Fermentation medium for efficiently producing single-stranded DNA and DNA nanostructure by phagocytosis method and application of fermentation medium

Abstract

The invention discloses a fermentation medium for efficiently producing single-stranded DNA and a DNA nanostructure by a phagocytosis method and application of the fermentation medium, and relates to the technical field of biology. The fermentation culture medium is obtained by using ammonium chloride as a single inorganic nitrogen source to replace a nitrogen source (NH4) 2HPO4 in a Riesenberg culture medium. The invention also provides a method for efficiently producing single-stranded DNA (ssDNA) and a DNA nanostructure by a phagocytic grain method, high yield of the ssDNA can be promoted by adopting the fermentation culture medium, a nitrogen source is optimized and screened through the culture medium, and the invention finds that the yield of the single-stranded DNA can be increased by using ammonium chloride as a unique inorganic nitrogen source in the fermentation culture medium for production, and the yield of the single-stranded DNA can be increased by using the ammonium chloride as the unique inorganic nitrogen source in the fermentation culture medium. The yield of single-stranded DNA with triangular, hairpin rectangular, rod-like and quadrilateral DNA structures is increased to different extents by the fermentation culture medium, and a theoretical basis is provided for large-scale production of different types of ssDNA and DNA structures.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a fermentation medium for the efficient production of single-stranded DNA and DNA nanostructures using the phage particle method, and its application. Background Technology

[0002] With the widespread application of single-stranded DNA (ssDNA) and DNA nanostructures in gene therapy, nanotechnology, and other fields, scientists have an increasing demand for efficient production of customizable single-stranded DNA. Currently, common ssDNA synthesis methods mainly fall into three categories: chemical synthesis, enzymatic synthesis, and bio-fermentation. Chemical synthesis is the most widely used, as it allows for template-free ssDNA synthesis, providing a powerful tool for generating customized ssDNA sequences. However, this method is costly and is often used for shorter ssDNA sequences. Enzymatic synthesis can be further divided into enzymatic degradation, reverse transcription, asymmetric PCR, and rolling circle amplification (RAC), based on its principles and operational methods. Enzymatic synthesis of ssDNA is often cumbersome and suffers from low recovery efficiency during gel extraction. Compared to other methods, bio-based methods offer higher production efficiency and lower costs. Bio-fermentation utilizes the replication system of living cells to synthesize ssDNA, significantly reducing synthesis costs and making it the preferred method for large-scale ssDNA production. Bio-fermentation methods include phage synthesis and phage particle synthesis. Phage synthesis involves packaging target ssDNA into filamentous phages, which are then secreted extracellularly. Removing the protein coat yields pure ssDNA. Phage synthesis solved the problems of low production efficiency and high cost of ssDNA, laying the foundation for large-scale ssDNA production. However, the sequence and length of the ssDNA produced are essentially fixed, making user customization impossible and significantly limiting efficient ssDNA preparation. Therefore, scientists developed a method for producing customized ssDNA using phage particles. First, dsDNA ori, ssDNA ori, an antibiotic resistance gene, and a single-stranded DNA (scaffold strand) of a customized length and sequence are linked into a phage particle, which is then transformed into *E. coli*. Helper phages are used for infection. Once the helper phages enter *E. coli*, they preferentially initiate the replication of the ssDNA ori within the phage particle, forming a single strand. This single strand is then combined with the phage protein coat to form a phage particle, which is ultimately secreted extracellularly. Compared to the phage method, the phage particle method can produce ssDNA with different sequences and lengths, breaking through the size limitations of traditional ssDNA production based on the M13 phage genome, and providing a new approach for the efficient production of customizable ssDNA.

[0003] Currently, the maximum yield of ssDNA produced using the phage particle method at the shake-flask level can reach 4-5 mg / L; by scaling up production in a bioreactor, the maximum yield obtained is 140 mg / L. With the rapid development of cutting-edge fields such as gene editing, synthetic biology, and nucleic acid drugs, the demand for high-quality, large-scale ssDNA will continue to grow. Therefore, the large-scale and efficient preparation of ssDNA has become a critical issue that urgently needs to be addressed. Summary of the Invention

[0004] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide a fermentation medium for the efficient production of single-stranded DNA and DNA nanostructures by phage particle method and its application. This invention aims to determine the nitrogen source of the fermentation medium used in the production of ssDNA in order to improve the yield of ssDNA to meet application requirements. Specifically, this invention provides a culture medium formulation that can significantly improve the yield of different single-stranded DNA and DNA nanostructures and its application, specifically using the phage particle method to produce ssDNA, and achieving efficient preparation of ssDNA by optimizing the nitrogen source composition in the fermentation medium.

[0005] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a fermentation medium for the efficient production of single-stranded DNA and DNA nanostructures by phage particle method, wherein the fermentation medium is Riesenberg medium, and ammonium chloride is used as a single inorganic nitrogen source to replace the original nitrogen source (NH4)2HPO4 in Riesenberg medium.

[0006] Optionally, the concentration of ammonium chloride in the fermentation medium is 1.25 g / L to 5.25 g / L.

[0007] Optionally, the fermentation medium has the following components: 13.3 g / L KH2PO4, 3.25 g / L ammonium chloride, 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 2 Na • 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.

