Plasmid extraction method and equipment

Abstract

The embodiment of the invention provides a plasmid extraction method and equipment, and the method comprises the following steps: providing a cell lysis solution which comprises a reaction solution containing plasmids and a suspended matter suspended in the reaction solution; a first bubbling medium is introduced into the bubbling device, the first bubbling medium drives the suspended matter to float upwards from the bottom of the cell lysis solution through the bubbling device, and an upper-layer suspended matter and a lower-layer reaction solution are formed; separating the upper-layer suspended matters from the lower-layer reaction liquid to obtain the lower-layer reaction liquid containing plasmids.

Description

Cross-references

[0001] This application claims priority to Chinese application No. 2023112584690 filed on September 26, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention relates to the field of biotechnology, and in particular to a plasmid extraction method and device. Background Art

[0003] With the rise of gene and cell therapy, the research and production needs of plasmid extraction are expanding, and conventional plasmid extraction and purification methods can no longer meet the growing production needs. In terms of protein precipitate removal, the conventional practice is to renature the plasmid by alkaline lysis, and then use centrifugation or filtration to remove the precipitate to obtain the supernatant containing the plasmid. The centrifugation method has obvious operational disadvantages. It requires manual weighing, balancing, and closing the lid, and then manually opening the lid after the centrifugation. The number of centrifuge bottles that each centrifuge can accommodate is limited, and the centrifugation time is relatively long. For relatively large solutions, it is also necessary to divide them into multiple bottles for centrifugation, which has problems such as cumbersome operation, long time consumption, and high labor costs. Because the protein precipitate is viscous and delicate, it is difficult to remove in advance and is easy to clog the filter. The filtration time and consumables cost of the filtration method are usually high, which is not conducive to large-scale and automated production applications of plasmid extraction.

[0004] Therefore, it is necessary to provide a plasmid extraction method and equipment, which are simple to operate, short in time and low in cost. Summary of the invention

[0005] One or more embodiments of the present specification provide a plasmid extraction method, the method comprising: providing a cell lysis solution, the cell lysis solution comprising a reaction solution containing a plasmid and a suspended substance suspended in the reaction solution; introducing a first bubbling medium into the cell lysis solution, the first bubbling medium driving the suspended substance to float up from the bottom of the cell lysis solution to form an upper layer of suspended substance and a lower layer of reaction solution; separating the upper layer of suspended substance and the lower layer of reaction solution to obtain the lower layer of reaction solution containing the plasmid.

[0006] In some embodiments, the method further comprises: purifying the lower layer reaction liquid to obtain a purified lower layer reaction liquid containing the plasmid.

[0007] In some embodiments, purifying the lower layer reaction liquid to obtain a purified lower layer reaction liquid containing a plasmid includes: adding a filter aid material to the lower layer reaction liquid; filtering the lower layer reaction liquid after adding the filter aid material to obtain the purified lower layer reaction liquid containing a plasmid.

[0008] In some embodiments, providing the cell lysis solution comprises: providing cells; lysing the cells to obtain the cell lysis solution, wherein during the process of lysing the cells, a second bubbling medium is introduced into the cells.

[0009] In some embodiments, the method comprises passing a first bubbling medium into the bacterial lysate at a preset bubbling rate, wherein the preset bubbling rate is 0.5 L / min-20 L / min.

[0010] In some embodiments, the preset bubbling rate is 3 L / min-8 L / min.

[0011] In some embodiments, the first bubbling medium is selected from air, carbon dioxide, nitrogen, an inert gas, or a liquid medium with a lower density and insoluble in water. The liquid medium with a lower density and insoluble in water can be silicone oil.

[0012] In some embodiments, the second bubbling medium is selected from air, carbon dioxide, nitrogen, an inert gas, or a liquid medium with a lower density and insoluble in water. The liquid medium with a lower density and insoluble in water can be silicone oil.

[0013] One or more embodiments of the present specification provide a plasmid extraction device, the device comprising: a reaction chamber for accommodating a cell lysis solution, the cell lysis solution comprising a reaction solution containing a plasmid and a suspended substance suspended in the reaction solution; a bubbling device, located at the bottom of the reaction chamber, a first bubbling medium passing through the bubbling device to drive the suspended substance to float up from the bottom of the reaction chamber to form an upper layer of suspended substance and a lower layer of reaction solution; a drain port, used to discharge the upper layer of suspended substance or the lower layer of reaction solution from the reaction chamber.

[0014] In some embodiments, the bubbling device includes: a bubbling sieve plate, the bubbling sieve plate including a plurality of mesh holes; an inner cavity; and an air inlet, the bubbling medium enters the inner cavity from the air inlet and enters the reaction chamber from the plurality of mesh holes of the bubbling sieve plate.

[0015] In some embodiments, the ratio of the frit size of the bubbling frit to the cross-sectional area of ​​the reaction chamber is 20%-100%.

[0016] In some embodiments, the ratio of the frit size of the bubbling frit to the cross-sectional area of ​​the reaction chamber is 60%-100%.

[0017] In some embodiments, the mesh has a pore size less than or equal to 550 μm.

[0018] In some embodiments, the mesh has a pore size of 1 μm-10 μm.

[0019] In some embodiments, the distribution density of the mesh is greater than or equal to 30 meshes.

[0020] In some embodiments, the distribution density of the mesh is 1300-15000 mesh.

[0021] In some embodiments, the reaction chamber is a flexible reaction chamber.

[0022] In some embodiments, the apparatus further comprises: a clamp for clamping the outer shell of the flexible reaction chamber to block the communication between the flexible reaction chamber and the bubbling device.

[0023] In some embodiments, the device further comprises at least one inlet and outlet port for controlling the inflation degree of the flexible reaction chamber.

[0024] In some embodiments, the outer shell of the reaction chamber is provided with at least one transparent observation window.

[0025] In some embodiments, the liquid discharge port includes a pipette, a first end of which extends into the bottom of the reaction chamber, and the lower layer of reaction liquid is sucked into the pipette from the first end of the pipette and discharged from the reaction chamber from the second end of the pipette.

[0026] In some embodiments, the liquid discharge port is located at the bottom of the reaction chamber or the bottom of the bubbling device.

[0027] In some embodiments, the device further comprises at least one liquid adding guide device for adding reagents for preparing the cell lysate into the reaction chamber.

[0028] In some embodiments, the liquid adding guide device includes: a liquid adding port, through which the reagent is added into the reaction chamber; a liquid adding guide plate, used to make the reagent flow down along the wall of the reaction chamber; and a fixing hole, used to fix the liquid adding guide device on the reaction chamber.

[0029] One or more embodiments of the present specification provide a method for using a plasmid extraction device, the method comprising: adding a cell lysis solution to the reaction chamber; or, adding cells to the reaction chamber, and lysing the cells in the reaction chamber to obtain a cell lysis solution; the cell lysis solution comprises a reaction solution containing a plasmid and a suspended substance suspended in the reaction solution; a first bubbling medium is introduced into the cell lysis solution in the reaction chamber through the bubbling device, the first bubbling medium drives the suspended substance to float from the bottom of the cell lysis solution to form an upper layer of suspended substance and a lower layer of reaction solution; the upper layer of suspended substance or the lower layer of reaction solution is discharged from the reaction chamber through a discharge port to separate the upper layer of suspended substance from the lower layer of reaction solution to obtain the lower layer of reaction solution containing the plasmid.

[0030] In some embodiments, the method of use further comprises: during the process of lysing the cells, introducing a second bubbling medium into the cells in the reaction chamber through the bubbling device.

[0031] In some embodiments, the method of use further comprises: adding a reagent for preparing the cell lysate into the reaction chamber through at least one liquid adding port guide.

[0032] In some embodiments, the reaction chamber is a flexible reaction chamber; the method of use further includes: during the process of lysing the cells, clamping the outer shell of the flexible reaction chamber with a clamp to block the connection between the flexible reaction chamber and the bubbling device; during the process of introducing the first bubbling medium, canceling the clamping of the outer shell of the flexible reaction chamber by the clamp.

[0033] Some embodiments of the present specification include at least the following beneficial effects: (1) The suspended matter in the cell lysis solution is migrated and compressed to the liquid surface of the reaction solution through the bubbling device, so that the suspended matter and the reaction solution are separated in the reaction chamber in a simple and quick manner. The device has a simple structure, simple operation, short time consumption and low cost, and has wide applicability and practicality; the bubbling medium is introduced by physical means to separate the upper layer of suspended matter and the lower layer of reaction solution, thereby reducing the impact on the properties of the reaction solution and avoiding the impact on product quality; (2) By setting a filter to intercept the upper layer of suspended matter, a large amount of suspended matter can be prevented from entering the external filter, clogging the filter membrane, reducing the filtration speed of the external filter, and greatly shortening the filtration time. After intercepting the upper suspended matter, a smaller external filter can be used to filter a larger amount of lower reaction liquid. There is no need to replace the clogged filter multiple times when filtering a large amount of solution, thereby reducing the cost of using the expensive filter; (3) The outer shell of the flexible reaction chamber is clamped by a clamp to block the connection between the flexible reaction chamber and the bubbling device, which can effectively prevent the reagent from entering the bubbling screen plate of the bubbling device when preparing the cell lysate, causing residue or insufficient reaction, thereby helping to improve the effect of plasmid extraction; (4) Low-cost filter aid materials are used for filtration. The excellent liquid filterability brought by the porosity of the filter aid material makes it difficult for the protein-filter aid material mixture deposited on the external filter to be clogged, which not only increases the filtration efficiency, but also reduces the risk of clogging of the external filter and avoids the increase in the cost of consumables for replacing the external filter after clogging; (5) The technical solution of the present invention shortens the time consumption and improves the efficiency by 316%. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] This specification will be further described in the form of exemplary embodiments, which will be described in detail by the accompanying drawings. These embodiments are not restrictive, and in these embodiments, the same number represents the same structure, wherein:

[0035] Figure 1 It is one of the structural schematic diagrams of the plasmid extraction device shown in some embodiments of this specification;

[0036] Figure 2 is one of the side views of the plasmid extraction device according to some embodiments of the present specification;

[0037] Figure 3 This is the second structural schematic diagram of the plasmid extraction device according to other embodiments of this specification;

[0038] Figure 4 is a second side view of a plasmid extraction device according to other embodiments of the present specification;

[0039] Figure 5 , Figure 6 is a schematic diagram of a bubbling device according to some embodiments of the present specification;

[0040] Figure 7 is a schematic diagram of a bubbling device and a reaction chamber according to some embodiments of the present specification;

[0041] Figure 8 is a schematic diagram of a bubbling device, a reaction chamber and a holder according to some embodiments of the present specification;

[0042] Fig. 9 is a schematic diagram of a liquid adding guide device according to some embodiments of this specification;

[0043] Figure 10-13 is a schematic diagram of a plasmid extraction device according to some embodiments of the present specification;

[0044] Figure 14-15 is a schematic diagram of a plasmid extraction device according to other embodiments of this specification;

[0045] Fig.16 is an exemplary flow chart of a plasmid extraction method according to some embodiments of the present specification;

[0046] Fig.17 It is a comparison diagram of the results of cell lysates without bubbling treatment and after bubbling treatment shown in some embodiments of this specification;

[0047] Fig.18 is an electrophoresis diagram of a plasmid recovered according to the plasmid extraction method shown in some embodiments of this specification;

[0048] Fig.19 is an exemplary flow chart of a method for using a plasmid extraction device according to some embodiments of the present specification.

