A plasmid extraction device and method

Abstract

The present invention discloses a plasmid extraction device and method, belonging to the field of biotechnology, which includes a separation container, electrodes arranged in the separation container, a plasmid adsorption column, a stirrer, and a reagent delivery system. A filter membrane is provided in the separation container, and the filter membrane divides the separation container into a screening area and a lysis area that integrates fragmentation and precipitation; the reagent delivery system includes a liquid suction conduit and a liquid discharge conduit; the plasmid adsorption column is arranged between the liquid suction conduit and the liquid discharge conduit; the liquid suction conduit is used to introduce reagents into the plasmid adsorption column and / or the reagent delivery system; the liquid discharge conduit is used to introduce reagents into and / or outside the separation container. This device enables the entire process to achieve full automation from bacterial liquid sampling to plasmid collection, without the need for centrifugation throughout the process, solves the problems such as the necessity of manual sample transfer and centrifugation in the process of plasmid extraction by existing automated equipment, and realizes the true full automation of plasmid extraction.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology, and particularly to a plasmid extraction device and method. Background Art

[0002] Plasmids exist in many organisms such as bacteria and yeasts, and are very small circular DNA molecules that can replicate autonomously outside the cell chromosome. Plasmids can be artificially and purposefully added with known or unknown DNA fragments outside bacteria to form recombinant "plasmid DNA". Then, this newly synthesized recombinant "plasmid DNA" is introduced into bacteria and propagated as the bacteria multiply in large numbers. Then, a large number of such bacteria are collected, and the new recombinant "plasmid DNA" is extracted or separated from the bacteria by special methods, so as to obtain a relatively large amount (several micrograms or several grams) of biologically active new recombinant "plasmid DNA".

[0003] Recombinant "plasmid DNA" is mostly extracted through kits, and during its extraction process, repeated centrifugation and repeated rinsing are required. The complexity of the extraction process makes it difficult to automate the extraction of recombinant "plasmid DNA". Currently, existing plasmid extraction automation devices basically require manual participation in multiple centrifugation or other steps. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides a plasmid extraction device, which can realize the full-automatic process from bacterial liquid loading to plasmid collection, and does not require centrifugation throughout the process, solving problems such as the need for manual sample transfer and centrifugation during the plasmid extraction process of existing automation devices.

[0005] In order to achieve the above object, the present invention is realized through the following technical solutions:

[0006] A plasmid extraction device includes a separation container, electrodes for plasmid electrophoresis arranged in the separation container, a plasmid adsorption column, and a reagent delivery system, and further includes a controller and a stirrer arranged at the lower part of the separation container and automatically started and stopped by the controller; the electrodes are electrically connected to the controller;

[0007] A filter membrane is further arranged in the separation container; the filter membrane divides the separation container into a screening area and a lysis area integrating fragmentation and precipitation;

[0008] The reagent delivery system includes at least one liquid suction conduit and at least one liquid outlet conduit; the plasmid adsorption column is arranged between the liquid suction conduit and the liquid outlet conduit; the liquid suction conduit is used for introducing reagents into the plasmid adsorption column and / or the reagent delivery system; the liquid outlet conduit is used for introducing reagents into and / or outside the separation container;

[0009] A liquid control valve is provided on each of the liquid suction conduits and each of the liquid discharge conduits.

[0010] Furthermore, the liquid suction conduit at least includes:

[0011] A plasmid separation liquid suction conduit, which is connected to the screening area and transports the plasmid to the plasmid adsorption column;

[0012] A washing liquid conduit, which is used to introduce washing liquid to the plasmid adsorption column to wash the plasmid;

[0013] A liquid discharging conduit, which is used to introduce eluent to the plasmid adsorption column to release the plasmid;

[0014] An electrolyte suction conduit, which is used to transport electrolyte to the separation container and provide an electrophoresis environment for the separation container

[0015] A separation reagent suction conduit, which is used to transport lysis solution and precipitant to the lysis area to break bacteria and perform precipitation separation.