[0008] Secondly, the present invention provides an application of the fermentation medium described above in the efficient production of single-stranded DNA and DNA nanostructures using the phage particle method.

[0009] Thirdly, this invention provides a method for efficiently producing single-stranded DNA and DNA nanostructures using phage plasmids, comprising the following steps: Step 1: Transform the phage particles into the host bacteria to obtain host bacteria carrying the phage particles; Step 2: Add the host bacteria carrying the phage particles to the seed culture medium and culture them to prepare the seed solution; Step 3: Add the seed liquid to the fermentation medium for culturing; Step 4: When the host bacteria carrying the phage particles reach the preset cell density, helper phages are added for infection. Step 5: After culturing and lysis, single-stranded DNA and DNA nanostructures are obtained.

[0010] Optionally, step one includes the following specific steps: Phage particles were added to DH5α competent cells and mixed well. After being placed on ice for 30 min, the cells were heat-shocked at 42°C for 60 s to allow the phage particles to enter E. coli. After being placed on ice again for 5 min, antibiotic-free medium was added and the cells were cultured at 37°C for 1 h. The cultured bacterial solution was spread onto a solid medium plate containing the antibiotic ampicillin and incubated upside down at 37°C for 12 h. After clearly visible single colonies grew on the plate, single colonies were picked and cultured. The cultured bacterial solution after 6 h was sent for sequencing verification. After confirming that the sequencing results were correct by comparing them with the sequence of the correct phage particles, the bacterial strain containing the correct phage particles was inoculated into 5 mL of medium containing ampicillin and cultured at 37°C for 12 h. The phage particles were then extracted using a plasmid extraction kit and transformed into the host bacteria to obtain host bacteria carrying phage particles.

[0011] Optionally, step two includes the following specific steps: The host bacteria carrying the phage particles were added to the seed culture medium and cultured at 200 rpm and 37°C for 8-10 h to obtain the seed solution. Step three includes the following specific steps: Add the seed culture to the fermentation medium, calculate the inoculum size, and ensure the initial OD... 600 =0.03, incubated in a shaker at 250 rpm and 37°C.

[0012] Optionally, step four includes culturing in a shake flask or a bioreactor, wherein: When culturing in shake flasks, step four includes the following specific steps: When culturing in shake flasks, the bacterial cell density reaches OD200 after 2-2.5 hours following inoculation of the seed culture. 600When the infection ratio is 0.4~1, helper phages are added at a multiplicity of infection of 10 for infection. When culturing in a bioreactor, step four includes the following steps: The culture medium from step two was inoculated into a 5 L bioreactor. The dissolved oxygen level was controlled at 10%–25% and the aeration rate at 3.0–6.0 L / min. Culture was carried out for 5–5.5 h after inoculation with the culture medium from step two, until the cell density reached OD200. 600 =17~19 hours, with helper phages added at multiplicity 1 for infection.

[0013] Step five includes the following specific steps: After fermentation, the fermentation broth was transferred to a centrifuge tube, centrifuged, and the supernatant was collected. A mixed solution of PEG 8000 and PEG 1 was added to the supernatant and incubated on ice. The supernatant was discarded by centrifugation, and 200 µl of TE buffer was added to the centrifuge tube to resuspend the precipitate. The residual cell precipitate was removed by centrifugation, and the supernatant was transferred to a new centrifuge tube. Add lysis buffer PPB2 to the supernatant to lyse the phage particles, mix well, and incubate. Add neutralization buffer PPB3 to neutralize the solution and mix well. Incubate on ice, centrifuge, transfer the supernatant to a new centrifuge tube, repeat centrifugation, and transfer to a new centrifuge tube again. Add anhydrous ethanol to the supernatant, mix well, and incubate on ice. Centrifuge to remove the supernatant. Add 75% ethanol to wash, mix well, centrifuge to discard the supernatant, and allow to evaporate at room temperature until there is no alcohol odor. Add sterile water to the centrifuge tube to dissolve the single-stranded DNA, obtaining single-stranded DNA. The single-stranded DNA was prepared to a final concentration of 50 nM in a buffer containing 50 mM HEPES, 100 mM NaCl and 4 mM ZnCl2. The pH was adjusted to 7.0 and the mixture was incubated overnight at 37°C for 12 h. After enzyme digestion, the target fragment was recovered. The target fragment concentration was 50 nM in a buffer containing 5 mM Tris, 5 mM NaCl, 1 mM EDTA and 10 mM MgCl2. The mixture was treated at 65 °C for 15 min, and then the temperature was lowered from 60 °C to 45 °C at a rate of 1 °C / h to obtain the DNA origami structure.

[0014] Optionally, the host bacterium is male *Escherichia coli*. E. coli XL1-Blue F', with VCSM13 as the helper phage.

[0015] Optionally, the phage particle includes a plasmid with a sequence of DNA nanostructures that can self-assemble into triangular, hairpin, rectangular, rod-shaped, or quadrilateral shapes.

[0016] Optionally, the seed culture medium is: 16 g / L Tryptone, 10 g / L Yeast Extract, and 5 g / L NaCl.