[0049] Reference numerals: 100, reaction container; 110, reaction chamber; 111, fixed support foot; 112, liquid receiving tray; 113, observation window; 120, bubbling device; 121, bubbling screen plate; 122, inner chamber; 123, air inlet; 130, liquid discharge port; 131, liquid discharge switch; 140, clamp; 150, liquid addition port; 160, air inlet and outlet; 170, liquid addition guide; 171, liquid addition guide plate; 172 , fixing hole; 180, upper cover; 190, liquid adding tube; 200, frame; 210, lifting assembly; 220, stirring assembly; 221, clearance hole; 300, chassis; 311, first electric pump; 312, second electric pump; 313, third electric pump; 314, fourth electric pump; 320, indicator light and buzzer; 330, touch screen; 340, emergency stop button; 350, pressure sensor interface; 360, pump tube hook. DETAILED DESCRIPTION

[0050] In order to more clearly illustrate the technical solutions of the embodiments of this specification, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some examples or embodiments of this specification. For ordinary technicians in this field, this specification can also be applied to other similar scenarios based on these drawings without creative work. Unless it is obvious from the language environment or otherwise explained, the same reference numerals in the figures represent the same structure or operation.

[0051] It should be understood that the "system", "device", "unit" and / or "module" used herein are a method for distinguishing different components, elements, parts, portions or assemblies at different levels. However, if other words can achieve the same purpose, the words can be replaced by other expressions.

[0052] As shown in this specification and claims, unless the context clearly indicates an exception, the words "a", "an", "an" and / or "the" do not refer to the singular and may also include the plural. Generally speaking, the terms "comprise" and "include" only indicate the inclusion of the steps and elements that have been clearly identified, and these steps and elements do not constitute an exclusive list. The method or device may also include other steps or elements.

[0053] Flowcharts are used in this specification to illustrate the operations performed by the system according to the embodiments of this specification. It should be understood that the preceding or following operations are not necessarily performed precisely in order. Instead, the steps may be processed in reverse order or simultaneously. At the same time, other operations may also be added to these processes, or one or more operations may be removed from these processes.

[0054] The removal of protein precipitates produced after cell lysis has always been a difficult problem in the industry. Centrifugation is usually used to remove small volumes of precipitates, but the centrifugation method has obvious operational disadvantages. It requires manual weighing, balancing, and lid closing, and manual opening after centrifugation. In addition, the number of centrifuge bottles that each centrifuge can accommodate is limited, and the centrifugation time is relatively long. For relatively large solutions, they also need to be divided into multiple bottles for centrifugation. As for the method of directly filtering the solution after lysis and neutralization using a capsule filter or a hollow fiber filter, since the protein precipitate is viscous and delicate, it is difficult to remove in advance and is easy to clog the filter. Therefore, the filtration time and consumables cost of the direct filtration method are also relatively high.

[0055] Some embodiments of the present specification provide a plasmid extraction method and device, based on which a cell lysate is layered to form an upper suspended substance and a lower reaction liquid, and a purified reaction liquid can be obtained by separating the upper suspended substance from the lower reaction liquid and filtering the lower reaction liquid. The method is simple to operate, short in time and low in cost, and is conducive to large-scale and automated production applications of plasmid extraction.

[0056] Figure 1 It is one of the structural schematic diagrams of the plasmid extraction device shown in some embodiments of this specification; Figure 2 is one of the side views of the plasmid extraction device according to other embodiments of the present specification; Figure 3 This is the second structural schematic diagram of the plasmid extraction device according to other embodiments of this specification; Figure 4 This is a second side view of a plasmid extraction device according to some embodiments of this specification. Figure 1-Figure 4 The following embodiments are understood, but the drawings are only for illustration of some of the embodiments and do not constitute a limitation of the embodiments.

[0057] In some embodiments, the plasmid extraction device includes a reaction chamber 110, a bubbling device 120 and a drain port 130. The reaction chamber 110 is used to accommodate a cell lysate, which includes a reaction solution containing plasmids and a suspended substance suspended in the reaction solution. The bubbling device 120 is located at the bottom of the reaction chamber 110. The bubbling medium drives the suspended substance to float from the bottom of the reaction chamber 110 through the bubbling device 120. The floating suspended substance gathers on the surface of the reaction solution and is stratified with the reaction solution to form an upper layer of suspended matter and a lower layer of reaction solution. The drain port 130 is used to discharge the upper layer of suspended matter or the lower layer of reaction solution out of the reaction chamber 110. Among them, the reaction chamber 110, the bubbling device 120 and the drain port 130 constitute a reaction container 100.

[0058] The reaction chamber 110 is a chamber for containing cell lysis solution. The shape of the reaction chamber 110 is not limited, for example, the reaction chamber 110 can be a cylindrical chamber, a rectangular chamber or any other shape.

[0059] In some embodiments, the reaction chamber 110 may be an open chamber (eg, an opening is provided above the reaction chamber) or a closed chamber. Preferably, in order to reduce the cleaning workload and the risk of cross infection, the reaction chamber 110 may be designed as a disposable closed chamber.

[0060] In some embodiments, the cell lysate comprises an E. coli lysate. The E. coli lysate refers to a liquid having a suspended substance formed after the E. coli is subjected to a lysis treatment. In some embodiments, the cell lysate may include a reaction solution containing a plasmid and a suspended substance suspended in the reaction solution. Wherein, the reaction solution refers to a liquid component in the cell lysate. Exemplarily, in the E. coli lysis process, the bacterial cell wall can be destroyed by using a strong alkali solution, heating or lysozyme. For example, sodium dodecyl sulfate (SDS) and TritonX-100 can lyse the cell membrane. After being treated with lysozyme and SDS or Triton X-100, the bacterial chromosomal DNA will be entangled and attached to the cell fragments. At the same time, since the bacterial chromosomal DNA is much larger than the plasmid, it is susceptible to mechanical forces and nucleases and is cut into linear fragments of different sizes. When treated with strong heat or acid or alkali, the linear chromosomal DNA of the bacteria is denatured, and the two chains of the covalently closed circular DNA (Covalently closed circular DNA, referred to as cccDNA) will not separate from each other. When the external conditions return to normal, the linear chromosome DNA fragments are difficult to renature, but are entangled with the denatured proteins and cell fragments, while the plasmid DNA double strands return to their original state, re-form natural supercoiled molecules, and exist in the liquid phase in a dissolved state. The denatured linear chromosome DNA fragments are entangled with the denatured proteins and cell fragments, suspended in the liquid phase, and form a suspended substance. Therefore, the suspended substance includes the denatured linear chromosome DNA fragments, denatured proteins and cell fragments produced by the lysis treatment of Escherichia coli.

[0061] In some embodiments, the reaction chamber 110 may be a rigid reaction chamber. When the reaction chamber 110 is a rigid reaction chamber, the outer shell of the reaction chamber 110 may be a hard material such as a metal material.

[0062] In some embodiments, the reaction chamber 110 may be a flexible reaction chamber. When the reaction chamber 110 is a flexible reaction chamber, the outer shell of the reaction chamber 110 may be made of a flexible material such as plastic.

[0063] In some embodiments, the outer shell of the reaction chamber 110 may be made of a transparent material. In some embodiments, the outer shell of the reaction chamber 110 may be provided with at least one transparent observation window. Through the above-mentioned device, the operator can better observe the conditions in the reaction chamber 110 (e.g., the lysis of E. coli, the bubbling, the stratification, etc.).

[0064] The bubbling device 120 refers to a structure for generating bubbles. For example, the types of the bubbling device 120 may include a tank-type bubbling reactor, a bubbling tube reactor, a bubbling tower, a disc-type bubbler, etc. In some embodiments, the bubbling device 120 may pass a bubbling medium from the bottom of the reaction chamber 110 to the inside of the reaction chamber 110, and the bubbling medium drives the suspended matter to float from the bottom of the reaction chamber 110. The floating suspended matter gathers on the liquid surface of the reaction solution and is stratified with the reaction solution to form an upper layer of suspended matter and a lower layer of reaction solution. In some embodiments, the bubbling device 120 may be located inside or outside the reaction chamber.

[0065] Bubble medium refers to a substance capable of forming bubbles in a reaction solution. In certain embodiments, the bubble medium may include a gas, such as air, nitrogen, carbon dioxide, an inert gas, etc. In certain embodiments, the bubble medium may also include a liquid (less dense than water and insoluble in water), such as silicone oil, etc. Since the bubble medium is insoluble or poorly soluble (e.g., the solubility is very small) in the reaction solution, when the bubble medium enters the reaction solution through the mesh of the bubble sieve plate in the bubbling device, a spherical dispersed phase is formed in the reaction, and in this application, the dispersed phase formed by the gas bubble medium or the liquid bubble medium in the reaction solution is referred to as "bubble".

[0066] In some embodiments, the bubbling medium may include a first bubbling medium and a second bubbling medium. The first bubbling medium is used to separate the upper suspended matter from the lower reaction liquid. The second bubbling medium is used to mix the liquid during the E. coli lysis process. The first bubbling medium and the second bubbling medium may be the same or different, which may be determined based on actual conditions. For more information about the first bubbling medium and the second bubbling medium, please refer to Fig.16 and its related description.

[0067] In some embodiments, the bubbling device 120 may be disposed at the bottom of the reaction chamber 110. In some embodiments, the bubbling device 120 may be non-detachably connected to the reaction chamber 110, for example, integrally formed, welded, etc. In some embodiments, the bubbling device 120 may be detachably connected to the reaction chamber 110, for example, the bubbling device 120 may be snap-fitted to the bottom of the reaction chamber 110 by threads, buckles, etc. In some embodiments, the shape of the bubbling device 120 matches the shape of the reaction chamber 110. For example, when the reaction chamber 110 is a cylindrical cavity, the bubbling device 120 may be disc-shaped. For another example, when the reaction chamber 110 is a rectangular cavity, the bubbling device 120 may be strip-shaped. In some embodiments, the shape of the bubbling device 120 may also be different from the shape of the reaction chamber 110.

[0068] In some embodiments, the reaction chamber 110 and the bubbling device 120 may be separate and independent structures. In some embodiments, the bubbling device 120 may be pre-placed at the bottom of the reaction chamber 110, or may be placed at the bottom of the reaction chamber 110 before bubbling.