[0016] Furthermore, the liquid discharge conduit includes:

[0017] A waste liquid conduit, which is connected to the plasmid adsorption column and discharges the washed washing liquid out of the separation container;

[0018] A plasmid collection conduit, which is connected to the plasmid adsorption column and discharges the released plasmid out of the separation container for collection;

[0019] A conveying conduit, which is connected to the separation container and conveys electrolyte and reagent to the separation container; the conveying conduit includes an electrolyte discharge conduit communicated with the electrolyte suction conduit, and a separation reagent discharge conduit communicated with the separation reagent suction conduit and conveyed to the lysis area of the separation container.

[0020] Furthermore, the separation reagent suction conduit includes a lysis solution suction conduit and a precipitation reagent suction conduit.

[0021] Furthermore, the filter membrane includes a screening filter membrane and a blocking filter membrane, and the screening area is between the screening filter membrane and the blocking filter membrane.

[0022] Furthermore, the electrode includes a negative electrode plate and a positive electrode plate, the screening area is arranged close to the negative electrode plate; the blocking filter membrane is arranged between the negative electrode plate and the screening filter membrane; the lysis area is between the screening filter membrane and the positive electrode plate.

[0023] Furthermore, the stirrer is a magnetic stirrer, and the magnetic stirrer is electrically connected to the controller.

[0024] Furthermore, an infusion pump that is automatically started and stopped by the controller is provided on the reagent delivery system, and the liquid control valve is an electrically controlled valve whose timing switch is controlled by the controller.

[0025] Furthermore, the plasmid adsorption column is made of silica gel; the blocking filter membrane is a PVDF membrane with a pore size of 0.01 μm; the screening filter membrane is a PVDF membrane with a pore size of 0.45 μm.

[0026] The method for extracting plasmid using the plasmid extraction device of the present invention includes:

[0027] S1: Add the bacterial liquid into the lysis area, and the controller sequentially controls the electrolyte to enter the screening area and the lysis solution to enter the lysis area, and simultaneously starts the magnetic stirrer to stir;

[0028] S2: Add a precipitation reagent to the lysed bacterial liquid in S1, and simultaneously start the magnetic stirrer to stir;

[0029] S3: The controller controls the electrodes to start so that the plasmid electrophoreses and is screened and filtered into the screening area; then controls the liquid control valve in the separation liquid suction conduit to start, and adsorbs the plasmid to the plasmid adsorption column for adsorption;

[0030] S4: The controller sequentially controls the washing solution and the eluent to enter the plasmid adsorption column to wash and de-liquify the plasmid; after de-liquifying, collect through the plasmid collection conduit.

[0031] The plasmid extraction device and method of the present invention have the beneficial effects that:

[0032] The separated plasmid is initially separated in the separation container through the blocking filter membrane and the screening filter membrane, and then the separated plasmid is transported to the plasmid adsorption column by the infusion pump. After repeatedly washing and purifying the plasmid on the plasmid adsorption column, it is then analyzed. The entire process is controlled by the controller to time the various liquid control valves, enabling the entire process to have a specific cycle, and can realize full automation from bacterial liquid loading to plasmid collection, solving the problems that sample transfer and centrifugation must be carried out manually in the process of extracting plasmid by existing automated equipment, and realizing the true full automation of plasmid extraction. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0034] Figure 1 is the structural schematic diagram of the present invention;

[0035] Figure 2 is the control structural schematic diagram of the present invention;

[0036] Figure 3It is a schematic structural diagram of the liquid control valve in the present invention;

[0037] 1. Housing, 2. Negative electrode plate, 3. Blocking filter membrane, 4. Screening filter membrane, 5. Magnetic stirrer, 6. Positive electrode plate, 7. Plasmid separation liquid suction conduit, 8. Washing liquid conduit, 9. Dewatering conduit, 10. Electrolyte suction conduit, 11. Lysis solution suction conduit, 12. Precipitation reagent suction conduit, 13. Electrolyte outlet conduit, 14. Separation reagent outlet conduit, 15. Waste liquid conduit, 16. Plasmid collection conduit, 24. Infusion pump, 29. Plasmid adsorption column, 30. Lysis area, 31. Screening area, 32. Magnetic stirrer, 33. Valve body, 34. Flow-diverting pad, 35. Steel ball, 36. Blocking rubber ring, 37. Electromagnet, 38. Liquid inlet, 39. Liquid outlet;

[0038] Among them, 17, 18, 19, 20, 21, 22, 23, 25, 26, 27, 28 are all liquid control valves. Specific embodiments

[0039] The present invention will be further described in detail below with reference to specific embodiments and the accompanying drawings.

