Plasmid cracking device

Abstract

The plasmid cracking device comprises a liquid adding mechanism, a separating and mixing mechanism, a plurality of cracking parts and a switching part, the plurality of cracking parts are used for providing a place for cracking plasmids and a cracking solution; the place is connected with a liquid adding mechanism and a neutralizing mechanism; the neutralizing mechanism is used for adding a neutralizing solution into the cracked mixed solution; the switching part is connected with different cracking parts through a plurality of ports, and the switching part is configured to enable one or more of the cracking parts to connect the liquid adding mechanism and the neutralizing mechanism through switching. According to the utility model, the cracking place can be changed through the switching part, continuous operation can be realized, the problem of tedious operation is solved, the device is simple in structure, convenient to control and easy to realize automation, and the efficiency of screening the cracking time is greatly improved.

Description

Technical Field

[0001] The utility model relates to the technical field of biological cell genetic engineering, and particularly relates to a plasmid lysis device. Background Art

[0002] Since plasmids were discovered in the 1950s, they have influenced many fields of molecular biology and become an important genetic engineering tool. pDNA can be used for cloning, amplifying, and expressing target proteins; the production of viral vectors and mRNA also requires pDNA; GMP-grade pDNA can also be used in fields such as DNA vaccines, gene therapy, and cell therapy. Currently, the commonly used method is the traditional alkaline lysis method. Under alkaline conditions, the proteins, RNA, host DNA, and plasmids of Escherichia coli are released into the solution and denatured. After adding a neutralizing solution, plasmid DNA can renature and remain soluble, while a large amount of denatured proteins and host DNA bind together through hydrophobic interactions and precipitate. Escherichia coli is generally selected as the host cell for plasmid production, and the cells are lysed after fermentation culture to obtain plasmids. There are various plasmid forms in the lysed broth: supercoiled plasmid DNA (scDNA), open circular DNA (ocDNA), linear DNA, and plasmid DNA aggregates, etc. In addition, impurities such as host proteins (HCP), host nucleic acids (HCD), RNA, and endotoxins are also present. Insufficient lysis will reduce the plasmid recovery rate, while excessive lysis will damage plasmid molecules, making the broth viscous and difficult to filter, and will also lyse a large amount of bacterial cell walls into soluble endotoxins, increasing the pressure of subsequent purification. Therefore, it is necessary to precisely control the lysis parameters and timely separate the released plasmids from the bacterial debris.

[0003] The following problems will occur during the existing plasmid lysis:

[0004] 1. For ordinary lysis, it is usually thoroughly mixed by stirring. However, the shear force brought by stirring will break bacterial chrDNA into DNA fragments of the same size as plasmids, and the chemical and physical properties of these bacterial chrDNA fragments are similar to those of plasmid DNA fragments, making it difficult to remove them in subsequent separation and purification. In addition, once the stirring is uneven, it is easy to cause too high a local alkali concentration, and the high pH value will cause irreversible denaturation of plasmid DNA, thereby reducing the supercoiled ratio of plasmid DNA and its recovery rate.

[0005] 2. Ordinary lysis is more suitable for the preparation of laboratory-level samples. When it comes to large-scale preparation, when reaching a scale of hundreds of liters, the scalability of ordinary lysis is poor, difficult to control, and the lysis efficiency will be unstable. Content of the Utility Model

[0006] The purpose of the present utility model is to overcome the problems existing in the prior art and provide a plasmid lysis device. Through a switching part, the lysis site can be changed, enabling continuous operation, solving the problem of cumbersome operation in the prior art. Moreover, the device has a simple structure, is easy to control, and is easy to automate, greatly improving the efficiency of screening the lysis time.

[0007] To achieve the above technical objectives and reach the above technical effects, the present utility model is realized through the following technical solutions:

[0008] A plasmid lysis device, which includes the following structures:

[0009] A liquid adding mechanism for adding a bacterial suspension and a lysis solution into the plasmid lysis device;

[0010] A separation and mixing mechanism for separating plasmids in the initial bacterial suspension and mixing the plasmids with the lysis solution;

[0011] A lysis mechanism, including a lysis part providing a site for lysis of plasmids and the lysis solution, and a switching part communicating with the lysis part;

[0012] A neutralization mechanism for adding a neutralization solution to the lysed mixture;

[0013] A collection mechanism for collecting the neutralized mixture;

[0014] There are multiple lysis parts, and at least one switching part connects two lysis parts;

[0015] The lysis mechanism connects the liquid adding mechanism and the neutralization mechanism;

[0016] The separation and mixing mechanism is located between the liquid adding mechanism and the lysis mechanism.