[0069] In some embodiments, the bubble coverage area of ​​the bubbler 120 (e.g., the coverage area of ​​the mesh on the bubbler screen) can be the same as the cross-sectional area of ​​the reaction chamber. In some embodiments, the bubbler area can be smaller than the cross-sectional area of ​​the reaction chamber. For more information about the bubbler screen and the ratio of the bubbler screen size to the cross-sectional area of ​​the reaction chamber, see Figure 5-Figure 7 and its related description.

[0070] In some embodiments, the bubbling device 120 may include one or more bubbling devices, which may be specifically configured according to actual needs and are not limited here. In some embodiments, multiple bubbling devices 120 may be configured at different liquid level heights to achieve sufficient stratification of the suspended matter and the reaction liquid.

[0071] In some embodiments, the bubbling device 120 may include a bubbling screen plate 121, an inner cavity 122, and an air inlet 123. For more information about the structure of the bubbling device 120, see Figure 5-Figure 7 and its related description.

[0072] In some embodiments of the present specification, the suspended matter in the cell lysis solution is migrated and compressed to the liquid surface position of the reaction solution through a bubbling device, so that the suspended matter and the reaction solution are stratified in the reaction chamber, and the suspended matter and the reaction solution can be separated simply and quickly. The device has a simple structure, simple operation, short time consumption and low cost, and has wide applicability and practicality.

[0073] The liquid discharge port 130 may be used to discharge the upper suspended matter or the lower reaction liquid out of the reaction chamber 110 .

[0074] In some embodiments, the liquid discharge port 130 includes a pipette, a first end of which extends into the bottom of the reaction chamber 110 , and the lower layer of reaction liquid is sucked into the pipette from the first end of the pipette and discharged from the reaction chamber 110 from the second end of the pipette.

[0075] The pipette is a pipe for discharging the lower layer of reaction liquid. The first end of the pipette extends into the bottom of the reaction chamber 110 and contacts the reaction liquid in the reaction chamber 110. The second end of the pipette is located outside the reaction chamber 110. The second end of the pipette can be connected to an external container for receiving the sucked lower layer of reaction liquid.

[0076] In some embodiments, the pipette can be connected to an external suction device (eg, an electric pump, etc.) to automatically discharge the lower layer of reaction liquid from the reaction chamber 110 via the pipette. For more information about the pipette, see Figure 14-15 Related description.

[0077] In some embodiments of the present specification, by extending a pipette into the bottom of the reaction chamber, the lower layer of reaction liquid can be more thoroughly discharged from the reaction chamber, which is beneficial to improving the efficiency and effect of plasmid extraction.

[0078] In some embodiments, the drain port 130 may be located at the bottom of the reaction chamber 110 or the bottom of the bubbling device 120, so that the lower layer of reaction liquid, the cleaning liquid used to clean the reaction chamber, and / or the waste liquid and other liquids can be more conveniently discharged from the reaction chamber 110. Among them, the side of the reaction chamber 110 away from the liquid surface of the reaction liquid is the bottom of the reaction chamber 110, and the side of the bubbling device 120 close to the bottom of the reaction chamber 110 is the bottom of the bubbling device 120. In some embodiments, the liquid in the reaction chamber 110 can be discharged from the drain port 130 under the action of gravity. In some embodiments, the drain port 130 can be connected to a pump to automatically pump out the liquid from the drain port 130.

[0079] In some embodiments, the drain port 130 further includes a drain switch 131 for controlling the switch of the drain port. The drain switch 131 can be opened automatically or manually to allow the liquid to be discharged from the reaction chamber 110 through the drain port 130. The drain switch 131 can include an electric valve, a pneumatic valve, a switch knob, etc. In some embodiments, when the drain port 130 is located at the bottom of the bubbling device 120, after the drain switch 131 is closed manually or automatically, the drain switch 131 closes the bottom channel of the inner cavity 122, and at this time, the bubbling medium is introduced from the air inlet 123 of the bubbling device 120, and the bubbling medium overflows from the bubbling sieve plate 121, completing the process of bubbling and aggregating suspended matter. When the drain switch 131 is opened, the lower layer of the reaction liquid in the reaction chamber 110 passes through the bubbling sieve plate 121, enters the inner cavity 122, and is discharged from the drain port 130. The bubbling sieve plate 121 serves to prevent the suspended matter from being discharged together with the reaction liquid. For more information about the bubble screen 121, the inner cavity 122 and the air inlet 123, see Figure 5-Figure 7 and its related description.

[0080] In some embodiments, in order to further filter the lower layer of reaction liquid, a filter screen can be set at the drain port 130 to intercept the upper layer of suspended matter. The filter screen can be made of a polymer filter membrane, filter paper, filter cloth, etc. For more information on separating the upper layer of suspended matter and the lower layer of reaction liquid, see Fig.16 and its related description.

[0081] In some embodiments, the drain port 130 can be connected to an external filter. The external filter is disposed outside the reaction chamber 110. The external filter is used to filter the lower layer of the reaction solution to obtain a purified reaction solution. In some embodiments, the external filter can be a capsule filter, a hollow fiber filter, etc. This application does not limit the external filter.

[0082] In some embodiments, the drain port may be located at the top of the reaction chamber 110 to discharge the upper suspended matter.

[0083] In some embodiments, after the lower layer reaction liquid separated from the upper layer suspension is discharged from the reaction chamber 110 through the liquid discharge port 130 , it can be directly purified by an external filter to obtain a purified lower layer reaction liquid containing plasmids.

[0084] In some embodiments, before the lower layer reaction liquid separated from the upper layer suspension enters the external filter, a filter aid material may be added to the lower layer reaction liquid, and then the lower layer reaction liquid after the filter aid material is added is purified by the external filter to obtain a purified lower layer reaction liquid containing plasmids. For more information on purifying the lower layer reaction liquid, see Fig.16 and its related description.

[0085] In some embodiments of the present specification, by setting a filter to intercept the upper suspended matter, a large amount of suspended matter can be prevented from entering the external filter and clogging the filter membrane, reducing the filtration speed of the external filter, and greatly shortening the filtration time. After intercepting the upper suspended matter, a smaller external filter can be used to filter a large amount of lower layer reaction liquid, without having to replace the clogged filter multiple times when filtering a large amount of solution, thereby reducing the cost of using expensive filters.

[0086] In some embodiments, the plasmid extraction device further includes at least one liquid adding port 150 for adding reagents for preparing cell lysate into the reaction chamber 110 .

[0087] In some embodiments, the number of liquid adding ports 150 can be determined based on actual needs. For example, the number of liquid adding ports 150 can be set according to the type of reagent. It can be understood that when the number of liquid adding ports 150 is only one, the various reagents for preparing the E. coli lysis suspension (e.g., resuspension reagent, lysis reagent, and neutralization reagent, etc.) are all added to the reaction chamber 110 from the liquid adding port 150; when the number of liquid adding ports 150 is multiple, the various reagents for preparing the E. coli lysis suspension can be added to the reaction chamber 110 from different liquid adding ports 150.

[0088] In some embodiments, the liquid adding port 150 may be directly connected to the reaction chamber 110 , for example, the liquid adding port 150 may be directly disposed on a side wall or an upper end of the reaction chamber 110 .

[0089] In some embodiments, the liquid adding port 150 may be indirectly connected to the reaction chamber 110 based on other structures. In some embodiments, the liquid adding guide 170 includes the liquid adding port 150 , a liquid adding guide plate 171 and a fixing hole 172 .

[0090] The liquid adding guide plate 171 can make the reagent flow toward the wall of the reaction chamber 110, and then make the reagent flow down along the wall of the reaction chamber 110, so as to prevent the reagent from falling directly into the reaction chamber 110, causing splashing and impacting the reactants inside and affecting the experimental effect.

[0091] In some embodiments, the liquid adding guide 170 can be fixed to the side wall (e.g., inner wall or outer wall) of the reaction chamber 110 through the fixing hole 172. In some embodiments, the liquid adding guide 170 can be fixed to the upper cover of the reaction chamber 110 through the fixing hole 172 (for the description of the upper cover, see Figure 10-Figure 15 ) or an upper wall (for example, an upper wall surface when the reaction chamber 110 is a closed type).

[0092] In some embodiments, the plasmid extraction solid-liquid system may further include at least one air inlet and outlet 160. The air inlet and outlet 160 is disposed on the reaction chamber 110 and extends out of the reaction chamber 110. In some embodiments, the structure and arrangement of the air inlet and outlet 160 may be the same as or different from the structure and arrangement of the liquid adding port 150. In some embodiments, the air inlet and outlet 160 and the liquid adding port 150 may have the same structure, that is, the liquid adding port 150 may be used for both adding reagents and air inlet and outlet.

[0093] Gas can be introduced into the reaction chamber 110 and / or gas in the reaction chamber 110 can be exhausted through the inlet and outlet ports 160. In some embodiments, when preparing a cell lysate, the inflation degree of the flexible reaction chamber 110 can be controlled through the inlet and outlet ports 160 to avoid the risk of poor reaction effect due to insufficient inflation or bursting due to excessive inflation.

[0094] For an open reaction chamber, there is no need to set an inlet and outlet.

[0095] Figure 5 , Figure 6 is a schematic diagram of the structure of a bubbling device according to some embodiments of this specification; Figure 7 is a schematic diagram of a bubbling device and a reaction chamber according to some embodiments of this specification. Figure 5-Figure 7 The following embodiments are understood, but the drawings are only for illustration of some of the embodiments and do not constitute a limitation of the embodiments.

[0096] In some embodiments, the bubbling device 120 includes a bubbling screen 121, an inner cavity 122 and an air inlet 123. The bubbling screen 121 includes a plurality of meshes; the bubbling medium enters the inner cavity 122 from the air inlet 123 and enters the reaction chamber 110 from the plurality of meshes of the bubbling screen 121.

[0097] The bubbling screen plate 121 is the core structure of the bubbling device 120 .

[0098] When the cross-sectional area of ​​the reaction chamber 110 is constant, the larger the bubbling coverage area (the coverage area of ​​the mesh in the bubbling screen), the better the solid-liquid separation effect (for example, more suspended matter gathers on the liquid surface), and the shorter the time required for solid-liquid separation. When the bubbling medium rises in the reaction liquid, it tends to diffuse in all directions, and can bring up the suspended matter in the reaction liquid. Therefore, if the mesh of the bubbling screen plate 121 only covers a part of the cross-sectional area of ​​the reaction chamber 110, an ideal solid-liquid separation effect can also be achieved (for example, the solid-liquid separation time and the clarity of the lower layer of the reaction liquid are both within an acceptable range). In some embodiments, the ratio of the screen size (the coverage of the mesh) of the bubbling screen plate 121 to the cross-sectional area of ​​the reaction chamber 110 is greater than or equal to 20%. In some embodiments, the ratio of the sieve plate size (mesh coverage) of the bubbling sieve plate 121 to the cross-sectional area of ​​the reaction chamber 110 is 20%-60%, 20%-40%, 20%-100%, 40%-60%, 40%-100%, 60%-100%. In some embodiments, the ratio of the sieve plate size (mesh coverage) of the bubbling sieve plate 121 to the cross-sectional area of ​​the reaction chamber 110 can be at least one of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, etc., which can be set according to specific needs and is not limited here.