[0017] This invention has at least one of the following beneficial effects: 1. In this invention, through optimization and screening of nitrogen sources in the culture medium, it was found that using ammonium chloride as the sole inorganic nitrogen source in the fermentation medium can increase the yield of single-stranded DNA. This fermentation medium increases the yield of triangular, hairpin-shaped, rod-shaped, and quadrilateral single-stranded DNA to varying degrees, providing a theoretical basis for the large-scale production of different types of ssDNA.

[0018] 2. This invention also provides the application of fermentation medium obtained by using ammonium chloride as the sole inorganic nitrogen source in the efficient production of single-stranded DNA and DNA nanostructures by phage particle method. Using ammonium chloride as the sole inorganic nitrogen source to replace the nitrogen source (NH4)2HPO4 in the original fermentation medium (Riesenberg medium) can promote the efficient expression of phage proteins, thereby increasing the yield of single-stranded DNA. Therefore, the method of this invention can improve the yield of single-stranded DNA. Attached Figure Description

[0019] Figure 1 The results are from Example 1, where A represents bacterial growth, B represents single-stranded DNA yield, and C represents single-stranded DNA electrophoresis.

[0020] Figure 2 The results are from Example 2, where A represents bacterial growth, B represents single-stranded DNA yield, and C represents single-stranded DNA electrophoresis.

[0021] Figure 3 The results are from Example 3, where A represents bacterial growth, B represents the ssDNA yield curve, and C represents the single-stranded DNA electrophoresis diagram.

[0022] Figure 4 The results are from Example 4, where A represents bacterial growth, B represents the ssDNA yield curve, and C represents the single-stranded DNA electrophoresis diagram.

[0023] Figure 5 The results are from Example 5, where A represents bacterial growth, B represents the ssDNA yield curve, and C represents the single-stranded DNA electrophoresis diagram.

[0024] Figure 6 The results are from Example 6, where A represents bacterial growth, B represents the ssDNA yield curve, and C represents the single-stranded DNA electrophoresis diagram.

[0025] Figure 7 The results are from Example 7, where A represents bacterial growth, B represents the ssDNA yield curve, and C represents the single-stranded DNA electrophoresis diagram.

[0026] Figure 8 The image shows the AFM characterization of triangular, hairpin rectangular, rod-shaped, and quadrilateral single-stranded DNAs obtained in Example 8 after DNAzyme self-cutting, annealing, and folding. Detailed Implementation

[0027] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0028] The relevant experimental materials and methods involved in this invention are as follows: 1. Experimental Materials 1.1 Culture medium LB medium: 10 g / L Tryptone, 5 g / L Yeast Extract, 10 g / L NaCl.

[0029] Seed culture medium: 2×YT medium: 16 g / L Tryptone, 10 g / L Yeast Extract, 5 g / L NaCl.

[0030] Primary seed culture medium: 2×YT medium: 16 g / L Tryptone, 10 g / L Yeast Extract, 5 g / L NaCl.

[0031] Secondary seed culture medium and fermentation basal culture medium: 13.3 g / L KH2PO4, 3.25 g / L ammonium chloride, 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 2 Na • 2 H2O, 60 mg / L ferric citrate, 1.5 mg / L CuCl2•2 H2O, 2.5 mg / L Na2MoO4•2 H2O, 8 mg / L Zn(CH3COO)2•2 H2O.

[0032] Feeding medium: 750 g / L glucose, 20 g / L MgSO4×7H2O, 15 g / L ammonium chloride.

[0033] Riesenberg 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 (sterilely filtered), 15 mg / L MnCl2•4H2O, 3 mg / L H3BO3, 2.5 mg / L CoCl2•6H2O, 8.4 mg / L EDTA 2 Na • 2 H2O, 60 mg / L ferric citrate, 1.5 mg / L CuCl2•2 H2O, 2.5 mg / L Na2MoO4•2 H2O, 8 mg / L Zn(CH3COO)2•2 H2O.

[0034] Feeding medium: 750 g / L glucose, 20 g / L MgSO4×7H2O, 18.5 g / L (NH4)2SO4.

[0035] 1.2 Test strains and plasmids Host bacteria: Escherichia coli E. coli XL1-BlueF' (purchased from Shanghai Yuanye Biotechnology Co., Ltd.)

[0036] Helper phage: VCSM13 (purchased from Shanghai Rongmin Biotechnology Center).

[0037] Phagemids: In this invention, phagemids are four recombinant plasmids that respectively carry sequences that can self-assemble into triangular, hairpin rectangular, rod-shaped and quadrilateral DNA nanostructures. The synthesis method of the four recombinant plasmids includes: mixing the sequences (synthesized by GenScript) that respectively carry sequences that can self-assemble into triangular, hairpin rectangular, rod-shaped and quadrilateral DNA nanostructures with vector fragments and performing a seamless cloning ligation reaction, reacting at 50°C for 15 min to obtain recombinant plasmids.

[0038] It should be noted that this invention only uses the above-mentioned recombinant plasmids with DNA nanostructure sequences that can self-assemble into triangles, hairpin rectangles, rods, and quadrilaterals as examples to elaborate on the technical solution of this invention in detail, so as to prove that the culture medium and culture method of this invention can obtain ssDNA. It does not mean that only the above-mentioned recombinant plasmids can use the culture medium and culture method of this invention. Other phage particles purchased on the market are also applicable to the culture medium and culture method of this invention.

