Fully automated plasmid production system, and preparation method

Abstract

Disclosed in the present invention are a fully automated plasmid production system, and a preparation method. A bacterial suspension, an alkaline lysis solution, a neutralization solution are mixed in a flow mode by means of a static mixer, and the bacteria are continuously segmented and mixed, improving the alkaline lysis and neutralization efficiency, and further improving the plasmid yield. During plasmid DNA collection, a strategy combining precipitation and floating is adopted, so that the method is particularly suitable for large-scale and automated plasmid production. The method improves the DNA precipitation efficiency, allows for better layering between an intermediate clarified liquid layer and upper suspended impurities, remarkably reduces the turbidity of the intermediate clarified liquid, and facilitates subsequent separation and filtration. A DNA yield higher than that of a conventional method is obtained both before or after filtration.

Description

A fully automatic plasmid production system and preparation method Technical Field

[0001] The present application relates to the field of biopharmaceuticals, and in particular to a fully automated plasmid production system and preparation method. Background Art

[0002] With the rise of gene therapy and nucleic acid vaccines / drugs, the demand for GMP-grade plasmids has increased significantly. Currently, the main method for plasmid purification is alkaline lysis to extract plasmids. The basic principle is to use alkaline solution to lyse cells to release plasmids and other intracellular substances, remove proteins through adsorption by potassium dodecylsulfonate precipitation, and separate genomic DNA based on the size difference between chromosomal DNA and plasmid DNA. When using alkaline lysis to prepare plasmids on a small scale, it is generally done by manually adding and mixing by inversion. When preparing large quantities of plasmids, although there have been reports of equipment for related automated systems to solve the problems of difficult large-scale alkaline lysis operations, insufficient alkaline lysis, and low yields, they still cannot meet the needs in terms of controlling the alkaline lysis time, evaluating the alkaline lysis effect, and preventing errors during the operation.

[0003] The existing technology for extracting plasmid DNA mostly uses the phenol / chloroform method, ethanol precipitation method, nucleic acid adsorption column method and the like. Although the phenol / chloroform method has the advantage of inhibiting DNase degradation, the reagent toxicity is relatively high, and long-term operation has a significant impact on personnel health. In addition, the nucleic acid recovery rate is low, and the loss is large. Due to the large operating system and poor repeatability of operations by different personnel, it is difficult to carry out large-scale standardized production. The ethanol precipitation method increases the total volume of the system, and the use of ethanol for multiple precipitations results in a long process flow and low production efficiency; and DNA is dissolved to a certain extent in the solution, which affects the yield. The nucleic acid adsorption column method uses the affinity between nucleic acids and magnetic beads or solid phase column materials to remove impurities from the sample, thereby making the purified nucleic acid purer. This method has high requirements for samples and consumables, requires repeated centrifugation, and is not convenient for automated large-scale production operations.

[0004] Chinese patent application CN114561273A (published May 31, 2022) discloses a continuous alkaline lysis system for preparing plasmids and a method for preparing the same. This system utilizes an atomizer before lysis to address DNA contamination caused by shear forces from prolonged mixing and stirring, thereby improving plasmid yield. After adding a neutralizing solution, the system passes the solution through a continuous flow centrifugation device to collect the clarified liquid. Both the spiral lysis and mixing devices of this system are spiral coil structures, making it impossible to evaluate the mixing effect. The spiral lysis device only increases the contact time between the feed and liquid, without providing further mixing. This makes it difficult to effectively identify and control issues such as pH changes and bubble generation that may arise during production. Summary of the Invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a fully automatic plasmid production system based on the alkaline lysis method, which can monitor abnormalities in real time and efficiently precipitate and stratify the neutralized liquid. Another purpose of the present invention is to provide a method for automated and large-scale production of plasmids using the alkaline lysis method.

