Method for lysing bacterial cells

Abstract

The present disclosure provides a method for lysing bacterial cells, comprising mixing a lysis solution with a bacterial cell suspension while shaking, adding a buffer solution, and shaking the mixture. A method for isolating plasmids from the bacterial cell lysate is also provided.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to Chinese Patent Application No. 2020109496845, filed on September 10, 2020, the entire disclosure of which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to the field of biotechnology, particularly to methods for lysing bacterial cells.

Background Art

[0003] Gene therapy refers to introducing an exogenous therapeutic gene into target cells to correct or compensate for diseases caused by gene defects or abnormalities. Alternatively, the product expressed by the exogenous gene may act on the therapeutic target for treatment.

[0004] Plasmids are very useful as vectors in gene therapy. They can be packaged into viral vectors and used directly for in - vivo treatment, or used to modify target cells (such as T cells or stem cells) in vitro that can later be administered to patients for treatment.

[0005] The purity of plasmids (including supercoil content and endotoxin levels, etc.) can affect the packaging of viral vectors and the effectiveness of gene therapy. There are various types of plasmids, including open - circular plasmids, supercoiled plasmids, linear plasmids, replication intermediates, etc. Open - circular plasmids can lead to the inactivation of specific functional genes, thereby reducing the efficiency of viral packaging or the effectiveness in vivo. Therefore, the supercoil content is an important quality attribute of plasmids and an important control point in the plasmid purification process. The purification process can remove some of the open - circular plasmids, but minimizing the formation of open - circular plasmids is important for plasmid production.

[0006] The manipulated plasmid can be produced by fermentation of Escherichia coli (E. coli). Alkaline lysis is a common method for extracting plasmids from Escherichia coli. Open circular plasmids are mostly produced during the process of bacterial lysis. In the conventional process, a manual lysis method or a stirring lysis method is used. The efficiency of the former method varies depending on the operator. It is not easy to control, and the method generally does not conform to high throughput. The latter method may apply excessive shear force, which may cause great damage to the plasmid, especially large plasmids. This can easily lead to the formation of open circular plasmids.

[0007] Therefore, it is necessary to develop a simple, convenient and very efficient method for plasmid purification. Summary of the Invention Problems to be Solved by the Invention

[0008] One object of the present disclosure is to provide a method for lysing bacterial cells. This bacterial cell lysis method can greatly improve the mixing of bacterial cells and the lysate, so that plasmids can be released from the bacterial cells, and the yield and quality of the plasmids are improved. Means for Solving the Problems

[0009] In a first aspect of the present disclosure, a method for lysing bacterial cells is provided. The method can include the following steps: (a) adding a lysate to a bacterial cell suspension to obtain a first mixture, and shaking the first mixture at a rotation speed in the range of about 10 rpm to about 30 rpm, or about 10 rpm to about 20 rpm for about 1 minute to about 10 minutes, about 1 minute to about 8 minutes, or about 3 minutes to about 8 minutes, and incubating the first mixture to obtain a preliminary bacterial cell lysate; and (b) Add a buffer to the preliminary bacterial cell lysate to obtain a second mixture, and shake the second mixture at a rotational speed in the range of about 10 rpm to about 70 rpm, about 30 rpm to about 55 rpm, or about 40 rpm to about 50 rpm for about 1 minute to about 10 minutes, or about 3 minutes to about 8 minutes, and incubate the second mixture to obtain a bacterial cell lysate.

[0010] In certain embodiments, the lysis solution is an alkaline lysis buffer.

[0011] In one embodiment, the lysis solution contains NaOH and / or SDS.

[0012] In certain embodiments, the lysis solution contains NaOH having a concentration in the range of about 50 mM to about 400 mM, about 100 mM to about 400 mM, about 100 mM to about 300 mM, about 100 mM to about 200 mM, about 200 mM to about 400 mM, about 200 mM to about 300 mM, about 50 mM to about 300 mM, about 50 mM to about 200 mM, about 5 mM to about 100 mM, about 150 mM to about 250 mM, about 150 mM to about 200 mM, about 200 mM to about 250 mM, about 50 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, or about 300 mM.

[0013] In certain embodiments, the lysis solution contains SDS having a concentration in the range of about 0.5% (w / v) to about 2% (w / v), about 0.5% (w / v) to about 1.5% (w / v), about 0.5% (w / v) to about 1% (w / v), about 1% (w / v) to about 2% (w / v), about 1% (w / v) to about 1.5% (w / v), about 1.5% (w / v) to about 2% (w / v), about 0.5% (w / v), about 1% (w / v), about 1.5% (w / v) or about 2% (w / v).

[0014] In one embodiment, in step (a), while shaking the bacterial cell suspension, add the lysis solution to the bacterial cell suspension at a flow rate in the range of about 50 ml / min to about 400 ml / min, about 50 ml / min to about 300 ml / min, about 50 ml / min to about 200 ml / min, or about 150 ml / min to about 200 ml / min.

[0015] In one embodiment, in step (a), the first mixture is incubated for about 1 minute to about 60 minutes, about 1 minute to about 8 minutes, or about 1 minute to about 5 minutes.