[0099] Therefore, when the same reaction chamber 110 is used to process the same volume of cell lysate, the area of ​​the bubbling screen plate can be reduced to reduce costs.

[0100] When processing the same volume of cell lysate, the diameter of the bubbling sieve plate 121 remains unchanged, and the smaller the cross-sectional area of ​​the reaction chamber 110, the better the solid-liquid separation effect. This is also determined by the relatively larger coverage area of ​​the bubbling sieve plate 121; in addition, when processing the same volume of liquid, if the cross-sectional area of ​​the reaction chamber 110 is smaller, the liquid level is higher, and the disturbance of the upper suspended matter when discharging the lower reaction liquid is also smaller.

[0101] In some embodiments, the bubble screen 121 may include a plurality of meshes.

[0102] The mesh refers to the through holes distributed on the bubble screen plate 121. The aperture (diameter) of the mesh can be determined in a variety of ways. In some embodiments, the aperture of the mesh can be an empirical value, an experimental value, or a simulated value.

[0103] The mesh aperture can affect the floating speed of the suspended matter and the stratification effect of the suspended matter and the reaction liquid. The smaller the mesh aperture, the smaller the bubbles generated when the bubbling medium passes through the mesh, the slower the bubble floating speed, and the easier it is to bring the suspended matter to the liquid surface during the floating process. There will be no obvious disturbance phenomenon in the liquid phase, and the smaller the impact force caused when the liquid surface is broken, the calmer the liquid surface, and the more stable the aggregation state of the suspended matter will be. On the contrary, the larger the mesh aperture, the larger the bubbles generated when the bubbling medium passes through the mesh. When the bubbles are larger, the floating is faster. During the floating process, it is easy to "exclude" the suspended matter in the path to both sides and cannot be brought to the liquid surface for aggregation. It is also easy to produce disturbance phenomenon in the liquid phase, accelerate the convection of the liquid, which is not conducive to the aggregation of suspended matter. The impact force caused by the large bubbles when they break at the liquid surface is obvious, and even the liquid surface will appear tumbling state, breaking the aggregation of suspended matter. In some embodiments, the mesh aperture is less than or equal to 550μm. In some embodiments, the mesh aperture is 0.5 μm-10 μm, 0.5 μm-100 μm, 0.5 μm-500 μm, 10 μm-100 μm, 10 μm-500 μm, 100 μm-500 μm, etc. In some embodiments, the mesh aperture can be at least one of 0.5 μm, 1 μm, 5 μm, 10 μm, 15 μm, 20 μm, 40 μm, 50 μm, 60 μm, 80 μm, 100 μm, 120 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, etc., which can be set according to specific needs and is not limited here.

[0104] The mesh distribution density can affect the stratification speed of the suspended matter and the reaction liquid. The greater the mesh distribution density, the greater the probability of the suspended matter contacting the bubbles, and the bubbles generated when the bubbling medium passes through the mesh can bring the suspended matter to the liquid surface faster. In some embodiments, the mesh distribution density is greater than or equal to 30 meshes. In some embodiments, the mesh distribution density is 30-1300 mesh, 30-5000 mesh, 30-10000 mesh, 1300-5000 mesh, 1300-10000 mesh, 1300-15000 mesh, 5000-10000 mesh, 5000-15000 mesh, etc. In some embodiments, the mesh distribution density can be at least one of 30 mesh, 40 mesh, 50 mesh, 100 mesh, 150 mesh, 200 mesh, 250 mesh, 300 mesh, 400 mesh, 500 mesh, 600 mesh, 800 mesh, 1000 mesh, 1200 mesh, 1500 mesh, 2000 mesh, 2500 mesh, 3000 mesh, 4000 mesh, 5000 mesh, 6000 mesh, 7000 mesh, 8000 mesh, 9000 mesh, 10000 mesh, 12000 mesh, 13000 mesh, 14000 mesh, 15000 mesh, etc., which can be set according to specific needs and is not limited here.

[0105] In some embodiments, the bubbling device 120 may include one or more bubbling sieve plates 121. The sieve plate size, mesh aperture, and mesh distribution density of the multiple bubbling sieve plates 121 may be the same or different. The multiple bubbling sieve plates 121 may be arranged at different liquid level heights along the height direction of the reaction chamber 110. For example, the multiple bubbling sieve plates 121 may be connected to the inner side wall of the reaction chamber. For another example, the multiple bubbling sieve plates 121 may be connected to the stirring rod of the stirring assembly.

[0106] In some embodiments, the material of the bubbling screen plate 121 is not limited. For example, the material of the bubbling screen plate 121 includes metal sintered porous material, machined microporous metal material, polymer porous material, dense fiber porous material, etc.

[0107] The inner cavity 122 refers to a cavity structure for accommodating the bubbling medium in the bubbling device 120. In some embodiments, the cross-section of the inner cavity 122 may be rectangular, circular, or semicircular, etc., which is not limited in the present application.

[0108] The air inlet 123 refers to a passage for the bubbling medium to enter the inner cavity 122 .

[0109] In some embodiments, the bubbling medium enters the inner cavity 122 from the air inlet 123, overflows from the multiple meshes of the bubbling screen plate 121, forms bubbles and enters the reaction chamber 110. The bubbles float up under the action of buoyancy and drive the suspended matter to float up continuously. The floating suspended matter gathers on the liquid surface of the reaction liquid and is stratified with the reaction liquid, thereby forming an upper layer of suspended matter and a lower layer of reaction liquid.

[0110] In some embodiments of the present specification, the bubbling medium forms bubbles through the bubbling device and enters the reaction chamber, driving the suspended matter to float up from the bottom of the reaction chamber, thereby achieving stratification of the suspended matter and the reaction liquid. The method is simple in principle, and the bubbling device has a simple structure and is easy to implement.

[0111] Figure 8 is a schematic diagram of a bubbling device, a reaction chamber and a holder according to some embodiments of this specification. Figure 8 The following embodiments are understood, but the drawings are only for illustration of some of the embodiments and do not constitute a limitation of the embodiments.

[0112] In some embodiments, the plasmid extraction solid-liquid separation device further includes a clamp 140 for clamping the outer shell of the flexible reaction chamber to block the communication between the flexible reaction chamber and the bubbling device 120. The clamp 140 is disposed in the reaction container 100.

[0113] For example, when preparing a cell lysate, the outer shell of the flexible reaction chamber can be clamped by the clamp 140 to block the connection between the flexible reaction chamber and the bubbling device 120, so that the E. coli can fully react with a variety of reagents, and avoid the reagents from entering the bubbling screen plate 121 of the bubbling device 120 to cause residues or inadequate reactions. When the preparation of the cell lysate is completed, the clamp 140 can be loosened to connect the flexible reaction chamber and the bubbling device 120, and the bubbling device 120 introduces a bubbling medium into the flexible reaction chamber, so that the bubbling medium drives the suspended matter to float from the bottom of the flexible reaction chamber through the bubbling device 120, and the suspended matter after floating gathers on the surface of the reaction solution and is stratified with the reaction solution to form an upper layer of suspended matter and a lower layer of reaction solution.

[0114] In some embodiments of the present specification, the outer shell of the flexible reaction chamber is clamped by a clamp to block the connection between the flexible reaction chamber and the bubbling device, which can effectively prevent the reagent from entering the bubbling screen plate of the bubbling device when preparing the cell lysis solution, causing residue or insufficient reaction, thereby helping to improve the effect of plasmid extraction.

[0115] In some embodiments, when the reaction chamber 110 is a rigid reaction chamber, a retractable partition may be provided between the rigid reaction chamber and the bubbling device 120 to block the communication between the rigid reaction chamber and the bubbling device 120 .

[0116] Figure 10-Figure 15 is a schematic diagram of a plasmid extraction device according to some embodiments of this specification. Figure 10-Figure 15 The following embodiments are understood, but the drawings are only for illustration of some of the embodiments and do not constitute a limitation of the embodiments.

[0117] See also Figure 10-Figure 15 In some embodiments, the plasmid extraction device may further include a rack 200 and a chassis 300 .

[0118] In some embodiments, the rack 200 includes a lifting assembly 210 and a stirring assembly 220. The lifting assembly 210 is used to lift the stirring assembly 220. The stirring assembly 220 is used to stir and mix the reagents in the reaction chamber 110 to prepare a cell lysate. In some embodiments, the stirring assembly 220 includes at least one stirring blade. In some embodiments, the stirring assembly 220 includes at least two stirring blades. Preferably, three stirring blades may be included. The type of stirring blade may include but is not limited to an anchor stirring blade.

[0119] In some embodiments, the stirring assembly 220 is connected to the upper cover 180 of the reaction chamber 110. The stirring assembly 220 and the upper cover 180 can be raised and lowered under the control of the lifting assembly 210. When the lifting assembly 210 lowers the stirring assembly 220 and the upper cover 180 to the lowest position, the stirring assembly 220 is inside the reaction chamber 110 and the upper cover 180 closes the reaction chamber 110, and the stirring assembly 220 can stir inside the reaction chamber 110. When the lifting assembly 210 does not lower the stirring assembly 220 and the upper cover 180 to the lowest position, the stirring assembly 220 is not completely inside the reaction chamber 110, that is, the upper cover 180 does not close the reaction chamber 110, and the stirring assembly 220 stops stirring at this time. The upper cover 180 can prevent foreign matter from falling into the reaction chamber 110 and the reagent from splashing out.

[0120] In some embodiments, the bubbling device 120 can be disposed on the stirring assembly 220. The bubbling device 120 is fixedly connected to the stirring assembly 220. In some embodiments, a multi-layer bubbling device including a plurality of bubbling sieve plates can be disposed on the stirring assembly 220, and each layer of the bubbling sieve plates is located at a different height of the stirring assembly 220. When the bubbling device 120 rotates with the stirring assembly 220, the bubbling area can be increased, the bubbling effect can be optimized, and the suspended matter can be fully floated and aggregated. For more information about the bubbling device 120, see Figure 1-Figure 4 and its related description.

[0121] In some embodiments, the suction tube (i.e. Fig.12 , Fig.13 The pipette 190 shown in the figure passes through the upper cover 180 and is connected to the lifting assembly 210. At least one stirring blade in the stirring assembly 220 is arranged around the clearance hole 221. The clearance hole 221 surrounds the pipette 190, that is, the pipette 190 passes through the clearance hole 221, the upper cover 180 and is connected to the lifting assembly 210. The clearance hole 221 has no contact with the pipette 190. When the stirring assembly 220 is in operation, it rotates around the pipette 190, and the pipette 190 does not rotate. The pipette 190 and the lifting assembly 210 are relatively fixed.

[0122] In some embodiments, the lifting assembly 210 and the stirring assembly 220 are respectively connected to the chassis 300 for communication so as to perform corresponding functions under the control of the chassis 300 .