[0006] Technical solution: In order to achieve the above-mentioned purpose of the invention, the present invention provides a fully automatic plasmid production system comprising: a primary confluence section, whose input end is respectively connected to the bacterial suspension input pipeline and the lysate input pipeline, and is output to the secondary confluence section; a secondary confluence section, whose input end is respectively connected to the primary confluence output pipeline and the neutralization liquid input pipeline, and is output to a collection device; the collection device includes a first interface connected to the secondary confluence output pipeline, a second interface connected to the precipitation liquid input pipeline, and a third interface connected to the supernatant input pipeline; a PLC controller, used to obtain the operating parameters of each of the input pipelines and / or output pipelines and issue control instructions; wherein, the outlet end of each confluence section is respectively provided with a static vortex mixer.

[0007] The fully automatic system provided by the present invention mainly includes a bacterial suspension input pipeline, a lysate input pipeline, a neutralization liquid input pipeline, a primary confluence output pipeline, a secondary confluence output pipeline, a precipitate input pipeline, a floatation liquid input pipeline, etc., for different media to flow through. Specifically, the operating parameters obtained by the PLC controller include but are not limited to: flow parameters and bubble parameters of the bacterial suspension input pipeline, the lysate input pipeline, and the neutralization liquid input pipeline, pH parameters of the primary confluence output pipeline and the secondary confluence output pipeline, and flow parameters of the precipitate input pipeline and the floatation liquid input pipeline. The control instructions issued by the PLC controller include but are not limited to start signals, stop signals, speed change signals, reversal signals, etc. sent to the peristaltic pumps of each pipeline respectively.

[0008] As a preferred embodiment of the present invention, a peristaltic pump and at least one flow meter are respectively provided on the bacterial suspension input pipeline, the lysis solution input pipeline, and the neutralization solution input pipeline. Each peristaltic pump input is also provided with a bubble detector, which is communicatively connected to the PLC controller. The PLC controller is provided with an early warning module. When the early warning module receives a signal from the bubble detector indicating that a bubble has been detected, it sends a stop signal to the peristaltic pump.

[0009] As a further optimization of the present invention, a pH sensor is provided at the output end of the static mixer and is in communication with the PLC controller. When the early warning module receives a threshold-exceeding signal from the pH sensor, it sends a stop signal to the peristaltic pump.

[0010] As a further optimization of the present invention, the static vortex mixer comprises a shell tube and a plurality of single spiral plates disposed within the shell tube, wherein adjacent spiral plates have opposite torsional directions. More specifically, the static vortex mixer is an SK-type static suspender, which provides a residence mixing time of 20 seconds to 2 minutes for the materials of the present invention.

[0011] Furthermore, the precipitating solution described herein refers to a reagent capable of forming a reversible precipitate with nucleic acids, preferably including but not limited to calcium chloride, ethanol, isopropanol, phenol / chloroform, formamide, acetic acid, and the like. The supernatant solution, which reacts with the precipitating solution to generate bubbles, includes but is not limited to sodium bicarbonate and potassium bicarbonate. As a preferred embodiment of the present invention, the precipitating solution is a calcium chloride solution, and the supernatant is a sodium bicarbonate solution.

[0012] The prior art does not offer a fully automated, large-scale production solution for precipitation and clarification of the neutralized solution for plasmid alkaline extraction. The present invention proposes, for the first time, an automated production method utilizing the interaction of a precipitating solution and a floating solution. The bubbles (primarily carbon dioxide) generated by the interaction between the two effectively propel the precipitate in the collection device upward. After standing, the clarified liquid layer in the middle is a solution containing the plasmid, while the bottom layer is a small amount of precipitate that has not completely floated. This method eliminates the need for consumables or centrifugation, nor does it require the introduction of complex reagents into the system. Precipitation and clarification can be achieved through automated control, while ensuring the yield and quality of plasmid extraction.

[0013] In order to further improve the sedimentation effect and the floating effect, the second interface is located at a liquid level height of about 90% of the distance from the bottom of the collecting device, and the third interface is located at a liquid level height of about 10% of the distance from the bottom of the collecting device.