[0017] Furthermore, the switching part includes one or a combination of two valves, namely a six-way valve and a three-way valve.

[0018] Furthermore, multiple lysis parts are connected in series or in parallel.

[0019] Furthermore, the lysis time of different lysis parts is controlled by the length of the lysis part and the pumping speed of the liquid adding mechanism.

[0020] Furthermore, the separation and mixing mechanism is a hollow fiber column.

[0021] Furthermore, the device also includes a control mechanism for controlling the connection of each port of the switching part to different lysis parts to change the lysis time, and the control mechanism controls the liquid adding actions of the liquid adding mechanism and the neutralization mechanism.

[0022] The beneficial effects of the present utility model are:

[0023] 1. The utility model can change different cracking sites through a switching part, obtain different cracking times, and improve the screening efficiency.

[0024] 2. The device of the utility model integrates the plasmid cracking device on one piece of equipment through a switching part, avoids the time for replacing pipelines, and shortens the screening time.

[0025] 3. The device of the utility model can realize automatic control, is applicable to large-scale cracking, and is easier to control the cracking effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a schematic diagram of a series connection mode of a cracking part of the device of the utility model;

[0027] Figure 2 It is a schematic diagram of another series connection mode of a cracking part of the device of the utility model;

[0028] Figure 3 It is a schematic diagram of a parallel connection mode of a cracking part of the device of the utility model;

[0029] Figure 4 It is a schematic diagram of another parallel connection mode of a cracking part of the device of the utility model;

[0030] Figure 5 It is a schematic diagram of changing the cracking time limit through a peristaltic pump in the device of the utility model;

[0031] Figure 6 It is a schematic diagram of the operation action process of the device of the utility model.

[0032] Explanation of the reference numerals in the figure: 1, 3 - storage bottles; 2, 4 - peristaltic pumps; 5 - hollow fiber column; 6 - hollow fiber column exhaust hole; 7 - first switching part; 8 - cracking part; 9 - neutralization part three-way valve; 10 - neutralization mechanism peristaltic pump; 11 - neutralization liquid storage bottle; 12 - collection mechanism; 13 - second switching part; 14 - variable speed pump. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0033] The following further describes the utility model in detail with reference to the drawings and embodiments. It can be understood that the specific embodiments described herein are only used to explain the utility model, rather than limiting the utility model. In addition, it should be noted that only some structures related to the utility model are shown in the drawings for the convenience of description, rather than all structures.

[0034] A plasmid cracking device, the device includes:

[0035] A liquid adding mechanism for adding a bacterial suspension and a cracking liquid into the plasmid cracking device;

[0036] Separation and mixing mechanism, used for separating plasmids in the initial bacterial suspension and mixing the plasmids with the lysis solution;

[0037] Lysis mechanism, including a lysis part providing a lysis site for the plasmids and the lysis solution, and a switching part communicating with the lysis part;

[0038] Neutralization mechanism, used for adding a neutralization solution to the lysed mixture;

[0039] Collection mechanism, used for collecting the neutralized mixture;

[0040] There are multiple lysis parts, and at least one switching part connects two lysis parts;

[0041] The lysis mechanism connects the liquid adding mechanism and the neutralization mechanism;

[0042] The separation and mixing mechanism is located between the liquid adding mechanism and the lysis mechanism.

[0043] For the plasmid lysis device of the present utility model, the bacterial suspension to be lysed and the pre-prepared lysis solution are added through the liquid adding mechanism, and then mixed and separated in the separation and mixing mechanism; the mixed mixture after mixing enters the lysis mechanism for lysis; the lysed mixture reacts with the neutralization solution of the neutralization mechanism to stop lysis, and the neutralized mixture enters the collection mechanism for collection.

[0044] Among them, the lysis mechanism includes a lysis part and a switching part. There are multiple lysis parts, which can provide multiple lysis sites. Through the switching part, it is switched to the required lysis part, or at least two lysis parts are connected to form a new lysis site; the lysis times provided by different lysis sites are different; the switching part switches the mixture to enter different lysis sites for different lysis times, so as to achieve the purpose of screening the lysis time.

[0045] The liquid adding mechanism includes a peristaltic pump and a storage bottle, and the storage bottle is used to hold the bacterial suspension and the lysis solution respectively.