[0123] In some embodiments, at least one electric pump and a pump tube are disposed in the chassis 300. In some embodiments, each liquid filling port 150 may correspond to an electric pump, and multiple liquid filling ports 150 may also share one electric pump.

[0124] In some embodiments, the at least one electric pump includes a first electric pump 311 , a second electric pump 312 , a third electric pump 313 , and a fourth electric pump 314 .

[0125] In some embodiments, one end of the pump tube (hereinafter referred to as the first pump tube) extends into the reagent barrel, and the other end is connected to the liquid adding port 150. The first electric pump 311, the second electric pump 312, and the third electric pump 313 can add the reagent into the reaction chamber 110 through the liquid adding port 150 through the first pump tube.

[0126] In some embodiments, one end of the pump tube (hereinafter referred to as the second pump tube) can be connected to the liquid suction tube 190 or the liquid discharge port 130, and the other end can be connected to the external filter. The fourth electric pump 314 can suck out or discharge the reaction liquid to the external filter through the second pump tube.

[0127] In some embodiments, the chassis 300 further includes an indicator light and a buzzer 320, a touch screen 330, an emergency stop button 340, a pressure sensor interface 350, a pump tube hook 360, etc. The indicator light is used to indicate the status of the device, for example, the chassis 300 is turned on, the chassis 300 is running, the chassis 300 is shut down, the chassis 300 device is faulty, the external filter pressure is too high, etc. The buzzer is used to indicate a fault condition, for example, the chassis 300 is faulty, the pressure in the reaction chamber 110 is too high, the pressure in the external filter is too high, etc. The emergency stop button 340 is used to directly shut down the chassis 300. The pump tube hook 360 is used to fix the pump tube to prevent the pump tube from falling out due to vibration during the addition and discharge process.

[0128] In some embodiments, the chassis 300 is used to control the operating parameters of the lifting assembly 210 and the stirring assembly 220. For example, the lifting speed, lifting height, and lifting time of the lifting assembly 210 can be controlled through the touch screen 330 of the chassis 300 and the program installed in its system. For example, the stirring speed, stirring direction, and stirring time of the stirring assembly 220 can be controlled through the touch screen 330 of the chassis 300 and the program installed in its system. For another example, the liquid adding speed, liquid adding time, and liquid adding amount of the electric pump can be controlled through the touch screen 330 of the chassis 300 and the program installed in its system. For another example, the bubbling time and bubbling rate of the bubbling device 120 can be controlled through the touch screen 330 of the chassis 300 and the program installed in its system.

[0129] In some embodiments, the pressure sensor interface 350 can be connected to an external filter. The software monitors the pressure in the external filter in real time according to the feedback of the pressure sensor. When the pressure is higher than a certain high pressure threshold, the software issues an instruction to stop the fourth electric pump 314. When the pressure drops to a certain low pressure threshold, the software issues an instruction to continue the fourth electric pump 314. Through real-time monitoring, reliable filtration can be automatically completed to avoid the pump tube bursting due to excessive pressure inside the external filter.

[0130] See also Figure 10-Figure 15In some embodiments, the reaction vessel 100 may further include a fixed support foot 111. The fixed support foot 111 is used to support the reaction chamber 110. In some embodiments, the fixed support foot 111 may be connected to the bottom of the reaction chamber 110 and supported or fixed on the ground or the chassis of the rack 200. In some embodiments, the fixed support foot 111 may be connected to the side wall or upper wall of the reaction chamber 110 and fixed to the side wall or top of the rack 200. In some embodiments, the reaction vessel 100 may further include a liquid receiving tray 112. The liquid receiving tray 112 is located below the reaction chamber 110, for example, in the space where the fixed support foot 111 raises the reaction chamber 110. The liquid receiving tray 112 can receive the reaction liquid leaked from the drain port 130, etc.

[0131] In some embodiments, the reaction vessel 100 may further include a feeding port (not shown in the figure), and the Escherichia coli cells may be added to the reaction chamber 110 through the feeding port. In some embodiments, the Escherichia coli cells may also be added to the reaction chamber 110 through the liquid adding port 150. For example, the Escherichia coli cells may be mixed with a portion of the resuspension reagent for initial resuspension, added to the reaction chamber 110 through the liquid adding port 150, and then the remaining resuspension reagent may be added to the reaction chamber 110 through the liquid adding port. In some embodiments, the Escherichia coli cells may be added to the reaction chamber 110 through the opening of the open reaction chamber. For example, the upper cover may be lifted by a lifting assembly, and the Escherichia coli cells may be added to the reaction chamber 110 through the opening above the reaction chamber.

[0132] Some embodiments of this specification also provide a plasmid extraction method, which includes the steps shown in process 1600. Fig.16 is an exemplary flow chart of the plasmid extraction method according to some embodiments of this specification. Fig.16 As shown, process 1600 may include steps 1610 to 1630. In some embodiments, process 1600 may be based on Figure 1-Figure 15 The plasmid extraction is performed using the solid-liquid separation device shown.

[0133] Step 1610, provide cell lysis solution.

[0134] For instructions on cell lysis buffer, see Figure 1-Figure 15 and its related description.

[0135] In some embodiments, the cell lysate can be prepared in advance and then placed in the reaction chamber. In some embodiments, the cell lysate can be prepared in the reaction chamber.

[0136] In some embodiments, providing a cell lysis solution comprises: providing cells; lysing the cells to obtain a cell lysis solution, wherein during the lysing of the cells, a second bubbling medium is introduced into the cells.

[0137] In some embodiments, cells include but are not limited to bacteria. In some embodiments, in addition to bacteria, cells may also include fungi and mammalian cells, etc. In some embodiments, cell lysates include but are not limited to bacterial lysates.

[0138] The following takes the lysis of Escherichia coli cells as an example to illustrate the method of obtaining a bacterial lysis solution. In some embodiments, providing an Escherichia coli lysis suspension includes: obtaining Escherichia coli cells; and lysing the Escherichia coli cells to obtain an Escherichia coli lysis suspension.

[0139] In some embodiments, the E. coli cells containing the plasmid can be placed in a culture medium for fermentation to obtain a fermentation broth; the fermentation broth is centrifuged to obtain E. coli cells. For example, the E. coli cells containing the plasmid can be placed in a culture medium and shaken overnight at 37° C. to reach an appropriate growth density to obtain a fermentation broth. The fermentation broth is centrifuged at 4000-5500 rpm for 5-10 minutes, and the supernatant is discarded to obtain the E. coli cells.

[0140] In some embodiments, the centrifugation method includes, but is not limited to, centrifugation using a conventional angle rotor centrifuge, centrifugation using a conventional horizontal rotor centrifuge, and centrifugation using a continuous flow centrifuge.

[0141] In some embodiments, the method of lysing the Escherichia coli cells includes alkaline lysis, thermal lysis, or other methods. The present application does not limit the method of lysing the Escherichia coli cells. The alkaline lysis method includes steps S1-S3:

[0142] S1: adding a resuspension reagent (e.g., 1% glucose, 50 mM / L EDTA, pH 8.0, 25 mM / L Tris-HCl, pH 8.0) to the Escherichia coli cells, mixing and resuspending the Escherichia coli cells to obtain an Escherichia coli resuspension solution;

[0143] S2: adding a lysis reagent (e.g., 0.2 mM / L NaOH, 1% SDS) to the E. coli heavy suspension, mixing and lysing the E. coli cells to obtain an E. coli cell lysis solution;

[0144] S3: adding a neutralizing agent (eg, 5 mol / L KAc (potassium acetate), pH 4.8) to the E. coli cell lysate, mixing and neutralizing the E. coli cell lysate to obtain a bacterial lysate.

[0145] In the above steps S1-S3, the glucose in the resuspension reagent can prevent the suspended Escherichia coli from quickly settling to the bottom of the reaction chamber; EDTA can inhibit the activity of DNase (deoxyribonuclease) and microbial growth. The NaOH in the lysis reagent is strongly alkaline. In a strongly alkaline environment, the cell membrane changes from a double-layer membrane structure to a microcapsule structure, that is, NaOH can dissolve cells and release DNA. SDS (sodium dodecyl sulfate) is used in combination with NaOH to enhance the strong alkalinity of NaOH, and SDS acts as an anionic surfactant to destroy the lipid bilayer membrane. The potassium acetate in the neutralization reagent can replace the sodium ions in SDS with potassium ions to form water-insoluble PDS (potassium dodecyl sulfate), which promotes protein precipitation; acetic acid can neutralize the alkalinity of NaOH.

[0146] In some embodiments, the volume ratio of the resuspension reagent, the lysis reagent and the neutralization reagent is in the range of 1-10: 1-10: 1-10. Preferably, the volume ratio of the resuspension reagent, the lysis reagent and the neutralization reagent is 1: 1: 1. In some embodiments, the volume ratio of the resuspension reagent to the Escherichia coli cell lysis solution is 1: 10.

[0147] In some embodiments, in step S1, the E. coli cells may be first contained in a centrifuge bottle or other container, a portion of the resuspension reagent may be added for initial resuspension, the initial resuspension liquid may be added to the reaction chamber, and the remaining resuspension reagent may be further added for stirring and resuspension.

[0148] The thermal lysis method includes suspending E. coli cells in a buffer containing Triton X-100 (polyethylene glycol octylphenyl ether) and lysozyme that can digest cell walls, heating to 100° C. to lyse them and obtain a bacterial lysate. The thermal lysis method is only an exemplary description and does not constitute a limitation on the implementation method.

[0149] In some embodiments, in step S2, during the lysis of the Escherichia coli cells, a second bubbling medium may be introduced into the Escherichia coli cells through a bubbling device.

[0150] For instructions on the bubbling device and reaction chamber, please refer to Figure 1-Figure 15 and its related description.

[0151] The second bubbling medium refers to a substance introduced into the bubbling device for the purpose of mixing the liquid during the lysis of the E. coli cells. In some embodiments, the second bubbling medium can be a gas medium such as air, carbon dioxide, nitrogen, an inert gas, etc. In some embodiments, the second bubbling medium can also be a liquid medium with a density less than that of water and insoluble in water, such as silicone oil, etc.

[0152] In some embodiments, in order to promote the mixing degree of E. coli cells and multiple reagents, during the lysis of E. coli cells, the plasmid extraction device can pass a second bubbling medium into the E. coli cells through a bubbling device. This can have three effects. The first aspect is to promote liquid mixing through bubbling and enhance the effect of E. coli lysis; the second aspect is to greatly reduce the adverse effects of reagents entering the bubbling device on E. coli lysis. For example, during the lysis of E. coli, the reagents added first enter the bubbling device, resulting in these reagents being unable to react chemically with the reagents added later, thereby affecting the lysis effect; the third aspect is to prevent the suspended matter produced after the reagents in the bubbling device complete the chemical reaction from clogging the mesh of the bubbling device.