[0014] Furthermore, the input end of the bacterial suspension input pipeline is connected to the bacterial suspension container, the input end of the lysate input pipeline is connected to the lysate container, and the input end of the neutralization liquid input pipeline is connected to the neutralization liquid container; optionally, the bacterial suspension container and the neutralization liquid container have a temperature control device that keeps the internal temperature constant at 2-8°C.

[0015] Furthermore, the primary confluence part and the secondary confluence part are joints having one outlet and at least two inlets. As a preferred embodiment of the present invention, the primary confluence part and the secondary confluence part are three-way joints.

[0016] Furthermore, both ends of the input and output lines are equipped with quick-connect connectors. The quick connectors described herein enable rapid assembly between the input and output lines and their respective containers through any combination of snap-fit, threaded, keyway, or pin fit. These quick connectors are preferably disposable, such as MPC quick-connect connectors, MPX quick-connect connectors, MPU quick-connect connectors, SIP connectors, Luer connectors, CPC connectors, and sterile connectors.

[0017] The present invention provides an automated plasmid alkaline lysis preparation method, comprising mixing a suspension with bacterial sludge, first fluidically mixing with a lysis solution, and then fluidically mixing with a neutralizing solution; after the fluidic mixing is completed, adding a precipitating solution to obtain a precipitated liquid; adding a supernatant to the bottom of the precipitated liquid, wherein the supernatant has the characteristic of reacting with the precipitating liquid to generate bubbles; then, allowing the supernatant to stand at 2-8°C for 4-24 hours, obtaining an intermediate clear liquid layer for subsequent purification. The subsequent purification steps include concentration and diafiltration, gel chromatography, affinity chromatography, ion exchange chromatography, concentration and liquid exchange, etc.

[0018] The flow mixing of the present invention refers to mixing by a suspender during the flow of feed liquid, more specifically, mixing by a static mixer, especially an SK type static mixer. It is only necessary to maintain a certain flow rate and flow rate. During the static mixer, different fluids can be fully dispersed and twisted by the design of the internal structure and the fluid flow state so that they are evenly mixed. When the fluid flows through the SK type static mixer, it is continuously cut by the single spiral plate inside it. At the same time, due to the rotation and distortion of the single spiral plate, the flow direction of the fluid is forced to change continuously, producing convection and eddy motion. The divided fluid merges between the two units and is then divided again. The fluid is continuously dispersed, convected, vortexed, and merged to achieve a uniformly mixed effect. This mixing method is relatively gentle to the contact of the bacterial suspension and the lysate and neutralizer, and avoids the situation where the local concentration is too high or the mixing is uneven, and avoids the destruction of the DNA double helix structure to the greatest extent. The mixing time of the present invention in the static mixer is 20 seconds to 2 minutes.

[0019] The method provided by the present invention can efficiently clarify and neutralize the feed liquid by allowing the precipitate and the supernatant to stand after the precipitate and the supernatant are added in sequence, and the intermediate clarified liquid layer has an average turbidity of no more than 15 NTU.

[0020] As a further optimization of the present invention, the precipitating solution is calcium chloride, and the final concentration after addition is 0.5-1.5 M; the supernatant is sodium bicarbonate, and the final concentration after addition of sodium bicarbonate is 1-10 g / L.

[0021] Furthermore, the suspension is an aqueous solution containing Tris-HCl, EDTA and glucose, with a pH of 7.5±0.2, and is stored at 2-8°C; the lysis solution is an aqueous solution containing sodium hydroxide and SDS, which can be stored at room temperature; and the neutralization solution is an aqueous solution of potassium acetate and glacial acetic acid, which is stored at 2-8°C.