[0046] The separation and mixing mechanism is a hollow fiber column. The peristaltic pumps in the liquid adding mechanism pump the bacterial suspension and the lysis solution in the storage bottle into the hollow fiber column respectively for mixing and separation.

[0047] The neutralization mechanism includes a peristaltic pump and a neutralization solution storage bottle; when the lysis for the specified time is completed, the peristaltic pump of the neutralization mechanism adds the neutralization solution to the plasmid lysis device to mix with the lysed mixture, and the lysis solution stops lysing the plasmids after mixing.

[0048] The collection mechanism collects the neutralized mixture and analyzes and tests it after collection.

[0049] Multiple lysis parts are connected in series or in parallel.

[0050] Such as Figure 1and Figure 2 As shown, multiple cracking parts are connected in series, and the cracking time of each cracking part is the same; among them Figure 1 The switching part is a six-way valve. By switching a single six-way valve, the cracked mixed liquid can enter the cracking part or enter the no-load pipeline. By switching the six-way valve, one or more cracking parts can be connected, so that the cracking time each time is different; Figure 2 The switching part is a three-way valve. The three-way valve adjacent to the cracking part determines whether it is connected to the next three-way valve. If not, the other adjacent three-way valve makes the cracked liquid enter the contact part with the neutralizing liquid.

[0051] Figure 1 As shown, there are three six-way valves connected in series. Each six-way valve has two switching states. One is to connect the cracking part, and the other is to connect the no-load pipeline. Although Figure 1 There are only three cracking parts connected in series, but as long as it is switched according to the technical solution of the present invention, as long as multiple cracking parts are connected in series in series, it belongs to the protection scope of the present invention; similarly Figure 2 In the case shown, if the number of series connections is increased, it also belongs to the protection scope of the present invention.

[0052] Such as Figure 3 and Figure 4 As shown, multiple cracking parts are connected in parallel. They are switched to each branch through a three-way valve or a six-way valve. There is a switching part on each branch, and there is 1 or 2 cracking parts on each branch. The cracking time between each cracking part in the cracking mechanism is different, and a no-load pipeline is arranged on one of the branches.

[0053] Such as Figure 3 As shown, it is switched to the parallel branches through a three-way valve; such as Figure 4 As shown, it is switched to the parallel branches through a six-way valve.

[0054] Figure 3 、 Figure 4 As shown, only two branches are connected in parallel. The way of adding multiple parallel branches can be continued by adding a three-way valve or a six-way valve; and except for the no-load pipeline, the cracking time of each cracking part is different.

[0055] The lysis section in the utility model can be specifically a lysis place, such as a pipeline; the lysis time of the lysis section in some embodiments is the same, and the lysis time of the lysis section in some embodiments is different; considering that the required lysis time is within a certain range, in order to improve the efficiency of screening the lysis time, several time points can be selected, such as 20s, 40s, 60s, and 80s; the lysis time can be determined by the peristaltic pump speed in the liquid adding mechanism and the length of the lysis section; in the embodiment of series lysis, the time of the lysis section is the same, for example, 20s, two series are 40s, three series are 60s, and four series are 80s... In the embodiment of parallel lysis, the time of the lysis section is different, and the length of the lysis section pipeline can be extended exponentially, and the pump speed of the peristaltic pump of the liquid adding mechanism is coordinated to make the time of each lysis section different, such as 20s, 40s, 60s, and 80s.

[0056] like Figure 5 As shown, the lysis time is controlled by adjusting the speed of the lysis solution in the plasmid lysis device; wherein the switching part is a six-way valve, the six-way valve can be switched to an empty pipeline or a lysis part, and the time of entering the lysis part is controlled by adjusting the pump speed of the peristaltic pump of the liquid adding mechanism; the faster the speed, the shorter the lysis time, and the slower the speed, the longer the lysis time.

[0057] The plasmid lysis device comprises a control mechanism, and the device is turned on and the neutralization liquid is collected by inputting a connection state or a planned lysis time for screening through an input end of the control mechanism.

[0058] like Figure 6 As shown, the operation process of the plasmid lysis device of the utility model is as follows:

[0059] Step a: Control the switching part to switch to the first lysis part, perform lysis for the first lysis time, and collect the neutralization solution;

[0060] Step b: controlling the switching part to switch to the second lysis part, performing lysis for a second lysis time, and collecting the neutralization solution;

[0061] Step c: repeat step b until the desired screening time is completed;

[0062] Step d: Analyze the collected multiple neutralization samples to obtain the optimal lysis time.