[0039] 2. Test Methods The shake flask culture method includes the following steps: S1. Add the phage to DH5α competent cells, mix well, place on ice for 30 min, then heat shock at 42℃ for 60 s to allow the phage to enter E. coli. Place on ice again for 5 min, then add antibiotic-free LB medium and incubate at 37℃ for 1 h. Spread the cultured bacterial solution onto solid LB medium plates containing the antibiotic ampicillin and incubate upside down at 37℃ for 12 h. After clearly visible single colonies grow on the plates, pick single colonies for further culture. Send the cultured solution for 6 h for sequencing verification. After confirming that the sequencing results are correct by comparing them with the sequence of the correct phage, inoculate the strain containing the correct phage into 5 mL of medium containing ampicillin and incubate at 37℃ for 12 h. Then, extract the phage using a plasmid extraction kit and transform it into the host bacterium XL1-Blue, which is then preserved to obtain the host bacterium XL1-Blue carrying the phage.

[0040] S2. In shake flask culture, host bacteria XL1-Blue carrying phage particles were added to 2×YT medium and cultured at 200 rpm and 37℃ for 8-10 h as seed culture. S3. In shake flask culture, the seed culture is added to the fermentation basal medium, the inoculum size is calculated and the initial OD600 is made up to 0.03, and the culture is carried out in a shaker at 250 rpm and 37°C. S4. When culturing in shake flasks, it takes 2-2.5 hours after inoculation with the seed culture for the bacterial cell density to reach OD. 600 =0.4~1; at this time, helper phages are added for infection at a multiplicity of infection of approximately 10; S5. After fermentation, take 1.6 ml of fermentation broth into a 2 mL centrifuge tube, centrifuge at 12000 rcf for 10 min, and collect the supernatant. Add 300 µl of a mixed solution of 240 g / L PEG 8000 and 180 g / L NaCl to the supernatant and incubate on ice for 30 min. Centrifuge the above solution at 8000 rcf for 10 min, discard the supernatant, and invert the centrifuge tube on the lab bench to remove as much supernatant as possible. Add 200 µl of TE buffer to the centrifuge tube to resuspend the precipitate. You can mix it using a pipette or a vortex mixer. Centrifuge at 6000 rcf for 15 min to remove residual cell pellet, and transfer the supernatant to a new 2 mL centrifuge tube using a pipette. S6. Add 400 µl of lysis buffer PPB2 to the supernatant to lyse the phage particles, gently invert to mix, and incubate for about 60 s; add 300 µl of neutralization buffer PPB3 to neutralize the solution, and gently invert to mix; after incubating on ice for 15 min, centrifuge the sample at 13000 rcf for 10 min, transfer the supernatant to a new 2 mL centrifuge tube, repeat the centrifugation once, and transfer to a new 2 mL centrifuge tube again; add 1 mL of anhydrous ethanol to the supernatant, mix using a vortex mixer, and incubate on ice for 30 min; centrifuge at 16000 rcf for 30 min, and discard the supernatant; add 400 µl of 75% ethanol to wash, mix using a vortex mixer, and then centrifuge at 16000 rcf for 30 min, discarding as much supernatant as possible, and allow to evaporate at room temperature until there is no alcohol odor; add 20 µl of sterile water to the centrifuge tube to dissolve the single-stranded DNA, obtaining single-stranded DNA; S7. In a buffer containing 50 mM HEPES, 100 mM NaCl and 4 mM ZnCl2, the final concentration of single-stranded DNA was 50 nM. The pH was adjusted to 7.0 and incubated overnight at 37°C for 12 h. After enzyme digestion, the target fragment was recovered. S8, in a buffer containing 5 mM Tris, 5 mM NaCl, 1 mM EDTA and 10 mM MgCl2, the target fragment concentration was 50 nM; treated at 65℃ for 15 min, and then the temperature was reduced from 60℃ to 45℃ at a rate of 1℃ / h to obtain the DNA origami structure.

[0041] The method of culturing in a bioreactor includes the following steps: S1. Add the phage to DH5α competent cells, mix well, place on ice for 30 min, then heat shock at 42℃ for 60 s to allow the phage to enter E. coli. Place on ice again for 5 min, then add antibiotic-free LB medium and incubate at 37℃ for 1 h. Spread the cultured bacterial solution onto solid LB medium plates containing the antibiotic ampicillin and incubate upside down at 37℃ for 12 h. After clearly visible single colonies grow on the plates, pick single colonies for further culture. Send the cultured solution for 6 h for sequencing verification. After confirming that the sequencing results are correct by comparing them with the sequence of the correct phage, inoculate the strain containing the correct phage into 5 mL of medium containing ampicillin and incubate at 37℃ for 12 h. Then, extract the phage using a plasmid extraction kit and transform it into the host bacterium XL1-Blue, which is then preserved to obtain the host bacterium XL1-Blue carrying the phage.