[0022] The present invention provides a fully automatic system for plasmid production. The system uses a static mixer to mix a bacterial suspension, an alkaline lysis solution and a neutralization solution in a fluidic manner, continuously breaks and mixes the bacterial cells, improves the efficiency of alkaline lysis and neutralization, and thus improves the plasmid yield. Through parameter setting and the synchronous start-up of multiple pumps, the addition amount of various materials can be controlled. At the same time, there is pH monitoring during the process, which makes the production process more stable and minimizes the batch-to-batch differences caused by human operation. The strategy of combining precipitation and floating phases is adopted when collecting plasmid DNA, which solves the problem that the flocculants in the precipitation process co-precipitate part of the DNA together, resulting in a low yield. It is particularly suitable for large-scale, automated plasmid production. This method can improve the efficiency of DNA precipitation, better stratify the middle clarified liquid layer and the upper suspended impurities, significantly reduce the turbidity of the middle layer clarified liquid, and is conducive to subsequent separation and filtration. Whether before or after filtration, a higher DNA yield than the traditional method is obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG1 is a schematic diagram of the system structure of Example 1 of the present invention;

[0024] FIG2 is a schematic diagram of the system principle of Example 1 of the present invention;

[0025] FIG3 is a flow chart of the preparation method of Example 1 of the present invention. DETAILED DESCRIPTION

[0026] The present invention will be further described below with reference to the accompanying drawings and examples. Example 1

[0027] As shown in Figures 1 and 2, this embodiment provides a fully automatic plasmid production system, including a bacterial suspension container 1, a lysis solution container 2, a neutralization solution container 3, a collection device 4, a precipitation solution container 5, a supernatant container 6, and a PLC controller 7.

[0028] The liquid outlet of the bacterial suspension container 1 is connected to the first tee 10 via a bacterial suspension input line 8, and the liquid outlet of the lysate container 2 is connected to the first tee 10 via a lysate input line 9. The outlet of the first tee 10 is provided with a first static suspender 11 and is connected to the second tee 14 via a primary confluence output line 12. The liquid outlet of the neutralizer container 3 is connected to the second tee 14 via a neutralizer input line 13. The outlet of the second tee 14 is provided with a second static vortex mixer 15 and is connected to the first interface of the collecting device 4 via a secondary confluence output line 16. The second interface of the collecting device 4 is connected to the sedimentation liquid container 5 via a sedimentation liquid input line 17, and the second interface of the collecting device 4 is connected to the floatation liquid container 6 via a floatation liquid input line 18. To further improve the sedimentation and floatation effects, the second interface is located at a liquid level of approximately 90% of the height of the collecting device from the bottom, and the third interface is located at a liquid level of approximately 10% of the height of the collecting device from the bottom.

[0029] The first static suspender 11 and the second static suspender 15 are both SK type static suspenders, which have a shell tube and a plurality of single spiral plates arranged in the shell tube. The twisting directions of adjacent spiral plates are opposite, providing a residence mixing time of 20 seconds to 2 minutes for the material.

[0030] All pipes are disposable high-density polyethylene (HDPE). The diameter and length of the pipes can be adjusted based on the scale of production. In this example, the pipes used are 90-110 cm long and 7.6 mm in diameter. Both ends of the pipes are equipped with CPC sterile quick-connect fittings to prevent contamination of the feed liquid.

[0031] As can be seen from Figure 1, the PLC controller is connected to all peristaltic pumps, flow meters, bubble detectors, and pH sensors of each pipeline respectively to obtain the operating parameters of each input pipeline and / or output pipeline, and to issue a control instruction. The peristaltic pump speed is 30-45ml / min. The bacterial suspension input pipeline 8, the lysate input pipeline 9, the neutralizer input pipeline 13, the sedimentation liquid input pipeline 17, and the supernatant input pipeline 18 are all provided with a peristaltic pump and a flow meter. In addition, the bacterial suspension input pipeline 8, the lysate input pipeline 9, and the neutralizer input pipeline 13 are also provided with a bubble detector. The first-level confluence output pipeline 12 and the second-level confluence output pipeline 16 are provided with a pH sensor. The PLC controller is provided with an early warning module. After the early warning module receives the signal that the bubble detector sends and monitors the bubbles, it sends a stop signal to the peristaltic pump. After the early warning module receives the signal that the pH sensor sends and exceeds the threshold value, it sends a stop signal to the peristaltic pump.