[0063] If the screening time exceeds 2, repeat step b to obtain the third neutralization sample, the fourth neutralization sample, and so on. Analyze the collected neutralization samples to obtain the optimal lysis time.

[0064] Before step a, configure the bacterial suspension, lysis solution, and neutralization solution; the bacterial suspension in the present utility model includes: 50 mM anhydrous glucose, 25 mM tromethamine, 10 mM disodium edetate, pH 7.5; the lysis solution includes: 0.2 M sodium hydroxide, 1% (W / V) sodium dodecyl sulfate; the neutralization solution includes: 3 M potassium acetate, 2 M glacial acetic acid, pH 5.5.

[0065] After configuring the solutions, turn on the instrument and pre-rinse the device in advance; control the entire lysis ratio through the peristaltic pump speed such that the bacterial suspension: lysis solution: neutralization solution = 1:1.2:1.4.

[0066] When in series mode, it is not necessary to stop the liquid addition mechanism from adding liquid, but when in parallel mode, it is necessary to stop the liquid addition mechanism from adding liquid.

[0067] When in series mode, in step b, before controlling the switching part, stop the liquid addition mechanism from adding liquid, and then turn on the liquid addition after switching.

[0068] Furthermore, after restarting the liquid addition, pre-rinse the device.

[0069] Furthermore, stopping the liquid addition mechanism from adding liquid includes stopping the addition of the lysis solution, or stopping the addition of both the lysis solution and the bacterial suspension.

[0070] Regarding the switching of the control switching part, there are differences in the switching of different connection methods.

[0071] As Figure 1 shown, the liquid addition mechanism adds the bacterial suspension to be lysed and the pre-configured lysis solution, and then mixes and separates them in the hollow fiber column; the mixed solution after mixing enters the Figure 1 shown lysis mechanism for lysis; the mixed solution after lysis reacts with the neutralization solution of the neutralization mechanism to stop lysis, and the neutralized mixed solution enters the collection mechanism for collection.

[0072] In some embodiments, the speed of the peristaltic pump of the liquid addition mechanism is coordinated with the length of the lysis part such that Figure 1 shown, the time of the lysis part is 20 s; switch all six-way valves to connect to the empty pipeline, and the control mechanism can detect that the liquid in the empty pipeline reaches the collection mechanism. Once it detects that the liquid reaches the collection mechanism, the control mechanism controls the switching part to switch: except for the first six-way valve switching to the lysis part, the other six-way valves still maintain the connection to the empty pipeline, and open the neutralization mechanism, and connect through the three-way valve to flush and neutralize the lysis solution, start timing, discard the neutralization solution in the first few seconds, and collect the neutralization solution around the 20th second; switch to the second six-way valve to connect to the lysis part, repeat the above operation, and through the accumulation of time, the neutralization solution at 20 s, 40 s, 60 s, and 80 s can be collected. After analysis, the optimal lysis time can be obtained.

[0073] Figure 2 The operation steps in Figure 1 are the same. The difference is that a three-way valve is switched to connect each lysis section in series.

[0074] Figure 3 and Figure 4 each have different lysis section times. After each lysis, before the switching section is switched, the peristaltic pump of the liquid adding mechanism stops adding liquid.

[0075] The device of the present utility model is integrated together, reducing the occupied space, avoiding the time for replacing pipelines, shortening the screening time, improving the screening efficiency, and the solution after lysis by this device can directly enter the analytical instrument for analysis. This device can be used as a module in combination with inspection instruments such as high performance liquid chromatography to further improve the efficiency of plasmid lysis in the screening stage.

[0076] Example 1

[0077] According to the method of Figure 2 , multiple lysis sections are connected in series and operated according to the process of the present utility model. Using plasmids (8000 - 9000bp) for virus packaging cultivated from Escherichia coli, the obtained bacterial suspension is configured into a solution as follows:

[0078] The bacterial suspension includes: 50 mM anhydrous glucose, 25 mM tromethamine, 10 mM disodium edetate, pH 7.5.

[0079] The lysis solution includes: 0.2 M sodium hydroxide, 1% (W / V) sodium dodecyl sulfate.

[0080] The neutralization solution includes: 3 M potassium acetate, 2 M glacial acetic acid, pH 5.5.

[0081] The whole lysis ratio is controlled by the peristaltic pump speed as bacterial suspension: lysis solution: neutralization solution = 1:1.2:1.4.