[0042] S2. Add the host bacteria to 2×YT medium and culture at 200 rpm and 37℃ for 8-10 h to obtain the primary seed culture. S3. Add the primary seed culture to the secondary seed culture medium (composition same as the fermentation basal medium), calculate the inoculum size, and ensure the initial OD... 600 =0.03, and cultured in a shaker at 250 rpm and 37℃ for 10-12 h as secondary seed culture; S4. Inoculate the secondary seed culture into a 5 L bioreactor, control the dissolved oxygen at 10%–25% and the aeration rate at 3.0–6.0 L / min. In the 5 L bioreactor, it needs to be cultured for 5–5.5 h until the cell density reaches OD0.05. 600 =17~19; At this time, helper phages are added to carry out infection with an infection multiplicity of approximately 1; S5. After fermentation, take 1.6 ml of fermentation broth into a 2 mL centrifuge tube, centrifuge at 12000 rcf for 10 min, and collect the supernatant. Add 300 µl of a mixed solution of 240 g / L PEG 8000 and 180 g / L NaCl to the supernatant and incubate on ice for 30 min. Centrifuge the above solution at 8000 rcf for 10 min, discard the supernatant, and invert the centrifuge tube on the lab bench to remove as much supernatant as possible. Add 200 µl of TE buffer to the centrifuge tube to resuspend the precipitate. You can mix it using a pipette or a vortex mixer. Centrifuge at 6000 rcf for 15 min to remove residual cell pellet, and transfer the supernatant to a new 2 mL centrifuge tube using a pipette. S6. Add 400 µl of lysis buffer PPB2 to the supernatant to lyse the phage particles, gently invert to mix, and incubate for about 60 s; add 300 µl of neutralization buffer PPB3 to neutralize the solution, and gently invert to mix; after incubating on ice for 15 min, centrifuge the sample at 13000 rcf for 10 min, transfer the supernatant to a new 2 mL centrifuge tube, repeat the centrifugation once, and transfer to a new 2 mL centrifuge tube again; add 1 mL of anhydrous ethanol to the supernatant, mix using a vortex mixer, and incubate on ice for 30 min; centrifuge at 16000 rcf for 30 min, and discard the supernatant; add 400 µl of 75% ethanol to wash, mix using a vortex mixer, and then centrifuge at 16000 rcf for 30 min, discarding as much supernatant as possible, and allow to evaporate at room temperature until there is no alcohol odor; add 20 µl of sterile water to the centrifuge tube to dissolve the single-stranded DNA, obtaining single-stranded DNA; S7. In a buffer containing 50 mM HEPES, 100 mM NaCl and 4 mM ZnCl2, the final concentration of single-stranded DNA was 50 nM. The pH was adjusted to 7.0 and incubated overnight at 37°C for 12 h. After enzyme digestion, the target fragment was recovered. S8, in a buffer containing 5 mM Tris, 5 mM NaCl, 1 mM EDTA and 10 mM MgCl2, the target fragment concentration was 50 nM; treated at 65 °C for 15 min, and then the temperature was reduced from 60 °C to 45 °C at a rate of 1 °C / h to obtain the DNA origami structure.

[0043] During cultivation in a 5 L bioreactor, the control system automatically adjusts the stirring speed and aeration rate, and automatically controls the acid and alkali replenishment to maintain the pH value within the reactor at the set value. Odion displacement (OD) of the bacterial culture is measured every two hours. 600 The ssDNA was isolated and purified according to the extraction method described in Molecular Cloning: A Laboratory Manual, and the relevant data were recorded.

[0044] The present invention will be further described in detail below with reference to specific embodiments, but the present invention is not limited to the following specific embodiments.

[0045] Example 1: Shake-flask horizontal screening of nitrogen sources in fermentation medium to improve triangular ssDNA yield 1. Objective: To investigate the effects of different inorganic nitrogen sources on single-stranded DNA production by replacing the original nitrogen source in Riesenberg medium.

[0046] 2. Experimental Procedure: The nitrogen source in the initial fermentation medium (Riesenberg medium) was diammonium hydrogen phosphate (4 g / L). Following the principle of equal nitrogen content, five different inorganic nitrogen sources were used to replace diammonium hydrogen phosphate: ammonium sulfate, ammonium chloride, sodium nitrate, sodium nitrite, and potassium nitrate. Shake flask experiments were conducted to investigate the effects of different nitrogen sources on cell growth and single-stranded DNA yield.

[0047] When investigating nitrogen sources, maintain the same culture conditions and uniformly control the initial bacterial concentration (OD). 600 =0.03, when the bacterial concentration grows to OD 600 When the value was 0.5, the VCSM13 helper phage was used for infection, with a multiplicity of infection (MOI) of 10. Phage particles with self-assembling triangular DNA nanostructures were used. The flasks were shaken at 250 rpm and 37°C for 14 h to determine the bacterial concentration and single-stranded DNA yield.

[0048] 3. Experimental Results: The results are shown in [the table below]. Figure 1 .

[0049] 4. Results Analysis: Depend on Figure 1 As shown in A, the OD values ​​in the experimental groups using ammonium nitrate, sodium nitrite, and potassium nitrate as nitrogen sources were higher. 600 Not reached the level of OD infection 600 (0.5), so only the other three groups were infected and single-stranded DNA was extracted.

[0050] Yield results after single-stranded DNA extraction are as follows: Figure 1 As shown in B, it was found that the yield per bacterial cell and per unit volume of single-stranded DNA produced using ammonium chloride as a nitrogen source was much higher than that of other groups, with the yield per unit volume reaching more than 10 mg / L.