[0040] Embodiment 1

[0041] A plasmid extraction device, as Figure 1 , Figure 2 and Figure 3 shown, includes a separation container, electrodes for plasmid electrophoresis provided in the separation container, a plasmid adsorption column 29 made of silica gel, and a reagent delivery system, and also includes a controller and a magnetic stirrer 32 that is automatically started and stopped by the controller. A filter membrane is provided in the separation container, and the filter membrane divides the separation container into a screening area 31 and a lysis area 30 that integrates fragmentation and precipitation;

[0042] The reagent delivery system includes a suction conduit and an outlet conduit; the plasmid adsorption column 29 is provided between the suction conduit and the outlet conduit; the suction conduit is used to introduce the reagent into the plasmid adsorption column 29 and the reagent delivery system; the outlet conduit is used to introduce the reagent into and outside the separation container.

[0043] In this embodiment, a liquid control valve is provided on each suction conduit and each outlet conduit, and the liquid control valve is an electrically controlled valve that is controlled by the controller to be switched on and off at a fixed time. For example, an Oriental An (DFA) intelligent timer switch is used to control the start and stop at a specified interval, so as to realize the automation of the entire process. An infusion pump 24 that is automatically started and stopped by the controller is provided on the reagent delivery system, and the controller can also be set to start and stop the infusion pump 24 at a fixed time.

[0044] The liquid control valve includes a valve body 33 and an electromagnet 37 arranged outside the valve body. Liquid inlets 38 and outlets 39 are provided at both ends of the valve body 33. A magnetic steel ball 35 is arranged at the center inside the valve body 33. A plugging rubber ring 36 is arranged inside the valve body near the liquid inlet, and a flow-dispersing pad 34 is arranged inside the valve body near the liquid outlet; the electromagnet 37 is arranged near the liquid inlet. The diameter of the steel ball is smaller than the inner diameter of the valve body.

[0045] The electronically controlled valve starts the electromagnet 37 through the controller, so that the steel ball is adsorbed by the magnetic force and combines with the plugging rubber ring 36 to close the valve and block the liquid. The controller turns off the electromagnet 37, and at the same time starts the infusion pump 24. The fluid in the infusion pump 24 enters from the liquid inlet, and after entering, the fluid drives the steel ball 37 to move towards the liquid outlet direction. A flow-dispersing pad 34 is provided at the liquid outlet, which can enable the liquid to pass smoothly.

[0046] The magnetic stirrer 32 drives the magnetic stir bar 5 to stir the internal bacterial liquid, so that the lysis solution and the bacterial liquid are fully mixed to achieve full lysis; at the same time, during the subsequent precipitation process, the lysed bacterial liquid and the precipitant can also be fully mixed to achieve full precipitation separation. The magnetic stirrer is electrically connected to the controller, and the start and stop of the magnetic stirrer are controlled by the controller at regular intervals, so that it is turned on in a specific process.

[0047] Exemplarily, the liquid suction catheter at least includes:

[0048] The plasmid separation liquid suction catheter 7 is connected to the screening area 31 and transports the plasmid to the plasmid adsorption column 29;

[0049] The washing solution catheter 8 is used to introduce the washing solution to the plasmid adsorption column 29 to wash the plasmid;

[0050] The de-liquid catheter 9 is used to introduce the eluent to the plasmid adsorption column 29 to separate the plasmid;

[0051] The electrolyte suction catheter 10 is used to transport the electrolyte to the separation container and provide an electrophoresis environment for the separation container

[0052] The separation reagent suction catheter is used to transport the lysis solution and the precipitant to the lysis area 30 to break the bacteria and perform precipitation separation.