[0016] In one embodiment, in step (b), the second mixture is incubated for about 1 minute to about 60 minutes, about 1 minute to about 8 minutes, or about 3 minutes to about 8 minutes.

[0017] In one embodiment, in step (b), the buffer solution contains an acetate buffer solution (such as a potassium acetate buffer solution).

[0018] In one embodiment, in step (b), while shaking the preliminary bacterial cell lysate, the buffer solution is added to the preliminary bacterial cell lysate at a flow rate in the range of about 50 ml / min to about 400 ml / min, about 50 ml / min to about 300 ml / min, about 50 ml / min to about 200 ml / min, or about 150 ml / min to about 200 ml / min.

[0019] In one embodiment, the bacterial cell suspension contains a buffer solution.

[0020] In one embodiment, the bacterial cell suspension is prepared by suspending bacterial cells in a suspension buffer solution and shaking the suspended bacterial cells to obtain a bacterial cell suspension.

[0021] In one embodiment, the suspension buffer solution contains a Tris buffer solution.

[0022] In one embodiment, the suspended bacterial cells are shaken at a rotational speed in the range of about 10 rpm to about 70 rpm, or about 10 rpm to about 20 rpm.

[0023] In one embodiment, the suspended bacterial cells are shaken for about 1 minute to about 5 minutes.

[0024] In certain embodiments, a first oscillator is used in step (a), a second oscillator is used in step (b), and a third oscillator is used when preparing the bacterial cell suspension.

[0025] In one embodiment, the first oscillator, the second oscillator, and the third oscillator are the same oscillator or different oscillators.

[0026] In one embodiment, the first oscillator is a 3D or 2D oscillator and / or the second oscillator is a 3D or 2D oscillator.

[0027] In one embodiment, the third oscillator is a 3D or 2D oscillator.

[0028] In one embodiment, the first oscillator is a back-and-forth oscillator, a left-and-right oscillator, and / or a waveform oscillator.

[0029] In one embodiment, the second oscillator is a back-and-forth oscillator, a left-and-right oscillator, and / or a waveform oscillator.

[0030] In one embodiment, the third oscillator is a back-and-forth oscillator, a left-and-right oscillator, and / or a waveform oscillator.

[0031] In one embodiment, the first oscillator includes a first tray.

[0032] In one embodiment, the second oscillator includes a second tray.

[0033] In one embodiment, the third oscillator includes a third tray.

[0034] In one embodiment, in step (a), the first mixture is placed on the first tray for shaking, and the first tray forms an angle of about 10 to about 20 degrees with respect to the horizontal plane.

[0035] In one embodiment, the bacterial cell suspension is placed in a liquid dispensing bag.

[0036] In one embodiment, in step (b), the preliminary bacterial cell lysate is placed in a second tray for shaking, and the second tray forms an angle of about 10 to about 20 degrees with respect to the horizontal plane.

[0037] In one embodiment, the preliminary bacterial cell lysate is placed in a liquid dispensing bag.

[0038] In one embodiment, the bacterial cells are engineered bacterial cells containing at least one plasmid.

[0039] In one embodiment, the plasmid is a P1 plasmid.

[0040] In one embodiment, the bacterial cells are Escherichia coli Top10.

[0041] In a second aspect of the present disclosure, a method for preparing a plasmid is provided. The method can include the following steps: (a) lysing bacterial cells using the method for lysing bacterial cells to obtain a bacterial cell lysate, wherein the bacterial cells contain a plasmid, and (b) isolating the plasmid from the bacterial cell lysate.

[0042] It should be understood that within the scope of the present disclosure, the above technical features of the present disclosure and the technical features (such as embodiments) specifically described below can be combined with each other to form new or preferred technical solutions. Due to space limitations, this will not be repeated here. BRIEF DESCRIPTION OF THE DRAWINGS

[0043]

Figure 1

Figure 2

Figure 3

Embodiments for Carrying out the Invention

[0044] The present disclosure provides a method for bacterial cell lysis. Compared with the conventional manual lysis method or stirring lysis method, the bacterial cell lysis method of the present disclosure can significantly improve the sufficient mixing of bacterial cells and the lysing solution, thereby releasing plasmids from bacterial cells, increasing the yield of plasmids, and increasing the concentration of plasmids and the content of supercoils.

