Fermentation production method for improving yield of customized functionalized single-stranded DNA
By combining the phagemid method with temperature control optimization during fermentation, the efficient preparation problem of customized functionalized single-strand DNA is solved, high yield and low cost production is achieved, and the application scope of DNA nanostructures is expanded.
Patent Information
- Application Number
- CN202410609758.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-05-16
- Publication Date
- 2025-08-01
AI Technical Summary
The prior art is difficult to efficiently prepare customized functionalized single-stranded DNA, and the production cost is high, limiting the large-scale application of DNA nanostructures in a wider field.
The phage pellet method is used to produce customized functionalized single-strand DNA, and the culture conditions are optimized during the fermentation process, such as promoting bacterial growth at the high temperature stage and adjusting to low temperature for auxiliary phage infection, combined with optimized fermentation medium and temperature control, efficient preparation is achieved.
It significantly improves the output of customized functionalized single-stranded DNA, reduces production costs, and lays the foundation for large-scale production and functional applications.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of fermentation, and more particularly to a fermentation production method for increasing the yield of customized functional single-stranded DNA (ssDNA). Specifically, it uses the phagemid method to produce customized functional ssDNA, and optimizes fermentation culture conditions such as culture medium and infection temperature, etc., to achieve efficient preparation of ssDNA. Background Art
[0002] DNA nanostructures are specific structures formed by self-assembling DNA molecules using the principle of base complementary pairing, which was initially proposed by Seeman in the 1980s. In 2006, the emergence of DNA Origami technology opened up a new way for the preparation of DNA nanostructures. Using a long single-stranded DNA as the "scaffold strand" and multiple short oligonucleotide "staple strands", they are folded into a predetermined geometric structure through the precise principle of base complementary pairing. These structures not only play a key role in basic scientific research, but also show great application potential and development prospects in practical applications. Currently, DNA nanostructures have been widely applied in many aspects such as drug delivery, tumor-targeted therapy, bioimaging, and biosensing.
[0003] DNA nanostructures are folded by the scaffold strand and staple strands through base complementary pairing. The staple strands mainly rely on chemical synthesis. Therefore, it is particularly important to increase the yield of the scaffold strand ssDNA. The genome of M13 phage has a stable single-stranded structure and an appropriate length, and is widely used as the scaffold strand for natural DNA Origami technology. After obtaining the circular single-stranded genome of M13 phage, adding chemically synthesized staple strands can assemble into nanostructures. In current research, high-yield ssDNA can be obtained through optimizing culture conditions and expanding the production scale. However, the final obtained ssDNA sequence and size are limited by the genome of M13 phage itself, which cannot meet the requirements of customizing ssDNA sequence and length. And a large amount of chemically synthesized staple strands need to be added when folding DNA nanostructures, increasing the production cost and limiting the large-scale application of DNA nanostructures in a wider range of fields.
[0004] In recent years, scientists have proposed that ssDNA can be produced using the phagemid method. Using the phagemid method can produce ssDNA with customized sequences and lengths, opening up a new way for constructing more complex and larger-sized DNA nanostructures, reducing the production cost, and expanding the application scope of DNA nanostructures. Currently, the maximum yield of producing customized ssDNA using the phagemid method at the shake flask level can reach 4 - 5 mg / L; by expanding the production scale through a bioreactor, the maximum yield obtained is 140 mg / L.
[0005] To further promote the application of DNA nanostructures, our research group successfully inserted functional aptamer sequences into ssDNA sequences without affecting the self-assembly of DNA nanostructures. After self-assembly, the resulting DNA nanostructures possess functions such as tumor targeting, expanding the application of DNA nanostructures in biomedicine and other fields. However, to date, the fermentation optimization of customized functionalized ssDNA has not been reported.
[0006] With the increasing demand for functionalized DNA nanostructures, the efficient and large-scale production of customized functionalized ssDNA has become a key issue that needs to be addressed in this field. This invention aims to establish a production process for efficiently preparing customized functionalized ssDNA while reducing production costs and energy consumption to meet application needs. Summary of the Invention
[0007] In light of this, the present invention provides a fermentation production method for increasing the yield of customized functionalized ssDNA. Specifically, this method utilizes a phagemid method to produce customized functionalized ssDNA and optimizes fermentation conditions, such as the culture medium and infection temperature, to achieve efficient ssDNA production. This method significantly increases the yield of customized functionalized ssDNA, offers simple process control, and is highly operable. It provides a theoretical basis for large-scale production of functionalized ssDNA and lays the foundation for the low-cost preparation and functionalized application of DNA nanostructures.