[0153] During the lysis process of E. coli, the gas in the reaction chamber can be discharged by controlling the opening and closing of the inlet and outlet ports, and / or gas can be introduced into the reaction chamber to control the expansion degree of the reaction chamber, thereby avoiding the risk of poor reaction effect due to insufficient expansion and bursting due to excessive expansion.

[0154] In some embodiments of the present specification, during the lysis of Escherichia coli cells, a second bubbling medium is introduced into the Escherichia coli cells through a bubbling device, so that the liquid can be mixed more fully, thereby facilitating faster and better formation of a bacterial lysis solution.

[0155] Step 1620, introducing a first bubbling medium into the bubbling device, the first bubbling medium passes through the bubbling device from the bottom of the E. coli lysis suspension, driving the suspended matter to float up, the floating suspended matter gathers on the surface of the reaction liquid and is stratified with the reaction liquid to form an upper layer of suspended matter and a lower layer of reaction liquid.

[0156] For a description of the suspension and the reaction solution, see Figure 1-Figure 13 and its related description.

[0157] The first bubbling medium refers to a substance introduced into the bubbling device to promote the floating of suspended matter. In some embodiments, the first bubbling medium can be a gas medium such as air, carbon dioxide, nitrogen, an inert gas, etc. In some embodiments, the first bubbling medium can also be a liquid medium with a density less than that of water and insoluble in water, such as silicone oil, etc. The first bubbling medium and the second bubbling medium can be the same or different, and can be specifically set according to actual conditions.

[0158] In certain embodiments, in order to ensure that the suspended matter can float and gather at the liquid surface of the reaction solution, the first bubbling medium can be introduced into the bubbling device (or bacterial lysate) at a preset bubbling rate. Wherein, the preset bubbling rate can be an empirical value, an experimental value or a simulated value, etc. In certain embodiments, the preset bubbling rate is 0.5L / min-20L / min. In certain embodiments, the preset bubbling rate is 0.5L / min-5L / min, 3L / min-5L / min, 3L / min-8L / min, 5L / min-8L / min, 5L / min-20L / min, 8L / min-20L / min, etc. In some embodiments, the preset bubbling rate can be at least one of 0.5L / minute, 3L / minute, 4L / minute, 5L / minute, 6L / minute, 7L / minute, 8L / minute, 10L / minute, 12L / minute, 15L / minute, 18L / minute, 20L / minute, etc., which can be set according to specific needs and is not limited here.

[0159] The preset bubbling rate can affect the time and effect of suspended matter aggregation. If the preset bubbling rate is too slow, the suspended matter aggregation time will be prolonged, or even the suspended matter cannot be effectively aggregated, and the expected experimental effect of rapid removal of suspended matter cannot be achieved. If the preset bubbling rate is too fast, the aggregation of suspended matter will be disturbed, affecting the effect of suspended matter aggregation.

[0160] In some embodiments, the first bubbling medium (e.g., air) overflows through the multiple mesh holes of the bubbling screen plate of the bubbling device to form a large number of small bubbles, and the bubbles float up under the action of buoyancy. The floating bubbles will drive the local liquid to move upward, thereby driving the suspended matter near the liquid in the bubble generation area (the bottom of the Escherichia coli lysis suspension) to float up, or the bubbles will collide with the suspended matter, causing the suspended matter to float up. The floating suspended matter gathers on the surface of the reaction liquid and is stratified with the reaction liquid, thereby forming an upper layer of suspended matter and a lower layer of reaction liquid.

[0161] Fig.17 It is a comparison chart of the results of Escherichia coli lysate without bubbling treatment and that with bubbling treatment shown in some embodiments of this specification.

[0162] like Fig.17As shown, there is a significant difference in the state of the suspended matter between the E. coli lysate without bubbling treatment (left picture) and the E. coli lysate with bubbling treatment (left picture). In the E. coli lysate without bubbling treatment, the suspended matter is dispersed in the reaction solution; while in the E. coli lysate with bubbling treatment, most of the suspended matter migrates to the liquid surface of the reaction solution and gathers into a tight "cake shape", and the reaction solution below can present a clearer state. It can be seen that the bubbling treatment is conducive to scooping up the suspended matter from above or extracting the clarified solution from below, effectively avoiding the traditional problems such as the complicated steps of subpackaging and centrifugation and the easy clogging of the filter caused by a large amount of suspended matter entering the filter.

[0163] Step 1630, separating the upper suspended matter and the lower reaction liquid to obtain the lower reaction liquid containing the plasmid.

[0164] In some embodiments, the upper suspended matter and the lower reaction liquid can be separated based on a variety of methods to obtain a lower reaction liquid containing a plasmid. In some embodiments, the upper suspended matter can be discharged first, and then the lower reaction liquid can be discharged through the discharge port to separate the upper suspended matter and the lower reaction liquid to obtain a lower reaction liquid containing a plasmid. For example, the method of discharging the upper suspended matter includes: manually scooping up the upper suspended matter using a filter screen or a colander or other tool. For another example, the method of discharging the upper suspended matter also includes: sucking out the upper suspended matter in the reaction chamber through a suction device, the suction tube of the suction device can extend from the top of the reaction chamber (for example, a suction tube provided on the top) into the interior of the reaction chamber and contact the upper suspended matter, and the upper suspended matter can be sucked out of the reaction chamber through the suction tube when the suction device is in operation.

[0165] In some embodiments, the upper suspended matter can be separated from the lower reaction liquid without discharging the upper suspended matter to obtain the lower reaction liquid containing the plasmid. For example, when the lower reaction liquid in the reaction chamber is discharged through the discharge port, the upper suspended matter can be intercepted to separate the upper suspended matter and the lower reaction liquid to obtain the lower reaction liquid containing the plasmid. In some embodiments, the method of intercepting the upper suspended matter includes setting a filter screen at the discharge port to intercept the upper suspended matter. For the description of the discharge port, the pipette, and the filter screen, please refer to Figure 1-Figure 15 and its related description.

[0166] In some embodiments, after obtaining the lower layer reaction solution containing the plasmid, the lower layer reaction solution can be further purified to obtain a purified lower layer reaction solution containing the plasmid.

[0167] In some embodiments, after the lower layer of reaction liquid is separated from the upper layer of suspended matter, it enters the external filter through the drain port and can be directly purified by the external filter to obtain the purified lower layer of reaction liquid containing the plasmid. For more information about the external filter, see Figure 1-Figure 4 and its related description.

[0168] In some embodiments, purifying the lower layer reaction liquid may further include: adding a filter aid material to the lower layer reaction liquid; filtering the lower layer reaction liquid after adding the filter aid material to obtain a purified lower layer reaction liquid containing the plasmid. In some embodiments, the filter aid material may be added to the lower layer reaction liquid before the lower layer reaction liquid separated from the upper layer suspended matter enters the reaction chamber through the drain port.

[0169] Filter-aid materials refer to materials with certain liquid filterability. For example, filter-aid materials can be one or a combination of diatomaceous earth, silicon carbide, activated carbon, foamed metal, microporous resin, perlite, cellulose, asbestos, graphite powder, sawdust, magnesium oxide, gypsum, acid clay, etc.

[0170] In some embodiments, a filter-aid material is added to the lower layer of the reaction liquid and mixed evenly to obtain a mixed solution, and the mixed solution is transported to an external filter for purification to obtain a purified lower layer of the reaction liquid containing the plasmid. Although the filter-aid material in the mixed solution is transported to the external filter for filtration together with the residual suspended matter, and the viscous suspended matter is easy to clog the external filter, since most of the suspended matter has been aggregated and drained after the bubbling treatment, the suspended matter deposited on the external filter is greatly reduced, and since the filter-aid material has been mixed with the suspended matter in advance, the excellent liquid filterability brought by the porosity of the filter-aid material makes the protein-filter-aid material mixture deposited on the external filter not easy to clog, which not only increases the filtration efficiency, but also avoids the increase in the cost of consumables for replacing the external filter after clogging.

[0171] In some embodiments, the mixed liquid may be delivered to the external filter by one or more methods such as pumping, negative pressure extraction, positive pressure extrusion, gravity flow, etc.

[0172] The features and performances disclosed in the embodiments of this specification are further described in detail below with reference to examples.

[0173] Example 1: A bubbling medium is introduced into an E. coli lysate (the volume of the E. coli lysate is 40 L) in a reaction chamber through a bubbling device. The E. coli lysate includes a reaction solution containing a plasmid and a suspended substance suspended in the reaction solution. The bubbling medium passes through the bubbling device from the bottom of the E. coli lysate to drive the suspended substance to float up, forming an upper layer of suspended substance and a lower layer of reaction solution. When the E. coli lysate shows obvious stratification (for example, Fig.17 When the temperature is 0.50 °C (as shown in the figure), stop introducing the bubbling medium. Separate the upper suspended matter from the lower reaction liquid.

[0174] The ratio of the area of ​​the bubbling sieve plate of the bubbling device to the cross section of the reaction chamber is 20%, the mesh size of the bubbling sieve plate is 10 μm, the distribution density of the mesh is 1300 meshes, and the bubbling rate of the bubbling medium is 3 L / min.

[0175] The flotation completion time of Example 1 (from the start of introducing the bubbling medium to the stop of introducing the bubbling medium) was 10 minutes.

[0176] Comparative Example 1: The difference from Example 1 is that the bubbling rate of the bubbling medium is 1 L / min. The floating completion time is 15 minutes.

[0177] Comparative Example 2: The difference from Example 1 is that the bubbling rate of the bubbling medium is 15 L / min. The floating completion time is 8 minutes.

[0178] Comparative Example 3: The difference from Example 1 is that the mesh size is 8 μm and the floating completion time is 5 minutes.

[0179] Comparative Example 4: The difference from Example 1 is that the distribution density of the mesh is 10 meshes. The floating completion time is 30 minutes.

[0180] Comparative Example 5: The difference from Example 1 is that the ratio of the area of ​​the bubbling sieve plate of the bubbling device to the cross section of the reaction chamber is 15%. The floating completion time is 15 minutes.

[0181] According to the test results of the above embodiments and comparative examples, it can be seen that the preset bubbling rate, mesh aperture, distribution density and the ratio of the cross-sectional area of ​​the bubbling screen plate of the bubbling device to the reaction chamber will affect the floating time of the suspended matter.

[0182] Fig.18 It is an electrophoresis diagram of the recovered plasmid shown in some embodiments of this specification.

[0183] like Fig.18 As shown, M1 is a DNA ladder molecular weight standard, lane 1 is a control experiment without bubbling treatment using a centrifugation method (ie, a control group), and lane 2 is a bubbling treatment experiment (ie, an experimental group) shown in some embodiments of this specification.

