those skilled in the art.
Examples
As shown in fig. 1:
step S101: the gene sequence of the giant panda TDP protein is constructed on a pET-28a expression vector, then the recombinant plasmid is transferred into Escherichia coli BL21(DE3) cells, and the transformed bacterial liquid is taken from an LB culture medium and coated.
In certain embodiments, the gene sequence of the panda TDP protein of this step may be replaced with CAHS 106094 protein, loss of viability during lyophilization with CAHS 106094 protein shows the most significant protective effect, and it may be co-expressed with SAHS 33020 protein to further improve bacterial survival.
S102, selecting the monoclonal colony cultured on the previous day, inoculating the monoclonal colony into a liquid culture medium for amplification culture, measuring the light absorption value at intervals, and performing subsequent operation on the bacterial liquid with the light absorption value at the wavelength of 600nm being within the range of 0.4-0.6.
Step S103: converting according to the value of the light absorption value of the bacterial liquid at the wavelength of 600nm, centrifuging 10^9 cells per mL of bacterial liquid at 4000rpm/min for 5 minutes, pouring out the supernatant after the centrifugation is finished, blowing 100 microliters of 0.3% glucose solution to the bacterial precipitation, and transferring the mixed solution into a 15mL tube.
Step S104: the sample is put into a freeze dryer and frozen for 2 hours at-70 ℃, and then vacuum-dried for 6-12 hours under the condition of 1 Pa.
Step S105: the bottom solid was dissolved by adding 1mL of sterile water and the freeze dried samples were resuscitated.
Various other modifications and changes may be made by those skilled in the art based on the above-described technical solutions and concepts, and all such modifications and changes should fall within the scope of the claims of the present invention.
Claims (10)
1. A freeze-drying preservation method of genetically engineered bacteria is characterized by comprising the following steps:
constructing a gene sequence of the giant reed TDP protein on a pET-28a expression vector to obtain a recombinant plasmid into which a sequence for coding the giant reed TDP protein is introduced;
transferring the recombinant plasmid into an escherichia coli competent cell to obtain a transformed bacterial liquid, and coating the bacterial liquid on an LB culture medium;
selecting a monoclonal bacterial colony cultured on the previous day, inoculating the monoclonal bacterial colony into a liquid culture medium for amplification culture, detecting the light absorption value of bacterial liquid in the liquid culture medium, and obtaining the bacterial liquid to be operated, wherein the light absorption value at the wavelength of 600nm is 0.4-0.6;
centrifuging the bacteria liquid to be operated with 10^9 cells per milliliter according to the light absorption value of the bacteria liquid to be operated, removing supernatant after centrifugation is finished, and blowing off thalli sediment by using glucose solution to obtain mixed liquid;
and transferring the mixed solution into a test tube, putting the test tube into a freeze dryer, and sequentially freezing and vacuum-pumping to finish freeze-drying preservation.
2. The freeze-drying preservation method of genetically engineered bacteria of claim 1, wherein the type of the competent cells of Escherichia coli is BL21(DE 3).
3. The freeze-drying preservation method for genetically engineered bacteria according to claim 1, wherein the step of detecting the absorbance of the bacteria liquid in the liquid culture medium comprises measuring the absorbance of the bacteria liquid in the liquid culture medium a plurality of times at intervals.
4. The freeze-drying preservation method of the genetically engineered bacteria of claim 1, wherein the step of centrifuging the bacteria solution to be manipulated at 10^9 cells per ml comprises centrifuging at 4000rpm/min for 3-6 minutes.
5. The method for freeze-drying preservation of genetically engineered bacteria according to claim 1, wherein the step of blowing off the bacterial pellet with the glucose solution comprises taking 100 μ l of 0.3% glucose solution.
6. The freeze-drying preservation method of the genetically engineered bacteria according to claim 1, wherein the test tube is a 15ml test tube.
7. The freeze-drying preservation method for the genetically engineered bacteria according to claim 1, wherein the steps of freezing and vacuum-pumping in sequence comprise freezing at-50 ℃ to-90 ℃ for 1-3 hours.
8. The freeze-drying preservation method for genetically engineered bacteria according to claim 7, wherein the step of sequentially freezing and vacuum-pumping further comprises vacuum-pumping under a pressure of 1Pa for 6-12 hours.
9. The freeze-drying preservation method of the genetically engineered bacteria according to claim 1, characterized in that the method further comprises adding sterile water to the sample subjected to freeze-drying preservation to dissolve the bottom solid to complete the resuscitation.
10. The freeze-drying preservation method for genetically engineered bacteria according to claim 9, wherein the volume of the sterile water is 0.5-1.5 ml.
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| CN202111282307.1A CN113817609A (en) | 2021-11-01 | 2021-11-01 | Freeze-drying preservation method of genetically engineered bacteria |
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| CN202111282307.1A CN113817609A (en) | 2021-11-01 | 2021-11-01 | Freeze-drying preservation method of genetically engineered bacteria |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0259739A1 (en) * | 1986-09-10 | 1988-03-16 | Rhone-Poulenc Inc. | Improved stability of freeze-dried cultures |
| US20020164771A1 (en) * | 1997-02-12 | 2002-11-07 | Invitrogen Corporation | Methods for lyophilizing competent cells |
| CN109055227A (en) * | 2018-09-27 | 2018-12-21 | 广州市金因源生物技术有限公司 | The protective agent and its method for preserving of genetic engineering bacterium strain freeze-drying preservation |
-
2021
- 2021-11-01 CN CN202111282307.1A patent/CN113817609A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0259739A1 (en) * | 1986-09-10 | 1988-03-16 | Rhone-Poulenc Inc. | Improved stability of freeze-dried cultures |
| US20020164771A1 (en) * | 1997-02-12 | 2002-11-07 | Invitrogen Corporation | Methods for lyophilizing competent cells |
| CN109055227A (en) * | 2018-09-27 | 2018-12-21 | 广州市金因源生物技术有限公司 | The protective agent and its method for preserving of genetic engineering bacterium strain freeze-drying preservation |
Non-Patent Citations (2)
| Title |
|---|
| CHERIE HESGROVE ET AL.,: ""The biology of tardigrade disordered proteins in extreme stress tolerance"", 《CELL COMMUNICATION AND SIGNALING》, vol. 18, no. 178 * |
| THOMAS C. BOOTHBY ET AL.,: ""Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation"", 《MOL CELL》, vol. 65, no. 6, pages 979 - 982 * |
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