[0053] Exemplarily, the liquid outlet catheter includes:

[0054] The waste liquid catheter 15 is connected to the plasmid adsorption column 29 and exports the washed washing solution to the outside of the separation container;

[0055] The plasmid collection catheter 16 is connected to the plasmid adsorption column 29 and exports the separated plasmid to the outside of the separation container for collection;

[0056] A delivery catheter is connected to a separation container and delivers electrolyte and reagent to the separation container; the delivery catheter includes an electrolyte outlet catheter 13 communicating with the electrolyte suction catheter 10, and a separation reagent outlet catheter 14 communicating with the separation reagent suction catheter and delivering to the lysis zone 30 of the separation container.

[0057] Among them, the separation reagent suction catheter includes a lysis solution suction catheter 11 and a precipitation reagent suction catheter 12.

[0058] In this embodiment, the electrode includes a negative electrode plate 2 and a positive electrode plate 6, and the negative electrode plate 2 and the positive electrode plate 6 are mainly set at both ends of the separation container. The electrode is mainly used for electrolysis of the electrolyte, and at the same time promotes the directional movement of the plasmid after separation, so that the plasmid can pass through the screening filter membrane 4 and gather around the negative electrode plate 2 after separation, achieving preliminary separation.

[0059] In this embodiment, the filter membrane includes a screening filter membrane 4 and a blocking filter membrane 3. The blocking filter membrane 3 is a PVDF membrane with a pore size of 0.01 um; the screening filter membrane 4 is a PVDF membrane with a pore size of 0.45 um. The separation container mainly includes a lysis zone 30 and a screening zone 31, and the lysis zone 30 and the screening zone 31 are separated by the screening filter membrane 4. The screening zone 31 is arranged close to the negative electrode plate 2; the blocking filter membrane 3 is arranged between the negative electrode plate 2 and the screening filter membrane 4, mainly used to block the plasmid to prevent the plasmid from being adsorbed by the negative electrode plate 2.

[0060] Between the screening filter membrane 4 and the positive electrode plate 6 is the lysis zone 30, and a stirring device is provided in the lysis zone 30. In the present invention, magnetic stirring is preferably used. The lysis zone 30 is mainly used for the reaction of the plasmid. For example, a lysis solution is added, and the bacteria are lysed under the action of magnetic stirring. In addition, a precipitation reagent can also be added to precipitate the genome and protein dissolved in the electrolyte after lysis into flocculent precipitates, facilitating the separation between the genome, protein and plasmid by the screening filter membrane 4.

[0061] In this embodiment, an infusion pump 24 and a liquid control valve are provided on the reagent delivery system. The infusion pump 24 mainly provides a driving force to facilitate the flow of the liquid. The liquid control valve is mainly used to control the flow direction.

[0062] Example 2

[0063] A method for extracting plasmid using a plasmid extraction device, comprising the following steps:

[0064] (1) Add 200 ml of normally cultured bacterial solution to the lysis zone 30 in the separation container, and at the same time add RNase A to the bacterial solution until the final concentration of RNase A in the liquid is 30 ug / mL. Run the device. After the device is started, all timers start timing, and all liquid control valves are in the closed state to prevent the liquid from flowing.

[0065] (2) When the timer runs to the specified time, start the infusion pump 24 and operate the liquid control valve 22 and the liquid control valve 25 in combination to suck 10 ml of electrolyte solution (reagent 1) from the electrolyte liquid suction conduit 10, transport it through the electrolyte liquid discharge conduit 13, and supply it to the separation container to provide an electrophoresis environment; among them, the liquid control valve, the pump speed is 10 ml / min, the running time is 1.5 min, and the liquid level in the screening area is 1 / 5 higher than the liquid level in the lysis area;

[0066] (3) When the timer runs to the specified time, start the infusion pump 24 and operate the liquid control valve 21 and the liquid control valve 26 in combination for 1.5 min, suck 10 ml of lysis solution (reagent 2) from the lysis solution liquid suction conduit 11, transport it through the separation reagent liquid discharge conduit 14, and spray it into the bacterial solution in the separation container. When the timer runs to the specified time, start the magnetic stirrer 32, drive the stirrer 5 to rotate at a speed of 1 revolution per second for 1 min for mixing, and let it stand for 2 min, and the bacteria are lysed; plasmids, genomes, proteins, etc. in the bacteria are dispersed in the electrolyte solution.