[0051] Figure 1 In the figure, C represents the nucleic acid electrophoresis of single-stranded DNA produced from different nitrogen sources.

[0052] Example 2: Scale investigation in shake flasks of the effect of NH4Cl concentration in fermentation medium on the yield of triangular ssDNA. 1. Objective: After screening ammonium chloride as a nitrogen source for high yield of single-stranded DNA, the concentration of ammonium chloride was investigated to screen the optimal nitrogen source concentration for single-stranded DNA production.

[0053] 2. Experimental Procedure: When investigating the nitrogen source concentration, ammonium chloride concentration gradients of 1.25, 2.25, 3.25, 4.25, and 5.25 g / L were set. The same culture conditions, methods, and conditions were maintained as in Example 1, and the initial bacterial concentration (OD) was uniformly controlled. 600 =0.03, when the bacterial concentration grows to OD 600 When the value was 0.5, the bacteria were infected with helper phage VCSM13 with a multiplicity of infection (MOI) of 10. After 14 h of culture, the bacterial concentration and single-stranded DNA yield were measured.

[0054] 3. Experimental Results: The results are shown in [the table below]. Figure 2 .

[0055] 4. Results Analysis: bacterial growth status as follows Figure 2 As shown in A in the figure. The single-stranded DNA yield results after extraction are shown in the figure. Figure 2 As shown in B, the highest single-stranded DNA yield was found when the ammonium chloride concentration was 3.25 g / L.

[0056] Figure 2 In the figure, C represents the nucleic acid electrophoresis of single-stranded DNA produced by different concentrations of ammonium chloride.

[0057] Example 3: Using NH4Cl as a nitrogen source in the fermentation medium to improve the yield of triangular ssDNA in shake flasks. 1. Objective: To verify whether replacing the nitrogen source in the original Riesenberg medium with NH4Cl would increase the yield of ssDNA compared to production in the original medium.

[0058] 2. Experimental Procedure: The ammonium chloride concentration in the fermentation medium was 3.25 g / L, and the culture method and conditions were the same as in Example 1. The original Riesenberg medium cultured under the same conditions served as a control group.

[0059] 3. Experimental Results: The results are shown in [the table below]. Figure 3 .

[0060] 4. Results Analysis: Depend on Figure 3 As shown in A, the bacteria entered a stationary phase after 12 hours of cultivation, with the maximum cell density in the original culture medium being OD. 600 =4, when using NH4Cl as the nitrogen source, the maximum cell density is OD. 600 =4.7.

[0061] The yield curve of ssDNA produced using NH4Cl as a nitrogen source is shown below. Figure 3 In the original culture medium, the maximum yield of single-stranded DNA B was 4 mg / L. When NH4Cl was used as the nitrogen source, the maximum yield was 7 mg / L, which increased the yield by about 70%.

[0062] Figure 3 In the figure, C represents the nucleic acid electrophoresis of single-stranded DNA produced at different time points.

[0063] Example 4: Validation of NH4Cl as a nitrogen source in fermentation medium to improve the yield of triangular ssDNA in a 5 L bioreactor 1. Objective: To apply the optimized culture medium composition to a 5 L bioreactor to expand the culture scale and increase ssDNA yield.

[0064] 2. Experimental Procedure: By comparing results at the shake-flask level, it was found that the use of NH4Cl effectively promoted phage particle packaging and significantly increased ssDNA yield. Therefore, the effect of NH4Cl as a nitrogen source on ssDNA yield was further investigated in a 5 L reactor.

[0065] Specifically, the dissolved oxygen during fermentation was controlled at 20% and the aeration rate at 5.0 L / min, with the cell density reaching OD200. 600 =18; at this point, helper phages are added to infect the virus with an infection multiplicity of approximately 1.

[0066] The original Riesenberg medium was cultured under the same conditions as a control group.

[0067] 3. Experimental Results: The results are shown in [the table below]. Figure 4 .

[0068] 4. Results Analysis: The cell growth and ssDNA production curves are as follows: Figure 4As shown in A and B in the diagram. Compared to the original culture medium, the use of NH4Cl as a nitrogen source promoted bacterial growth, and the cells entered the stationary phase at 8 hours of culture, with the maximum cell density reaching OD. 600 =90, the maximum cell density was 110% higher than that of the original culture medium. The highest single-stranded DNA yield in the control group was only 150 mg / L, while the highest single-stranded DNA yield in the experimental group using ammonium chloride as a nitrogen source was 200 mg / L, which was significantly higher than that in the control group, with a yield increase of about 33%.

[0069] Figure 4 In the figure, C represents the nucleic acid electrophoresis of single-stranded DNA produced at different time points.

[0070] Example 5: Using NH4Cl as the nitrogen source in the fermentation medium at the shake-flask level to improve the yield of hairpin rectangular ssDNA 1. Objective: To verify whether replacing the nitrogen source in the original Riesenberg medium with NH4Cl would increase the yield of ssDNA compared to production in the original medium.

[0071] 2. Experimental procedure: The culture method and conditions are the same as in Example 1, except that the plasmid in Example 3 is replaced with a phage particle that can self-assemble into a hairpin rectangular DNA nanostructure.