[0082] Table 1: Screening results of Example 1

[0083]

[0084] Conclusion: A lysis time of 60 s ± 10 s can be selected.

[0085] Example 2

[0086] The experimental conditions are the same as those in Example 1, and the plasmid is a plasmid (4000 - 5000bp) for virus packaging cultivated from Escherichia coli. The results are as follows in the table:

[0087] Table 2: Screening results of Example 2

[0088]

[0089] Conclusion: The lysis time can be selected as 40s ± 10s.

[0090] Example 3

[0091] The experimental conditions are the same as those in Example 1, using Figure 4 a device with a parallel structure, and the plasmid is a plasmid for virus packaging cultured in Escherichia coli (5000 - 6000bp). The experimental results are shown in Table 3:

[0092] Table 3: Screening Results of Example 3

[0093]

[0094] Conclusion: The lysis time can be selected as 40s ± 10s.

[0095] Comparative Example 1

[0096] The experimental conditions are the same as those in Example 1, and a common device is used. The results are shown in Table 4:

[0097] Table 4: Screening Results of Comparative Example 1

[0098]

[0099] Conclusion: The lysis time can be selected as 60s ± 10s.

[0100] Comparative Example 2

[0101] The experimental conditions are the same as those in Example 2, and a common device is used. The results are shown in Table 5:

[0102] Table 5: Screening Results of Comparative Example 2

[0103]

[0104] Conclusion: The lysis time can be selected as 60s ± 10s.

[0105] Judging from the screened lysis time, the results between Example 1 and Comparative Example 1 are consistent, but there are differences between Example 2 and Comparative Example 2. After repeating the experiments on Example 2 and Comparative Example 2 twice respectively, it is found that the results obtained in the 3 experiments of Example 2 are all 40s ± 10s, while the 3 results of Comparative Example 2 are 60s ± 10s, 40s ± 10s, and 20s ± 10s respectively; using the time screened in Example 2 for large-scale lysis, the purity of the obtained sample is 95.2%, which is close to the screened result; therefore, in addition to being able to quickly screen the lysis time, the interval value of the screened result of the device of the present utility model has a guiding effect on the subsequent large-scale lysis.

[0106] Moreover, from the perspective of the duration used for screening, the device of the present utility model can greatly improve the efficiency of screening and cracking time.

[0107] In addition, it should be noted that unless otherwise specified or indicated, the terms "first", "second", etc. in the specification are only used to distinguish the various components, elements, steps, etc. in the specification, rather than to represent the logical relationship or sequential relationship, etc. between the various components, elements, steps.

[0108] The above are only the preferred embodiments of the present utility model and are not intended to limit the present utility model. For those skilled in the art, the present utility model can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present utility model shall be included within the protection scope of the present utility model.

Claims

1. A plasmid lysis device, characterized in that: The device comprises the following structures: A liquid adding mechanism, used for adding bacterial suspension and lysis solution into the plasmid lysis device; A separation and mixing mechanism, used to separate the plasmid in the initial bacterial suspension and mix the plasmid with the lysis solution; The lysis mechanism includes a lysis part providing a place for plasmid and lysis solution to be lysed, and a switching part connected to the lysis part; A neutralization mechanism, used for adding a neutralizing liquid to the mixed liquid after cracking; A collecting mechanism, used for collecting the mixed liquid after neutralization; There are multiple cracking parts, and at least one switching part connects two cracking parts; The cracking mechanism connects the liquid adding mechanism and the neutralizing mechanism; The separation and mixing mechanism is located between the liquid adding mechanism and the cracking mechanism.

2. The plasmid splitting device according to claim 1, characterized in that: The switching part includes one of a six-way valve and a three-way valve, or a combination of the two valves.

3. The plasmid lysis device according to claim 2, characterized in that: The plurality of cracking parts are connected in series or in parallel.

4. The plasmid lysis device according to claim 3, characterized in that: The lysis time of different lysis parts is controlled by the length of the lysis part and the pump speed of the liquid adding mechanism.

5. The plasmid splitting device according to claim 4, characterized in that: The separation and mixing mechanism is a hollow fiber column.

6. The plasmid lysis device according to claim 5, characterized in that: The device also includes a control mechanism for controlling each port of the switching part to be connected with different lysis parts to change the lysis time, and the control mechanism controls the liquid adding action of the liquid adding mechanism and the neutralization mechanism.

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