[0184] The experimental conditions are as follows:

[0185] Experimental group: 40L of E. coli lysate was subjected to bubbling treatment. When the suspended matter and the reaction liquid showed obvious stratification, the bubbling was stopped. The lower layer of reaction liquid was extracted into a container, and filter aid material was added to the lower layer of reaction liquid. The reaction liquid including the filter aid material was extracted into an external filter for filtration to obtain a purified lower layer of reaction liquid containing the plasmid. In the above process, the bubbling treatment took 10 minutes (i.e., the time for flotation to be completed), and the total time was 60 minutes. The bubbling treatment parameters are: the preset bubbling rate of the first bubbling medium is 5L / min; the mesh aperture is 50μm; the mesh distribution density is 280 mesh; the ratio of the sieve plate size (mesh coverage) of the bubbling sieve plate to the cross-sectional area of ​​the reaction chamber is 1:1.

[0186] Control group: Use centrifugation instead of bubbling treatment to centrifuge 40L of E. coli lysate (the same as that used in the experimental group). Pour the reaction solution into a container and add filter aids to the reaction solution. Draw the reaction solution including the filter aids to an external filter for filtration to obtain a purified reaction solution. The centrifugation parameters are: 5000rpm centrifugation for 15 minutes, using a centrifuge to complete 14 rounds of centrifugation of 40L of E. coli lysate suspension, the amount of liquid centrifuged each time is 500ml*6 bottles, and the 14 rounds of centrifugation steps take a total of 210 minutes, and the total time is 250 minutes.

[0187] The experimental results are as follows Fig.18 As shown in the figure, there is no significant difference between the experimental group and the control group. There are three clearly visible bands in both lanes, which are open circular DNA, linear DNA and supercoiled DNA from top to bottom. Among them, the supercoiled DNA band has the highest brightness and the largest area, indicating that the supercoiled plasmid DNA content is the highest in the recovered plasmids, and the experimental effect is better. In terms of the comparison of recovery quality, there is no significant difference between the bubbling method and the traditional centrifugation method.

[0188] For the purified reaction solution, the clarity of the control group was 0.15NTU, and the clarity of the bubbling experiment group was 0.11NTU. The acceptable standard is <10NTU, and the experimental results are all better than the acceptable standard. The bubbling treatment took 10 minutes and the total time was 60 minutes. The centrifugation step took a total of 210 minutes and the total time was 250 minutes. It can be seen that under the premise that the solid-liquid separation effect meets the requirements, the bubbling treatment has a shorter treatment time and a 316% increase in efficiency compared to the centrifugal treatment.

[0189] In some embodiments, see Figure 1 , Figure 2 , Figure 5 , Figure 6 as well as Figure 9-Figure 15 The plasmid extraction device includes a reaction container 100, a frame 200 and a chassis 300. The reaction container 100 includes a reaction chamber 110, a bubbling device 120 and a liquid discharge port 130.

[0190] The reaction chamber 110 is a rigid open reaction chamber. The bubbling device 120 is located at the bottom of the reaction chamber 110 and outside the reaction chamber 110. The bubbling device 120 includes a bubbling screen plate 121, an inner cavity 122 and an air inlet 123. The inner cavity 122 is connected to the air inlet 123 and the liquid discharge port 130. The bubbling device 120 can be connected to the bottom of the reaction chamber 110 by threading. The switch of the liquid discharge port 130 can be manually or automatically controlled by the liquid discharge switch 131.

[0191] The reaction container 100 may further include a pipette 190, an observation window 113, a fixed support leg 111, and a liquid receiving tray 112. The reaction chamber 110 may be supported by the fixed support leg 111, and the liquid receiving tray 112 is placed below the fixed support leg 111.

[0192] The reaction container 100 further includes three liquid adding guides 170. The liquid adding guides include a liquid adding port 150, a liquid adding guide plate 171 and a fixing hole 172. The liquid adding guides 170 are fixed to the reaction chamber 110 through the fixing holes 172.

[0193] The frame 200 includes a lifting assembly 210, an upper cover 180, and a stirring assembly 220. The stirring assembly 220, the upper cover 180, and the pipette 190 are connected to the lifting assembly 210, and the lifting assembly 210 can control the rotation of the stirring assembly 220, as well as the lifting and lowering of the stirring assembly 220, the upper cover 180, and the pipette 190. The bottom of the stirring assembly 220 includes a clearance hole 221. One end of the pipette 190 passes through the clearance hole 221, and the other end extends out of the upper cover 180 and is connected to the lifting assembly 210.

[0194] When the stirring assembly 220 rotates, the pipette 190 remains stationary.

[0195] The chassis 300 includes an indicator light and a buzzer 320 , a touch screen 330 , an emergency stop button 340 , a pressure sensor interface 350 , four electric pumps, and a pump tube hook 360 .

[0196] When the reaction vessel 100 is used to prepare bacterial lysate, the lifting assembly 210 can be controlled by the chassis 300 to move upward to lift the upper cover 180, and the Escherichia coli cells can be added to the reaction chamber 110. Subsequently, the lifting assembly 210 can be controlled to move downward to move the upper cover 180 to the top of the reaction chamber 110, that is, to close the cover. The first electric pump 311 is controlled by the chassis 300 to add the resuspended reagent from the resuspended reagent barrel to the reaction chamber 110 through the liquid adding port 150, the second electric pump 312 is controlled to add the lysed reagent from the lysed reagent barrel to the reaction chamber 110 through the liquid adding port 150, and the third electric pump 313 is controlled to add the neutralizing reagent from the neutralizing reagent barrel to the reaction chamber 110 through the liquid adding port 150. The stirring assembly 220 is controlled to stir, and / or the bubbling device 120 is controlled to bubble in the reaction chamber 110 to mix the liquid. After obtaining the bacterial lysate, the bubbling device 120 is controlled to bubble into the reaction chamber 110 to perform solid-liquid separation to obtain an upper suspended substance and a lower reaction liquid. The fourth electric pump 314 can be used to connect the pipette 190 or the drain port 130 to discharge the lower reaction liquid from the reaction chamber 110 through the drain port 130 or the pipette 190. The lifting assembly 210 can also be controlled to lift the upper cover 180 to manually fish out the upper suspended substance. When the lower reaction liquid is discharged from the reaction chamber 110 through the drain port 130 located at the bottom of the bubbling device 120, the bubbling screen plate 121 can block the suspended substance from being discharged together.

[0197] In some embodiments, see Figure 3 , Figure 4 , Figure 7 , Figure 8, the reaction chamber 110 of the reaction container 100 can be a flexible closed reaction chamber. The shape of the flexible reaction chamber can be changed. The clamp 140 can clamp the outer shell of the flexible reaction chamber to block the communication between the flexible reaction chamber and the bubbling device 120. When the flexible reaction chamber is used to prepare the bacterial lysate, the clamp 140 can be used to clamp the outer shell of the flexible reaction chamber to block the communication between the flexible reaction chamber and the bubbling device 120, so that Escherichia coli can fully react with a variety of reagents, and avoid the reagents from entering the bubbling screen 121 of the bubbling device 120 to cause residues or insufficient reactions. When the preparation of the bacterial lysate is completed, the clamp 140 can be loosened to connect the flexible reaction chamber and the bubbling device 120, and the bubbling device 120 introduces a bubbling medium into the flexible reaction chamber, so that the bubbling medium drives the suspended matter to float from the bottom of the flexible reaction chamber through the bubbling device 120, and the suspended matter after floating gathers on the surface of the reaction solution and is layered with the reaction solution to form an upper layer of suspended matter and a lower layer of reaction solution. The flexible reaction chamber 110 may further include an air inlet and outlet 160. The drain port 130 is located at the bottom of the flexible reaction chamber 110. The bubbling device 120 and the flexible reaction chamber 110 may be integrally formed or detachably connected. For example, the bubbling screen plate 121 is placed in the flexible reaction chamber 110 and at the bottom of the flexible reaction chamber, and the space formed by the bubbling screen plate 121 and the bottom of the flexible reaction chamber 110 can be used as an inner cavity 122. The reaction vessel 100 further includes three liquid addition ports 150, which are used to add a resuspension reagent, a lysis reagent, and a neutralization reagent, respectively. The reaction vessel further includes an air inlet and outlet 160. When preparing a bacterial lysate, the flexible reaction chamber 110 can be controlled to perform an inverted reciprocating motion to mix the liquid, or a bubbling medium can be introduced into the flexible reaction chamber 110 through the bubbling device 120 to mix the liquid.

[0198] In some embodiments, the flexible reaction chamber 110 can be placed in a rack 200, which includes a motion mechanism (not shown in the figure), which can control the flexible reaction chamber 110 to perform an inverted reciprocating motion. The three liquid adding ports 150 on the flexible reaction chamber 110 can be connected to three electric pumps respectively to add reagents into the flexible reaction chamber 110 through the liquid adding ports 150. The fourth electric pump can be connected to the discharge port 130 of the flexible reaction chamber 110 to extract the lower layer of reaction liquid.

[0199] In some embodiments of the present specification, by introducing the first bubbling medium into the bubbling device, the first bubbling medium passes through the bubbling device from the bottom of the bacterial lysate, drives the suspended matter to float, and the suspended matter after floating gathers at the liquid surface of the reaction solution and is stratified with the reaction solution to form an upper suspended matter and a lower layer of reaction solution, separates the upper suspended matter and the lower layer of reaction solution, and filters the lower layer of reaction solution containing the plasmid to obtain the purified lower layer of reaction solution containing the plasmid, the method has few steps, simple operation, short time consumption, and low cost, which is conducive to the large-scale and automated production application of plasmid extraction. The suspended matter in the bacterial lysate is a hydrophilic particle, which will not have a strong surface tension, and therefore will not be hydrophobic and adsorbed by the bubbles generated by the bubbling medium, but the bubbles generated by the bubbling medium float, and drive the reaction solution to flow upward in the process of floating, and directly collide with the suspended matter, and the suspended matter rises and gathers at the liquid surface under the action of the impact force and the upward flow of the liquid. Suspended matter has a certain viscosity due to its composition (e.g., protein, etc.). After being aggregated together, it can maintain an aggregated state on the liquid surface for a period of time without loosening due to its viscosity, thereby achieving the effect of solid-liquid separation. By physically passing a bubbling medium to separate the upper suspended matter and the lower reaction liquid, the influence on the properties of the reaction liquid can be reduced and the influence on the product quality can be avoided.

[0200] Some embodiments of this specification also provide a method for using a plasmid extraction device, which includes the steps shown in process 1900. Fig.19 is an exemplary flow chart of the method of using the plasmid extraction device according to some embodiments of this specification. Fig.19 As shown, process 1900 may include steps 1910 to 1930. In some embodiments, process 1900 may be Figure 1-Figure 15 The method for using the plasmid extraction solid-liquid separation device is shown.

[0201] Step 1910, add cell lysis solution to the reaction chamber; or, add cells to the reaction chamber, lyse the cells in the reaction chamber, and obtain cell lysis solution. For more information about cell lysis solution, please refer to the above related description.

[0202] In some embodiments, the method of use includes: during the process of lysing the cells, introducing a second bubbling medium into the cells in the reaction chamber through a bubbling device. For more information about the second bubbling medium, see the above related description.

[0203] In some embodiments, the method of use includes: adding a reagent for preparing a cell lysate into the reaction chamber through at least one liquid inlet flow guide. For more information about the liquid inlet flow guide and the reagent for preparing a cell lysate, please refer to the above related description.