[0072] The original Riesenberg medium was cultured under the same conditions as a control group.

[0073] 3. Experimental Results: The results are shown in [the table below]. Figure 5 .

[0074] 4. Results Analysis: like Figure 5 As shown in A, the highest bacterial concentration in the experimental group using ammonium chloride as the nitrogen source was slightly higher than that in the control group, and the OD... 600 The maximum value reached approximately 4.5. This is based on the single-stranded DNA yield curve, as shown in the figure. Figure 5 As shown in B, the highest single-stranded DNA yield in the control group was only 3 mg / L, while the highest single-stranded DNA yield in the experimental group using ammonium chloride as a nitrogen source was 4.5 mg / L, which was significantly higher than that in the control group, with a yield increase of about 50%.

[0075] Figure 5 In the figure, C represents the nucleic acid electrophoresis of single-stranded DNA produced at different time points.

[0076] Example 6: Improving rod-shaped ssDNA yield by using NH4Cl as a nitrogen source in the fermentation medium at the shake-flask level. 1. Objective: To verify whether replacing the nitrogen source in the original Riesenberg medium with NH4Cl would increase the yield of ssDNA compared to production in the original medium.

[0077] 2. Experimental procedure: The culture method and conditions are the same as in Example 1, except that the plasmid in Example 3 is replaced with a phage particle that can self-assemble into a rod-shaped DNA nanostructure.

[0078] 3. Experimental Results: The results are shown in [the table below]. Figure 6 .

[0079] 4. Results Analysis: like Figure 6 As shown in A, the bacteria entered a stationary phase after 7 hours of cultivation, with the maximum cell density in the original culture medium being OD. 600 =4, when using NH4Cl as the nitrogen source, the maximum cell density is OD. 600 =4.5.

[0080] The yield curve of ssDNA produced using NH4Cl as a nitrogen source is shown below. Figure 6 In the original culture medium, the maximum yield of single-stranded DNA B was 2 mg / L. When NH4Cl was used as the nitrogen source, the maximum yield was 2.3 mg / L, which increased the yield by about 15%.

[0081] Figure 6 In the figure, C represents the nucleic acid electrophoresis of single-stranded DNA produced at different time points.

[0082] Example 7: Improving the Yield of Quadrilateral ssDNA by Using NH4Cl as a Nitrogen Source in the Fermentation Medium at the Shake Flask Level 1. Objective: To verify whether replacing the nitrogen source in the original Riesenberg medium with NH4Cl would increase the yield of ssDNA compared to production in the original medium.

[0083] 2. Experimental procedure: The culture method and conditions were the same as in Example 1, except that the plasmid in Example 3 was replaced with a phage particle that could self-assemble into a quadrilateral DNA nanostructure.

[0084] 3. Experimental Results: The results are shown in [the table below]. Figure 7 .

[0085] 4. Results Analysis: like Figure 7 As shown in A, the bacteria entered a stationary phase after 12 hours of cultivation, with the maximum cell density in the original culture medium being OD. 600 =3.7, when using NH4Cl as the nitrogen source, the maximum cell density is OD 600 =4.2.

[0086] The yield curve of ssDNA produced using NH4Cl as a nitrogen source is shown below. Figure 7In the original culture medium, the maximum yield of single-stranded DNA B was 2.8 mg / L. When NH4Cl was used as the nitrogen source, the maximum yield was 3.7 mg / L, which increased the yield by about 30%.

[0087] Figure 7 In the figure, C represents the nucleic acid electrophoresis of single-stranded DNA produced at different time points.

[0088] Example 8 Characterization of different single-stranded DNA folds 1. Objective: To characterize the practicality of the triangular, hairpin-shaped, rod-shaped, and quadrilateral single-stranded DNA produced in Example 3, Example 5, Example 6, and Example 7 by autocutting, annealing, folding, and AFM.

[0089] 2. Experimental Results: The results are shown in [link to results]. Figure 8 .

[0090] 3. Results Analysis: Triangular, hairpin rectangular, rod-shaped, and quadrilateral single-stranded DNAs produced using ammonium chloride as a nitrogen source were subjected to DNAzyme self-cutting, annealing, and folding, followed by AFM characterization. It was found that all four shapes folded correctly, proving the feasibility of this method for producing single-stranded DNAs of different sizes.

[0091] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A fermentation medium for the efficient production of single-stranded DNA and DNA nanostructures using the phage particle method, characterized in that, The fermentation medium is Riesenberg medium, and ammonium chloride is used as the sole inorganic nitrogen source to replace the nitrogen source (NH4)2HPO4 in Riesenberg medium.

2. The fermentation medium according to claim 1, characterized in that, The concentration of ammonium chloride in the fermentation medium is 1.25 g / L to 5.25 g / L.

3. The fermentation medium according to claim 1, characterized in that, The fermentation medium has the following components: 13.3 g / L KH2PO4, 3.25 g / L ammonium chloride, 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 2 Na • 2 H2O, 60 mg / L ferric citrate, 1.5 mg / L CuCl2•2 H2O, 2.5 mg / L Na2MoO4•2 H2O, 8 mg / L Zn(CH3COO)2•2 H2O.