[0008] To achieve the above objectives, the present invention provides the following technical solutions:
[0009] (a) In the early stage of the fermentation process, the culture temperature is maintained at a high temperature (preferably, controlled within the range of 35°C to 40°C) to promote rapid growth of the bacteria;
[0010] (b) When the host bacteria grows to a certain cell density, adding helper phage to infect the host, and at the same time adjusting the culture temperature to a low temperature (preferably, controlled within the range of 25° C. to 33° C.) and continuing the culture;
[0011] (c) Isolation and purification of functionalized ssDNA from the fermentation broth.
[0012] In some specific embodiments, in step (b), adjusting the culture temperature to a low temperature is a quick process. By adjusting the incubator temperature or increasing the condensation water flow rate, the culture temperature can be adjusted to a low temperature (preferably, controlled within the range of 25°C to 33°C) in a shorter time.
[0013] In some specific embodiments, in step (a), in the early stage of the fermentation process, when culturing at the shake flask level, it refers to the time from inoculating the seed liquid and starting to culture for 2 - 2.5 h until the cell density reaches OD600 = 0.4 - 1; or when culturing in a 5 L bioreactor, it refers to the time from inoculating the seed liquid and starting to culture for 5 - 5.5 h until the cell density reaches OD600 = 17 - 19.
[0014] In some specific embodiments, in step (b), the certain cell density means that when culturing at the shake flask level, the cell density reaches OD600 = 0.4 - 1; or in a 5 L bioreactor, the cell density reaches OD600 = 17 - 19, and at this time, the helper phage is added for infection.
[0015] In some specific embodiments, when adding the helper phage, the helper phage infection is carried out at a multiplicity of infection of 1 - 15.
[0016] In some specific embodiments, the host bacterium is male Escherichia coli JM109 or E. coli XL1 - Blue, and the helper phage is VCSM13.
[0017] In some specific embodiments, the maximum yield of the customized functionalized ssDNA is: when culturing at the shake flask level, the maximum yield of the customized functionalized ssDNA is 20 mg / L; when culturing in a 5 L bioreactor, the maximum yield of the customized functionalized ssDNA is 200 mg / L.
[0018] The present invention provides a fermentation production method for increasing the yield of customized functionalized ssDNA, including: in the early stage of the fermentation process, maintaining the culture temperature at a high temperature (preferably, controlled within the range of 35°C - 40°C), when the host bacterium grows to the early exponential phase or when the host bacterium grows to a certain cell density (OD600 = 0.4 - 1 at the shake flask level; OD600 = 17 - 19 in a 5 L bioreactor), adding the helper phage for infection at a multiplicity of infection of 1 - 15, and at the same time adjusting the culture temperature to a low temperature (preferably, controlled within the range of 25°C - 33°C) and continuing the culture in a short time, and separating and purifying the functionalized ssDNA from the fermentation broth to increase the yield of the customized functionalized ssDNA (20 mg / L at the shake flask level; 200 mg / L in a 5 L bioreactor). The present invention greatly increases the yield of the functionalized ssDNA, reduces the production cost, has the characteristics of simple process control and strong operability, lays a foundation for the application of functionalized DNA nanostructures, and provides a theoretical basis for the large - scale production of functionalized ssDNA. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 A shows the cell growth conditions of two temperature control processes in 2×YT medium
[0020] Figure 1 B is the production curve of functionalized ssDNA under two temperature control processes in 2×YT medium
[0021] Figure 1 C is the agarose gel electrophoresis diagram of functionalized ssDNA at different time points under two temperature control processes in 2×YT medium
[0022] Figure 2 A is the growth of bacteria under two temperature control processes in TB medium
[0023] Figure 2 B is the production curve of functionalized ssDNA under two temperature control processes in TB medium
[0024] Figure 2 C is the agarose gel electrophoresis diagram of functionalized ssDNA at different time points under two temperature control processes in TB medium
[0025] Figure 3 A is the growth of bacteria under two temperature control processes in a 5L bioreactor
[0026] Figure 3 B is the production curve of functionalized ssDNA under two temperature control processes in a 5L bioreactor
[0027] Figure 3 C is the agarose gel electrophoresis diagram of functionalized ssDNA at different time points under two temperature control processes in a 5L bioreactor
[0028] Figure 3 D is the change curve of dissolved oxygen (DO) and oxygen uptake rate (OUR) during batch fermentation under two temperature control processes in a 5L bioreactor
[0029] Note: In the figure, "high temperature" refers to constant temperature culture at 35°C - 40°C; "low temperature" refers to continuing the culture at 25°C - 33°C after adding helper phage infection Detailed implementation manners
[0030] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention
[0031] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0032] Unless otherwise specified, 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 pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.