[0067] (4) When the timer runs to the specified time, start the infusion pump 24 and operate the liquid control valve 20 and the liquid control valve 26 in combination to suck 30 ml of precipitation reagent (reagent 3) from the precipitation reagent liquid suction conduit 12, transport it through the separation reagent liquid discharge conduit 14, and spray it into the bacterial solution in the separation container. Then, when the timer runs to the specified time, start the magnetic stirrer 32 to drive the stirrer 5 to rotate at a speed of 2 revolutions per second for 2 min for mixing, and let it stand for 2 min to precipitate the genome and proteins as flocculent precipitates;

[0068] (5) When the timer runs to the specified time, turn on the positive and negative electrodes of the external power supply. In the separation container, the negative electrode plate 2 and the positive electrode plate 6 interact to provide an electrophoresis environment. Set the voltage to 80 V (5 V / cm), turn on for 5 s and turn off for 1 s, and operate the intermittent current power supply for 5 min. According to the physical and chemical properties of plasmids, genomes, and some proteins, the plasmid electrophoresis passes through the screening filter membrane 4 and enters the screening area 31 between the blocking filter membrane 3 and the screening filter membrane 4 to achieve the purpose of separating plasmids;

[0069] (6) When the timer runs to the specified time, start the infusion pump 24 and open the liquid control valve 17 to operate for 1 min. Due to the obstruction of the screening filter membrane 4, the flow of the solution on both sides is extremely slow. Transport the solution containing plasmids in the separation container through the plasmid separation liquid suction conduit 7 through the plasmid adsorption column 29, and the plasmids are adsorbed, while the waste liquid is discharged through the waste liquid conduit 15;

[0070] (7) When the timer runs to the specified time, the infusion pump 24 and the liquid control valves 18, 23, and 28 start to operate in combination at a pump speed of 10 ml / min for 1.5 min; 10 ml of cleaning solution (reagent 4) is sucked from the cleaning solution conduit 8, passed through the plasmid adsorption column 29 for impurity cleaning, and the waste liquid is discharged through the waste liquid conduit 15;

[0071] (8) When the timer runs to the specified time, the infusion pump 24 and the liquid control valves 19, 23, and 27 start to operate in combination at a pump speed of 10 ml / min for 1 min; 3 ml of eluent (reagent 5) is sucked from the elution liquid conduit 9, transported through the plasmid adsorption column 29 for elution, and the eluent is discharged through the plasmid collection conduit 16 for final collection.

[0072] In the present invention, the electrolyte, lysis solution, precipitation reagent, cleaning solution, eluent, etc. are conventional reagents in plasmid extraction and are common knowledge to those skilled in the art. Therefore, their specific names are not elaborated herein.

[0073] The technical solutions provided by the embodiments of the present invention have been introduced in detail above. Specific examples are used herein to elaborate the principles and implementation manners of the embodiments of the present invention. The descriptions of the above embodiments are only applicable to help understand the principles of the embodiments of the present invention; at the same time, for those of ordinary skill in the art, according to the embodiments of the present invention, there will be changes in the specific implementation manners and application scopes. In summary, the content of this specification should not be construed as a limitation to the present invention.