[0204] In some embodiments, the reaction chamber is a flexible reaction chamber, and the method of use includes: during the process of lysing cells, clamping the outer shell of the flexible reaction chamber with a clamp to block the connection between the flexible reaction chamber and the bubbling device; during the process of introducing the first bubbling medium, canceling the clamping of the outer shell of the flexible reaction chamber by the clamp. For more information about the flexible reaction chamber and the clamp, please refer to the relevant description above.

[0205] Step 1920, a first bubbling medium is introduced into the cell lysate in the reaction chamber through a bubbling device, and the first bubbling medium drives the suspended matter to float up from the bottom of the cell lysate to form an upper layer of suspended matter and a lower layer of reaction solution. For more information on the formation of the upper layer of suspended matter and the lower layer of reaction solution, please refer to the above description.

[0206] Step 1930, discharge the upper suspended matter or the lower reaction liquid from the reaction chamber through the liquid discharge port to separate the upper suspended matter from the lower reaction liquid to obtain the lower reaction liquid containing the plasmid. For more information on separating the upper suspended matter from the lower reaction liquid, refer to the above description.

[0207] The basic concepts have been described above. Obviously, for those skilled in the art, the above detailed disclosure is only for example and does not constitute a limitation of this specification. Although not explicitly stated here, those skilled in the art may make various modifications, improvements and corrections to this specification. Such modifications, improvements and corrections are suggested in this specification, so such modifications, improvements and corrections still belong to the spirit and scope of the exemplary embodiments of this specification.

[0208] At the same time, this specification uses specific words to describe the embodiments of this specification. For example, "one embodiment", "an embodiment", and / or "some embodiments" refer to a certain feature, structure or characteristic related to at least one embodiment of this specification. Therefore, it should be emphasized and noted that "one embodiment" or "an embodiment" or "an alternative embodiment" mentioned twice or more in different positions in this specification does not necessarily refer to the same embodiment. In addition, certain features, structures or characteristics in one or more embodiments of this specification can be appropriately combined.

[0209] In addition, unless explicitly stated in the claims, the order of the processing elements and sequences described in this specification, the use of alphanumeric characters, or the use of other names are not intended to limit the order of the processes and methods of this specification. Although the above disclosure discusses some invention embodiments that are currently considered useful through various examples, it should be understood that such details are only for illustrative purposes, and the attached claims are not limited to the disclosed embodiments. On the contrary, the claims are intended to cover all modifications and equivalent combinations that are consistent with the essence and scope of the embodiments of this specification. For example, although the system components described above can be implemented by hardware devices, they can also be implemented only by software solutions, such as installing the described system on an existing server or mobile device.

[0210] Similarly, it should be noted that in order to simplify the description disclosed in this specification and thus help understand one or more embodiments of the invention, in the above description of the embodiments of this specification, multiple features are sometimes combined into one embodiment, figure or description thereof. However, this disclosure method does not mean that the features required by the subject matter of this specification are more than the features mentioned in the claims. In fact, the features of the embodiments are less than all the features of the single embodiment disclosed above.

[0211] In some embodiments, numbers describing the number of components and attributes are used. It should be understood that such numbers used in the description of the embodiments are modified by the modifiers "about", "approximately" or "substantially" in some examples. Unless otherwise specified, "about", "approximately" or "substantially" indicate that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may change according to the required features of individual embodiments. In some embodiments, the numerical parameters should take into account the specified significant digits and adopt the general method of retaining digits. Although the numerical domains and parameters used to confirm the breadth of their range in some embodiments of this specification are approximate values, in specific embodiments, the setting of such numerical values ​​is as accurate as possible within the feasible range.

[0212] Each patent, patent application, patent application publication, and other materials, such as articles, books, specifications, publications, documents, etc., cited in this specification are hereby incorporated by reference in their entirety. Except for application history documents that are inconsistent with or conflicting with the contents of this specification, documents that limit the broadest scope of the claims of this specification (currently or later attached to this specification) are also excluded. It should be noted that if the descriptions, definitions, and / or use of terms in the materials attached to this specification are inconsistent or conflicting with the contents described in this specification, the descriptions, definitions, and / or use of terms in this specification shall prevail.

[0213] Finally, it should be understood that the embodiments described in this specification are only used to illustrate the principles of the embodiments of this specification. Other variations may also fall within the scope of this specification. Therefore, as an example and not a limitation, alternative configurations of the embodiments of this specification may be considered consistent with the teachings of this specification. Accordingly, the embodiments of this specification are not limited to the embodiments explicitly introduced and described in this specification.

Claims

1. A plasmid extraction method, characterized in that: include: Providing a cell lysis solution, the cell lysis solution comprising a reaction solution containing a plasmid and a suspended substance suspended in the reaction solution; A first bubbling medium is introduced into the cell lysate, wherein the first bubbling medium drives the suspended matter to float upward from the bottom of the cell lysate to form an upper layer of the suspended matter and a lower layer of the reaction solution; The upper suspended substance and the lower reaction liquid are separated to obtain the lower reaction liquid containing the plasmid.

2. The method according to claim 1, characterized in that The method further comprises: The lower layer reaction liquid is purified to obtain a purified lower layer reaction liquid containing the plasmid.

3. The method according to claim 2, characterized in that The step of purifying the lower layer reaction liquid to obtain a purified lower layer reaction liquid containing the plasmid comprises: adding a filter aid material to the lower layer reaction liquid; The lower layer reaction liquid after adding the filter aid material is filtered to obtain the purified lower layer reaction liquid containing the plasmid.

4. The method according to claim 1, characterized in that Providing the cell lysate comprises: Provide cells; The cells are lysed to obtain the cell lysate, wherein a second bubbling medium is introduced into the cells during the process of lysing the cells.

5. The method according to claim 1, characterized in that The method comprises introducing the first bubbling medium into the cell lysate at a preset bubbling rate, wherein the preset bubbling rate is 0.5 L / min-20 L / min.

6. The method according to claim 5, characterized in that The preset bubbling rate is 3 L / min-8 L / min.

7. The method according to claim 1, characterized in that The first bubbling medium is selected from air, carbon dioxide, nitrogen, an inert gas, or a liquid medium having a density lower than that of water and being insoluble in water.

8. The method according to claim 1, characterized in that The second bubbling medium is selected from air, carbon dioxide, nitrogen, an inert gas, or a liquid medium having a density lower than that of water and being insoluble in water.

9. A plasmid extraction device, characterized in that: include: A reaction chamber, used to contain a cell lysis solution, wherein the cell lysis solution includes a reaction solution containing a plasmid and a suspended substance suspended in the reaction solution; A bubbling device is located at the bottom of the reaction chamber, and a first bubbling medium drives the suspended matter to float from the bottom of the reaction chamber through the bubbling device to form an upper layer of suspended matter and a lower layer of reaction liquid; The liquid discharge port is used to discharge the upper layer of suspended matter or the lower layer of reaction liquid out of the reaction chamber.

10. The device according to claim 9, characterized in that The bubbling device comprises: A bubbling sieve plate, wherein the bubbling sieve plate comprises a plurality of meshes; Lumen; An air inlet, wherein the first bubbling medium enters the inner cavity from the air inlet and enters the reaction cavity from the multiple mesh holes of the bubbling screen plate.

11. The device according to claim 10, characterized in that The ratio of the sieve plate size of the bubbling sieve plate to the cross-sectional area of ​​the reaction chamber is 20%-100%.

12. The device according to claim 11, characterized in that The ratio of the sieve plate size of the bubbling sieve plate to the cross-sectional area of ​​the reaction chamber is 60%-100%.

13. The device according to claim 10, characterized in that The mesh has a pore size less than or equal to 550 μm.

14. The device according to claim 13, characterized in that The mesh has a pore size of 1 μm-10 μm.

15. The device according to claim 10, characterized in that The distribution density of the mesh is greater than or equal to 30 meshes.

16. The device according to claim 15, characterized in that The distribution density of the mesh is 1300-15000 mesh.

17. The device according to claim 9, characterized in that The reaction chamber is a flexible reaction chamber.

18. The device according to claim 9, characterized in that The device also includes: The clamp is used to clamp the outer shell of the flexible reaction chamber to block the communication between the flexible reaction chamber and the bubbling device.

19. The device according to claim 9, characterized in that The device also includes at least one inlet and outlet port for controlling the expansion degree of the flexible reaction chamber.

20. The device according to claim 9, characterized in that The outer shell of the reaction chamber is provided with at least one transparent observation window.

21. The device according to claim 9, characterized in that The liquid discharge port comprises a liquid pipette, a first end of which extends into the bottom of the reaction chamber, the lower layer reaction liquid is sucked into the liquid pipette from the first end of the liquid pipette, and discharged from the reaction chamber from the second end of the liquid pipette.

22. The device according to claim 9, characterized in that The liquid discharge port is located at the bottom of the reaction chamber or the bottom of the bubbling device.

23. The device according to claim 9, characterized in that The device also includes at least one liquid adding guide device, which is used to add reagents for preparing the cell lysate into the reaction chamber.

24. The device according to claim 23, characterized in that The liquid adding guide device comprises: A liquid adding port, through which the reagent is added into the reaction chamber; A liquid adding guide plate, used to make the reagent flow down along the wall of the reaction chamber; The fixing hole is used to fix the liquid adding guide device on the reaction chamber.

25. A method for using a plasmid extraction device, characterized in that: The plasmid extraction device comprises a reaction chamber, a bubbling device and a liquid discharge port, and the use method comprises: Adding a cell lysis solution to the reaction chamber; or, adding cells to the reaction chamber, and lysing the cells in the reaction chamber to obtain a cell lysis solution; the cell lysis solution includes a reaction solution containing a plasmid and a suspended substance suspended in the reaction solution; A first bubbling medium is introduced into the cell lysate in the reaction chamber through the bubbling device, and the first bubbling medium drives the suspended matter to float up from the bottom of the cell lysate to form an upper layer of suspended matter and a lower layer of reaction solution; The upper suspended matter or the lower reaction liquid is discharged from the reaction chamber through a liquid discharge port to separate the upper suspended matter from the lower reaction liquid to obtain the lower reaction liquid containing the plasmid.

26. The method according to claim 25, characterized in that The method of use further comprises: During the process of lysing the cells, a second bubbling medium is introduced into the cells in the reaction chamber through the bubbling device.

27. The method according to claim 25, characterized in that The method of use further comprises: The reagent for preparing the cell lysate is added into the reaction chamber through at least one liquid adding port guide.

28. The method according to claim 25, characterized in that The reaction chamber is a flexible reaction chamber; the use method further comprises: During the cell lysis process, the outer shell of the flexible reaction chamber is clamped by a clamp to block the communication between the flexible reaction chamber and the bubbling device; During the process of introducing the first bubbling medium, the clamping of the outer shell of the flexible reaction chamber by the clamper is cancelled.

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