4. The application of the fermentation medium according to any one of claims 1 to 3 in the efficient production of single-stranded DNA and DNA nanostructures by the phage particle method.

5. A method for efficient production of single-stranded DNA and DNA nanostructures using phage particles, characterized in that, Includes the following steps: Step 1: Transform the phage particles into the host bacteria to obtain host bacteria carrying the phage particles; Step 2: Add the host bacteria carrying the phage particles to the seed culture medium and culture them to prepare the seed solution; Step 3: Add the seed liquid to the fermentation medium according to any one of claims 1 to 3 for cultivation; Step 4: When the host bacteria carrying the phage particles reach the preset cell density, helper phages are added for infection. Step 5: After culturing and lysis, single-stranded DNA and DNA nanostructures are obtained.

6. The method according to claim 5, characterized in that, Step one includes the following specific steps: Phage particles were added to DH5α competent cells and mixed well. After being placed on ice for 30 min, the cells were heat-shocked at 42°C for 60 s to allow the phage particles to enter E. coli. After being placed on ice again for 5 min, antibiotic-free medium was added and the cells were cultured at 37°C for 1 h. The cultured bacterial solution was spread onto a solid medium plate containing the antibiotic ampicillin and incubated upside down at 37°C for 12 h. After clearly visible single colonies grew on the plate, single colonies were picked and cultured. The cultured bacterial solution after 6 h was sent for sequencing verification. After confirming that the sequencing results were correct by comparing them with the sequence of the correct phage particles, the bacterial strain containing the correct phage particles was inoculated into 5 mL of medium containing ampicillin and cultured at 37°C for 12 h. The phage particles were then extracted using a plasmid extraction kit and transformed into the host bacteria to obtain host bacteria carrying phage particles.

7. The method according to claim 5, characterized in that, Step two includes the following specific steps: The host bacteria carrying the phage particles were added to the seed culture medium and cultured at 200 rpm and 37°C for 8-10 h to obtain the seed solution. Step three includes the following specific steps: The seed culture was added to the fermentation medium, the inoculum size was calculated, and the initial OD was adjusted accordingly. 600 =0.03, incubated in a shaker at 250 rpm and 37°C.

8. The method according to claim 5, characterized in that, Step four involves culturing in a shake flask or a bioreactor, wherein: When culturing in shake flasks, step four includes the following specific steps: When culturing in shake flasks, the bacterial cell density reaches OD200 after 2-2.5 hours following inoculation of the seed culture. 600 When the infection ratio is 0.4~1, helper phages are added at a multiplicity of infection of 10 for infection. When culturing in a bioreactor, step four includes the following specific steps: The culture medium from step two was inoculated into a 5 L bioreactor. The dissolved oxygen level was controlled at 10%–25% and the aeration rate at 3.0–6.0 L / min. Culture was carried out for 5–5.5 h after inoculation with the culture medium from step two, until the cell density reached OD200. 600 =17~19 hours, with helper phages added at multiplicity 1 for infection.

9. The method according to claim 5, characterized in that, Step five includes the following specific steps: After fermentation, the fermentation broth was transferred to a centrifuge tube, centrifuged, and the supernatant was collected. A mixed solution of PEG 8000 and PEG 1 was added to the supernatant and incubated on ice. The supernatant was discarded by centrifugation, and 200 µl of TE buffer was added to the centrifuge tube to resuspend the precipitate. The residual cell precipitate was removed by centrifugation, and the supernatant was transferred to a new centrifuge tube. Add lysis buffer PPB2 to the supernatant to lyse the phage particles, mix well, and incubate. Add neutralization buffer PPB3 to neutralize the solution and mix well; incubate on ice, centrifuge, transfer the supernatant to a new centrifuge tube, repeat centrifugation, and transfer to a new centrifuge tube again; Add anhydrous ethanol to the supernatant, mix well, and incubate on ice; centrifuge to remove the supernatant; add 75% ethanol to wash, mix well, centrifuge to remove the supernatant, and allow to evaporate at room temperature until there is no alcohol smell; add sterile water to the centrifuge tube to dissolve the single-stranded DNA to obtain single-stranded DNA. The single-stranded DNA was prepared to a final concentration of 50 nM in a buffer containing 50 mM HEPES, 100 mM NaCl and 4 mM ZnCl2. The pH was adjusted to 7.0 and the mixture was incubated overnight at 37°C for 12 h. After enzyme digestion, the target fragment was recovered. The target fragment concentration was 50 nM in a buffer containing 5 mM Tris, 5 mM NaCl, 1 mM EDTA and 10 mM MgCl2. The mixture was treated at 65 °C for 15 min, and then the temperature was lowered from 60 °C to 45 °C at a rate of 1 °C / h to obtain the DNA origami structure.

10. The method according to claim 5, characterized in that, The host bacterium is male Escherichia coli. E. coli XL1-BlueF'; Phage particles include recombinant plasmids carrying DNA nanostructure sequences that can self-assemble into triangular, hairpin, rectangular, rod-shaped, or quadrilateral shapes; The helper phage was VCSM13; The seed culture medium consisted of 16 g / L Tryptone, 10 g / L Yeast Extract, and 5 g / L NaCl.

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