Claims

1. A plasmid extraction device, comprising a separation container, electrodes arranged in the separation container for plasmid electrophoresis, a plasmid adsorption column, and a reagent delivery system, characterized in that: It also includes a controller and a stirrer arranged at the lower part of the separation container and automatically started and stopped by the controller; the electrode is electrically connected to the controller; A filter membrane is also arranged in the separation container; the filter membrane divides the separation container into a screening area and a lysis area integrating fragmentation and precipitation; The reagent delivery system includes at least one liquid suction conduit and at least one liquid discharge conduit; the plasmid adsorption column is arranged between the liquid suction conduit and the liquid discharge conduit; the liquid suction conduit is used to introduce reagents into the plasmid adsorption column and / or the reagent delivery system; the liquid discharge conduit is used to introduce reagents into and / or outside the separation container; A liquid control valve is arranged on each of the liquid suction conduits and each of the liquid discharge conduits; The filter membrane includes a screening filter membrane and a blocking filter membrane, and the screening area is between the screening filter membrane and the blocking filter membrane; The electrode includes a negative electrode plate and a positive electrode plate, and the screening area is arranged close to the negative electrode plate; the blocking filter membrane is arranged between the negative electrode plate and the screening filter membrane; the lysis area is between the screening filter membrane and the positive electrode plate; The liquid control valve includes a valve body and an electromagnet arranged outside the valve body. The two ends of the valve body are provided with a liquid inlet and a liquid outlet. A magnetic steel ball is arranged at the center of the valve body. A blocking rubber ring is arranged near the liquid inlet in the valve body, and a flow-dispersing pad is arranged near the liquid outlet in the valve body; the electromagnet is arranged near the liquid inlet.

2. The plasmid extraction device according to claim 1, characterized in that: The liquid suction conduit at least includes: A plasmid separation liquid suction conduit, which is connected to the screening area and transports the plasmid to the plasmid adsorption column; A washing liquid conduit, which is used to introduce washing liquid into the plasmid adsorption column to wash the plasmid; A de-liquid conduit, which is used to introduce eluent into the plasmid adsorption column to separate the plasmid; An electrolyte suction conduit, which is used to transport electrolyte to the separation container and provide an electrophoresis environment for the separation container. A separation reagent suction conduit, which is used to transport lysis solution and precipitant to the lysis area to break and precipitate bacteria.

3. The plasmid extraction device according to claim 2, wherein: The liquid discharge conduit includes: A waste liquid conduit, which is connected to the plasmid adsorption column and discharges the washed washing liquid to the outside of the separation container; A plasmid collection conduit, which is connected to the plasmid adsorption column and exports the separated plasmid to the outside of the separation container for collection; A delivery conduit, which is connected to the separation container and transports electrolyte and reagents to the separation container; the delivery conduit includes an electrolyte outlet conduit communicated with the electrolyte suction conduit, and a separation reagent outlet conduit communicated with the separation reagent suction conduit and transported to the lysis area of the separation container.

4. The plasmid extraction device according to claim 3, wherein: The separation reagent suction conduit includes a lysis solution suction conduit and a precipitation reagent suction conduit.

5. The plasmid extraction device according to claim 1, wherein: The stirrer is a magnetic stirrer, and the magnetic stirrer is electrically connected to the controller.

6. The plasmid extraction device according to claim 1, wherein: An infusion pump automatically started and stopped by the controller is arranged on the reagent delivery system, and the liquid control valve is an electrically controlled valve controlled by the controller to switch on and off at a fixed time.

7. The plasmid extraction device according to claim 1, wherein: The plasmid adsorption column is made of silica gel; the blocking filter membrane is a PVDF membrane with a pore size of 0.01um; the screening filter membrane is a PVDF membrane with a pore size of 0.45um.

8. A method for extracting plasmid using the plasmid extraction device according to any one of claims 1-7, characterized in that: Including; S1: Add the bacterial solution to the lysis area, and the controller sequentially controls the electrolyte to enter the screening area and the lysis solution to enter the lysis area, and at the same time starts the magnetic stirrer to stir; S2: Add a precipitation reagent to the lysed bacterial solution in S1, and at the same time start the magnetic stirrer for stirring; S3: The controller controls the electrodes to start, causing the plasmid to electrophorese and screen into the screening area; then controls the liquid control valve in the separation liquid suction conduit to start, adsorbing the plasmid onto the plasmid adsorption column for adsorption; S4: The controller sequentially controls the washing solution and the eluent to enter the plasmid adsorption column to wash and desorb the plasmid; after desorbing, collect it through the plasmid collection conduit.

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