[0032] In Figure 1 , the bacterial suspension container 1 , the neutralization liquid container 3 , and the collection device 4 are all placed in independent temperature control devices to maintain the liquid temperature at 2-8° C. Alternatively, the three containers can be placed in the same temperature control device. Example 2

[0033] This example provides an automated plasmid alkaline lysis preparation method. This method is based on the fully automated system provided in Example 1 and uses the pMDLg-pRRE plasmid produced by Escherichia coli DH5α competent cells as the test object.

[0034] 1. Solution Preparation

[0035] Prepare a bacterial suspension by mixing 30 g of bacterial sludge (16.6% fermentation broth wet weight, plasmid concentration 749 ng / μl) with the suspension. The suspension contains 3.0 g Tris, 2.66 g EDTA, and 9.91 g glucose. The volume is adjusted to 1.0 kg (1 L). Adjust the pH to 7.5 ± 0.2 with hydrochloric acid and store at 2–8°C.

[0036] Prepare lysis solution by weighing 8.0 g of sodium hydroxide and 10.0 g of SDS in proportion, adjust the volume to 1.0 kg (1 L), and store at room temperature.

[0037] Prepare the neutralization solution by weighing 294.5 g of potassium acetate and 120 g of glacial acetic acid in proportion, dilute to 1.1 kg (1 L), and store at 2-8°C.

[0038] To prepare the precipitate, weigh 744.5 g of calcium chloride dihydrate according to the proportion, adjust the volume to 1.4 kg (1 L), and store at room temperature.

[0039] Prepare the supernatant solution by weighing 80 g of sodium bicarbonate in proportion, making the volume to 1.05 kg (1 L), and storing at room temperature.

[0040] 2. Automated production

[0041] Please refer to Figure 3, where P1, P2, P3, P4, and P5 are peristaltic pumps for the bacterial suspension container 1, the lysate container 2, the neutralization liquid container 3, the sedimentation liquid container 5, and the floatation liquid container 6, respectively. In this embodiment, the control instructions sent by the PLC controller to the peristaltic pumps are start and stop signals.

[0042] S110: The PLC controller sends a start command to P1 and P2 to mix the bacterial suspension with the lysis solution;

[0043] S120: Based on the start times of P1 and P2, the PLC controller sets the delay parameter for the start of P3 (20 seconds to 2 minutes, depending on the characteristics of the liquid and the pipeline parameters). When the liquid in the first-stage confluence output pipeline 12 reaches the second three-way connection 14, a start command is issued to P3 for further fluid mixing with the neutralizing liquid.

[0044] S130: When P1, P2, and P3 complete the addition of the slurry, they start checking for abnormalities. If a signal indicating that bubbles have been detected is received from the bubble detector, the PLC controller sends a stop command to P1, P2, and P3, otherwise it continues to execute S140.

[0045] S140: After the flow mixing is completed, the PLC controller sends a start command to P4 to add precipitation liquid to obtain precipitation liquid.

[0046] S150: When P4 completes adding the slurry, the PLC controller sends a start command to P5, adding a floating solution to the bottom of the precipitate solution until the process is complete. The sodium bicarbonate in the floating solution reacts with the calcium chloride in the precipitate, generating a large number of bubbles that propel the mixture of calcium carbonate and flocculent precipitate upward. The plasmid DNA is located in the middle clear liquid layer, while the lower layer is mainly composed of a small amount of precipitate that has not been completely floated.

[0047] 3. Resting and post-processing

[0048] The liquid is stored in a collecting device and kept at a constant temperature of 2-8°C for 4-24 hours.

[0049] Obtain the intermediate clear liquid layer, filter it with a deep filtration module or a 0.22um filter, concentrate and filter it, and then carry out subsequent purification.