[0033] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.
[0034] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.
[0035] The relevant test materials and methods involved in the present invention:
[0036] 1 Test Materials
[0037] 1.1 Culture Media
[0038] 1.1.1 Seed Culture Medium: LB Medium: 10 g / L Tryptone, 5 g / L Yeast Extract, 10 g / L NaCl.
[0039] 1.1.2 2×YT Medium: 16 g / L Tryptone, 10 g / L Yeast Extract, 5 g / L NaCl.
[0040] 1.1.3 TB Medium: 12 g / L Tryptone, 24 g / L Yeast extract, 4 mL / L glycerol, 2.31 g / L KH2PO4, 12.54 g / L K2HPO4.
[0041] 1.1.4 Primary seed culture medium: LB medium: 10 g / L Tryptone, 5 g / L Yeast Extract, 10 g / L NaCl.
[0042] 1.1.5 Secondary seed culture medium and fermentation basal medium: 13.3 g / L KH2PO4, 4 g / L (NH4)2HPO4, 1.7 g / L citric acid, 6 g / L glucose, 1.2 g / L MgSO4·7H2O, 1 mmol / L thiamine hydrochloride, 15 mg / L MnCl2·4H2O, 3 mg / L H3BO3, 2.5 mg / L CoCl2·6H2O, 8.4 mg / L EDTA 2Na·2H2O, 60 mg / L ferric citrate, 1.5 mg / L CuCl2·2H2O, 2.5 mg / L Na2MoO4·2H2O, 8 mg / L Zn(CH3COO)2·2H2O.
[0043] 1.1.6 Feed medium: 750 g / L glucose, 20 g / L MgSO4×7H2O, 18.5 g / L (NH4)2SO4.
[0044] 1.2 Test strains and plasmids
[0045] 1.2.1 Host bacteria: Escherichia coli JM109 (purchased from Sangon Biotech (Shanghai) Co., Ltd.), Escherichia coli XL1-Blue (purchased from Shanghai Yuanye Bio-Technology Co., Ltd.).
[0046] 1.2.2 Helper phage: VCSM13 (purchased from Shanghai Rongmin Biotechnology Center).
[0047] 1.2.3 Phagemid: In the present invention, the phagemid is plasmid p3024-525-AS preserved by the National Biochemical Engineering Technology Research Center of East China University of Science and Technology. This plasmid carries a customized sequence that can self-assemble into a triangular DNA nanostructure and an aptamer sequence targeting tumor cells.
[0048] 2 Test methods
[0049] 2.1 Infection time: The cell density of Escherichia coli reaches OD ,
[0048] ,
[0052] ,
[0051] , , 600 ,
[0050] , ,
[0049] , = 0.4 - 1.
[0050] 2.2 Multiplicity of infection: 1 - 15.
[0051] 2.3 Culture temperature: High temperature (preferably controlled within the range of 35°C - 40°C); low temperature (preferably controlled within the range of 25°C - 33°C).
[0052] 2.4 Stirring rate: Shaking flask level: 150 - 250 rpm; 5L bioreactor: 400 - 1000 rpm.
[0053] 2.5 Medium pH value: 6.5 - 7.5.
[0054] 2.6 Dissolved oxygen (DO): 10% - 25%.
[0055] 2.7 Aeration rate: 3.0 - 6.0 L / min.