[0050] 4. pH detection

[0051] In this embodiment, pH sensors are located at the outlets of the first static suspension vessel 11 and the second static suspension vessel 15, respectively. The PLC controller pre-sets the pH thresholds for the two pH sensors. During the flow mixing process described in S110 and S120, if the pH sensor values ​​exceed the thresholds, a stop signal is issued to the peristaltic pumps P1, P2, and P3. Production can resume after the problem is manually corrected. Example 3

[0052] In order to verify the advantages of the production process of the present invention, this example provides a set of alkaline lysis processes based on the prior art as a control: the suspension is weighed in a ratio of Tris-HCl 7.9g and EDTA disodium salt 3.7g, added to 800mL of ultrapure water to fully dissolve, diluted to 1L with water for injection, and 1M NaOH is added to adjust the pH to 7.5±0.5. The lysate is weighed in a ratio of sodium hydroxide 8g and SDS 10g, added to 800ml of water for injection to fully dissolve, and diluted to 1L. The neutralizer is weighed in a ratio of potassium acetate 294g, added to 600ml of water for injection to fully dissolve, 100mL of glacial acetic acid is measured and added to the above solution to mix, and diluted to 1L with water for injection. The precipitate is weighed in a ratio of calcium chloride dihydrate 735g, added to 800ml of water for injection to fully dissolve, and diluted to 1L with water for injection. The experimental group adopts the method described in Example 2.

[0053] Experimental method: 80% and 20% of the sample volume (precipitation liquid) were taken from the experimental group and the control group, respectively, and two experiments were conducted with and without the addition of the supernatant. All samples were tested on the intermediate clarified liquid layer after standing for 4 hours and 24 hours and filtration. DNA concentration was determined by ultraviolet spectrophotometry. DNA purity was determined by spectrophotometry and the absorbance ratios of A260 / A280 and A260 / A230 were calculated. The average turbidity value was statistically calculated according to the turbidimetric method and the unit was NTU. Plasmid yield was determined by ultraviolet spectrophotometry using the DNA concentration and the ratio of the plasmid concentration before alkaline lysis to the plasmid concentration in the filtered liquid after alkaline lysis was calculated. The results of all experiments are shown in Table 1:

[0054] Table 1 Differences in plasmid alkaline lysis processes and the influence of supernatant

[0055]

[0056] Test conclusion

[0057] Regarding the floating phenomenon of impurities: regardless of the experimental group or the control group, the floating impurities after adding sodium bicarbonate are more compact than the floating impurities in the lysis solution without adding sodium bicarbonate. Shaking during the process of taking out the container will not cause the impurities to mix with the solution, which is more conducive to the separation of the intermediate clear solution and subsequent filtration.

[0058] Regarding turbidity: regardless of the experimental group or the control group, the turbidity of the intermediate clear solution after adding sodium bicarbonate was significantly lower than that of the intermediate clear solution without adding sodium bicarbonate.

[0059] Regarding DNA concentration: In general, whether before or after filtration, the alkaline lysis process provided by the present invention obtains a higher DNA concentration than that obtained by the prior art.

[0060] Regarding the alkaline lysis yield: the alkaline lysis process provided by the present invention makes the plasmid yield significantly higher than that of the prior art, and the yield after adding the supernatant (sodium bicarbonate) is higher than the yield without adding sodium bicarbonate, regardless of the experimental group or the control group.

[0061] The present invention ensures plasmid concentration and yield through a series of measures, including automated and precise control of the addition ratio of lysate, neutralizing solution, and feed solution, controlling the contact time of alkaline lysis, and optimizing the feed-liquid mixing effect using a static suspender. Furthermore, the pH value of the feed solution is monitored online using a pH sensor to avoid plasmid quality issues caused by excessive or localized addition of lysate. The combined precipitation and flotation phase strategy significantly improves the yield of plasmids recovered from the precipitate, making it more conducive to large-scale, automated production.

[0062] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A fully automated plasmid production system, characterized in that include: A primary confluence section, the input ends of which are respectively connected to the bacterial suspension input pipeline and the lysate input pipeline, and are transmitted to the secondary confluence section; A secondary confluence part, the input end of which is respectively connected to the primary confluence output pipeline and the neutralization liquid input pipeline, and is output to the collection device; The collecting device comprises a first interface connected to the secondary confluence output pipeline, a second interface connected to the sedimentation liquid input pipeline, and a third interface connected to the floatation liquid input pipeline; a PLC controller is used to obtain the operating parameters of each of the input pipelines and / or output pipelines and issue control instructions; wherein, a static vortex mixer is respectively provided at the outlet end of each confluence part.