[0056] During the cultivation in a 5L bioreactor, the stirring speed and aeration rate are automatically adjusted through its control system; the amount of acid and base added is automatically controlled to keep the pH value in the tank at the set value. Samples are taken every two hours to detect the OD value of the bacterial solution, and the customized functional ssDNA is isolated and purified according to the extraction method described in "Molecular Cloning Experiment Guide", and the relevant data is recorded. 600 value, and the customized functional ssDNA is isolated and purified according to the extraction method described in "Molecular Cloning Experiment Guide", and the relevant data is recorded.
[0057] The present invention will be further elaborated below in conjunction with some specific embodiments:
[0058] Example 1 Using 2×YT medium at shaking flask level to reduce the cultivation temperature and increase the yield of functional ssDNA
[0059] 1 Purpose: To verify whether reducing the cultivation temperature during the cultivation process will increase the yield of customized functional ssDNA compared with cultivating in a high-temperature environment in 2×YT medium.
[0060] 2 Experimental results: The results are shown in Figure 1 .
[0061] 3 Result analysis:
[0062] As Figure 1 can be seen, when the cultivation temperature is constant within the high-temperature range, the growth of the bacteria is faster, and the maximum cell density is OD 600 = 4.5; when the cultivation reaches 2 - 2.5 h, after adding the helper phage for infection and then adjusting the cultivation temperature to low temperature, the growth rate of the bacteria is lower, and it enters the stationary phase at the 14th h of cultivation, and the maximum cell density is OD 600 = 5.5.
[0063] The yield curve of customized functional ssDNA at different cultivation temperatures is as shown in Figure 1 . When the cultivation temperature is constant within the high-temperature range, the maximum yield of customized functional ssDNA is 10 mg / L; when the cultivation reaches 2 - 2.5 h, after adding the helper phage for infection and then adjusting the cultivation temperature to low temperature, the yield of customized functional ssDNA increases, and the maximum yield can reach 12 mg / L. Compared with the high-temperature constant cultivation, the yield of functional ssDNA increases by 20%.
[0064] Example 2: Using TB medium at the shake flask level to reduce the culture temperature and increase the yield of functionalized ssDNA
[0065] 1 Objective: To investigate whether lowering the culture temperature during culturing in TB medium can increase the yield of customized functionalized ssDNA compared to culturing in a high temperature environment.
[0066] 2 Experimental results: See Figure 2
[0067] 3. Result analysis:
[0068] From the growth curve Figure 2 (A) It can be seen that in TB medium, there is no significant difference in bacterial growth rate under the two different culture temperature conditions. However, when cultured in a high temperature environment for 12 hours, the maximum bacterial density is reached. At this time, OD 600 =7.8; at the 2nd to 2.5th hour of culture, add helper phage for infection and adjust the culture temperature to a low temperature. At the 16th hour of culture, the culture enters the stable period and the maximum cell density can reach OD 600 =11.5. According to the yield curve Figure 2 (B) It can be concluded that the maximum yield of customized functionalized ssDNA in TB medium during constant temperature culture was approximately 17 mg / L. After adding helper phage infection and adjusting the culture temperature to a lower temperature at 2-2.5 hours of culture, the maximum yield of customized functionalized ssDNA in TB medium reached approximately 20 mg / L. Under the same conditions, this yield was approximately 66.7% higher than the yield of customized functionalized ssDNA in 2×YT medium (12 mg / L).
[0069] Example 3 Optimized temperature control process was verified in a 5L bioreactor
[0070] 1. Objective: To apply the optimized temperature control process in a 5L bioreactor to expand the culture scale and increase the yield of customized functionalized ssDNA by lowering the culture temperature compared with high-temperature constant-temperature culture.
[0071] 2 Experimental results: See Figure 3 .
[0072] 3. Result analysis:
[0073] By comparing the shake flask level, it was found that adjusting the culture temperature to a low temperature environment after adding helper phage infection can effectively promote the packaging of phagemids and significantly increase the yield of customized functionalized ssDNA. Therefore, the effect of lowering the culture temperature on the yield of customized functionalized ssDNA was further investigated in a 5L reactor. In the 5L bioreactor, when the cell density reached OD 600= 17 - 19 h (cultured for 5 - 5.5 h), add helper phage for infection at a multiplicity of infection of 1 - 15, and at the same time adjust the culture temperature to low temperature and continue culturing.
[0074] The curves of bacterial growth and the yield of customized functional ssDNA are as Figure 3 shown. When the culture temperature was adjusted to low temperature after adding helper phage for infection, the growth of bacteria was promoted, and it entered the stationary phase at 14 h of culture. The maximum bacterial density could reach OD 600 = 78, and the maximum bacterial density increased by 95% compared with that in high-temperature constant-temperature culture. According to the online parameter curve Figure 3 D, it can be seen that when the culture temperature was adjusted to low temperature at 5 - 5.5 h of culture, the dissolved oxygen (DO) increased and the oxygen uptake rate (OUR) decreased.
[0075] From the curve of the yield of customized functional ssDNA, it can be seen that in high-temperature constant-temperature culture, the maximum yield was 150 mg / L; when helper phage was added for infection at 5 - 5.5 h of culture and the culture temperature was adjusted to low temperature at the same time, the yield of customized functional ssDNA was increased to 200 mg / L. The yield of customized functional ssDNA increased by about 30% compared with that in high-temperature constant-temperature culture; compared with the shake flask level (TB medium, 20 mg / L), the yield of customized functional ssDNA increased by 10 times.
[0076] In summary, after adding helper phage for infection, adjusting the culture temperature to low temperature (preferably, within the range of 25°C - 33°C) can effectively increase the yield of customized functional ssDNA, proving that the temperature control in the fermentation process provided by the present invention can significantly increase the yield of customized functional ssDNA.
[0077] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.
Claims
1. A fermentation production method for increasing the yield of customized functional single-stranded DNA, characterized in that, It includes the following parts: (a) In the early stage of the fermentation process, maintain the culture temperature at a high temperature (preferably, control it within the range of 35°C to 40°C) to promote the rapid growth of the host; (b) When the host bacteria grow to a certain cell density, add helper phage to infect the host, and at the same time adjust the culture temperature to a low temperature (preferably, control it within the range of 25°C to 33°C) and continue the culture; (c) Isolate and purify the functionalized ssDNA from the fermentation broth.
2. According to the preparation method described in claim 1, characterized in that, In step (b), adjusting the culture temperature to a low temperature is a rapid process. By adjusting the temperature of the incubator or increasing the flow rate of the condensed water, the culture temperature is adjusted to a low temperature (preferably, control it within the range of 25°C to 33°C) within a short time.
3. The preparation method according to claim 1, characterized in that, In step (a), the early stage of the fermentation process refers to the period during shake flask culture when, starting from the inoculation of the seed culture, it is necessary to culture for 2 to 2.5 hours until the cell density reaches OD 600 = 0.4 to 1; or during culture in a 5 L bioreactor, starting from the inoculation of the seed culture, it is necessary to culture for 5 to 5.5 hours until the cell density reaches OD 600 = 17 to 19.
4. According to the preparation method described in claim 1, characterized in that, In step (b), the certain cell density refers to that when cultured at the shake flask level, the cell density reaches OD 600 = 0.4 - 1; or in a 5 L bioreactor, the cell density reaches OD 600 = 17 - 19, and at this time, the helper phage is added for infection.
5. The preparation method according to claim 4, characterized in that, When adding the helper phage, the helper phage infects at a multiplicity of infection of 1 to 15.
6. According to the preparation method described in claim 1, it is characterized in that, The host bacteria are male Escherichia coli JM109 or E. coli XL1-Blue, and the helper phage is VCSM13.
7. According to the preparation method described in claim 1, characterized in that, The maximum yield of the customized functionalized ssDNA is: when cultured at the shake flask level, the maximum yield of the customized functionalized ssDNA is 20 mg / L; when cultured in a 5 L bioreactor, the maximum yield of the customized functionalized ssDNA is 200 mg / L.
8. A fermentation production method for increasing the yield of customized functional ssDNA, characterized in that, During the fermentation process, when the host bacteria grow to the early exponential phase or when the host bacteria grow to a certain cell density (OD 600 = 0.4 - 1 at the shake flask level; OD 600 = 17 - 19 in a 5 L bioreactor), infect with helper phage at a multiplicity of infection of 1 - 15, and at the same time adjust the culture temperature to a low temperature within a short period of time (preferably, controlled within the range of 25°C - 33°C), so as to increase the yield of customized functional ssDNA (20 mg / L at the shake flask level; 200 mg / L in a 5 L bioreactor).