2. A fully automatic plasmid production system according to claim 1, characterized in that: A peristaltic pump and at least one flow meter are respectively provided on the bacterial suspension input pipeline, the lysis solution input pipeline, and the neutralization solution input pipeline.

3. A fully automatic plasmid production system according to claim 1 or 2, characterized in that: The input end of each peristaltic pump is also provided with a bubble detector which is in communication connection with the PLC controller.

4. A fully automatic plasmid production system according to claim 3, characterized in that: The PLC controller is provided with an early warning module. When the early warning module receives a signal from the bubble detector indicating that bubbles have been detected, it sends a stop signal to the peristaltic pump.

5. A fully automatic plasmid production system according to claim 4, characterized in that: The output end of each static mixer is provided with a pH sensor which is in communication with the PLC controller.

6. A fully automatic plasmid production system according to claim 5, characterized in that: When the early warning module receives the threshold-exceeding signal from the pH sensor, it sends a stop signal to the peristaltic pump.

7. A fully automatic plasmid production system according to claim 1, characterized in that: The static vortex mixer comprises a shell tube and a plurality of single spiral plates arranged in the shell tube, and the twisting directions of adjacent spiral plates are opposite.

8. A fully automatic plasmid production system according to any one of claims 1-2, 4-7, characterized in that: The input end of the bacterial suspension input pipeline is connected to the bacterial suspension container, the input end of the lysate input pipeline is connected to the lysate container, and the input end of the neutralization liquid input pipeline is connected to the neutralization liquid container; the bacterial suspension container and the neutralization liquid container have a temperature control device that keeps the internal temperature constant at 2-8°C.

9. A fully automatic plasmid production system according to claim 8, characterized in that: The primary confluence part and the secondary confluence part are joints with one outlet and at least two inlets.

10. A fully automatic plasmid production system according to claim 9, characterized in that: Both ends of the input pipeline and the output pipeline are provided with quick-connect joints.

11. A method for preparing a plasmid by alkaline lysis automation, characterized in that: After the suspension is mixed with the bacterial mud, it is first mixed with the lysis solution in a flow-type manner, and then mixed with the neutralization solution in a flow-type manner; after the flow-type mixing is completed, a precipitation solution is added to obtain a precipitation liquid; a supernatant is added to the bottom of the precipitation liquid, and the supernatant has the characteristic of reacting with the precipitation liquid to generate bubbles; and then the supernatant is allowed to stand at 2-8°C for 4-24 hours to obtain an intermediate clear liquid layer for subsequent purification.

12. The method for preparing a plasmid by alkaline lysis according to claim 11, characterized in that: The intermediate clear liquid layer has an average turbidity of no more than 15 NTU.

13. The method for preparing a plasmid by alkaline lysis according to claim 12, characterized in that: The flow mixing is carried out in a static vortex mixer, and the mixing time is 20 seconds to 2 minutes.

14. The method for preparing a plasmid by alkaline lysis automation according to claim 12 or 13, characterized in that: The precipitating liquid is calcium chloride, and the floating liquid is sodium bicarbonate.

15. The method for preparing a plasmid by alkaline lysis according to claim 14, characterized in that: The final concentration of the calcium chloride after addition is 0.5-1.5M, and the final concentration of the sodium bicarbonate after addition is 1-10g / L.

16. The method for preparing a plasmid by alkaline lysis according to claim 15, characterized in that: The suspension is an aqueous solution containing Tris-HCl, EDTA and glucose, with a pH of 7.5±0.2 and stored at 2-8°C; the lysis solution is an aqueous solution containing sodium hydroxide and SDS; and the neutralization solution is an aqueous solution of potassium acetate and glacial acetic acid and stored at 2-8°C.

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