[0045] The present disclosure provides a method for lysing bacterial cells. The method can include the following steps: (a) Adding a lysing solution to a bacterial cell suspension to obtain a first mixture, and shaking the first mixture at a rotational speed in the range of about 10 rpm to about 30 rpm, about 10 rpm to about 20 rpm, about 10 rpm to about 25 rpm, about 10 rpm to about 15 rpm, about 15 rpm to about 30 rpm, about 15 rpm to about 25 rpm, about 15 rpm to about 20 rpm, about 20 rpm to about 30 rpm, about 20 rpm to about 25 rpm, about 25 rpm to about 30 rpm, about 10 rpm, about 15 rpm, about 20 rpm, about 25 rpm, or about 30 rpm, for about 1 minute to about 10 minutes, about 1 minute to about 5 minutes, about 1 minute to about 8 minutes, about 2 minutes to about 10 minutes, about 2 minutes to about 8 minutes, about 2 minutes to about 5 minutes, about 3 minutes to about 5 minutes, about 3 minutes to 8 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, or about 8 minutes, and incubating the first mixture (without shaking) to obtain a preliminary bacterial cell lysate; and, (b) Add the buffer to the preliminary bacterial cell lysate to obtain a second mixture, and subject the second mixture to about 10 rpm to about 70 rpm, about 30 rpm to about 55 rpm, about 10 rpm to about 65 rpm, about 10 rpm to about 60 rpm, about 10 rpm to about 55 rpm, about 10 rpm to about 50 rpm, about 10 rpm to about 45 rpm, about 10 rpm to about 40 rpm, about 10 rpm to about 35 rpm, about 10 rpm to about 30 rpm, about 15 rpm to about 70 rpm, about 15 rpm to about 65 rpm, about 15 rpm to about 60 rpm, about 15 rpm to about 55 rpm, about 15 rpm to about 50 rpm, about 15 rpm to about 45 rpm, about 15 rpm to about 40 rpm, about 15 rpm to about 35 rpm, about 15 rpm to about 30 rpm, about 20 rpm to about 70 rpm, about 20 rpm to about 65 rpm, about 20 rpm to about 60 rpm, about 20 rpm to about 55 rpm, about 20 rpm to about 50 rpm, about 20 rpm to about 45 rpm, about 20 rpm to about 40 rpm, about 20 rpm to about 35 rpm, about 20 rpm to about 30 rpm, about 25 rpm to about 70 rpm, about 25 rpm to about 65 rpm, about 25 rpm to about 60 rpm, about 25 rpm to about 55 rpm, about 25 rpm to about 50 rpm, about 25 rpm to about 45 rpm, about 25 rpm to about 40 rpm, about 25 rpm to about 35 rpm, about 30 rpm to about 70 rpm, about 30 rpm to about 65 rpm, about 30 rpm to about 60 rpm, about 30 rpm to about 55 rpm, about 30 rpm to about 50 rpm, about 30 rpm to about 45 rpm, about 30 rpm to about 40 rpm, about 35 rpm to about 70 rpm, about 35 rpm to about 65 rpm, about 35 rpm to about 60 rpm, about 35 rpm to about 55 rpm, about 35 rpm to about 50 rpm, about 35 rpm to about 45 rpm, about 35 rpm to about 40 rpm, about 40 rpm to about 70 rpm, about 40 rpm to about 65 rpm, about 40 rpm to about 60 rpm, about 40 rpm to about 55 rpm, about 40 rpm to about 50 rpm, about 40 rpm to about 45 rpm, about 45 rpm to about 70 rpm, about 45 rpm to about 65 rpm, about 45 rpm to about 60 rpm, about 45 rpm to about 55 rpm, about 45 rpm to about 50 rpm, about 30 rpm, about 35 rpm, about 40 rpm, about 45 rpm, about 50 rpm, about 55 rpm, or about 60 rpm for about 1 minute to about 10 minutes, about 1 minute to about 5 minutes, about 1 minute to about 8 minutes, about 2 minutes to about 10 minutes, about 2 minutes to about 8 minutes, about 2 minutes to about 5 minutes,Shake at a rotation speed in the range of about 3 minutes to about 5 minutes, about 3 minutes to about 8 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, or about 8 minutes, and incubate the second mixture (without shaking) to obtain a bacterial cell lysate.

[0046] In certain embodiments, the lysate contains one or more hydroxides. The hydroxide may be a hydroxide of an alkali metal such as sodium hydroxide (NaOH), potassium hydroxide (KOH), lithium hydroxide (LiOH). 2 ) or a hydroxide of an alkaline earth metal such as magnesium hydroxide (Mg(OH) 2 ).

[0047] In certain embodiments, the lysate contains one or more surfactants or a surfactant. The surfactant may be an anionic surfactant such as sodium dodecyl sulfate (SDS).

[0048] In one embodiment, in step (a), while shaking the bacterial cell suspension, add the lysate to the bacterial cell suspension at a flow rate in the range of about 50 ml / min to about 400 ml / min, about 50 ml / min to about 350 ml / min, about 50 ml / min to about 300 ml / min, about 50 ml / min to about 250 ml / min, about 50 ml / min to about 200 ml / min, about 100 ml / min to about 400 ml / min, about 100 ml / min to about 350 ml / min, about 100 ml / min to about 300 ml / min, about 100 ml / min to about 250 ml / min, about 100 ml / min to about 200 ml / min, about 150 ml / min to about 400 ml / min, about 150 ml / min to about 350 ml / min, about 150 ml / min to about 300 ml / min, about 150 ml / min to about 250 ml / min, about 150 ml / min to about 200 ml / min, about 200 ml / min to about 400 ml / min, about 200 ml / min to about 350 ml / min, about 200 ml / min to about 300 ml / min, about 200 ml / min to about 250 ml / min, about 150 ml / min, about 200 ml / min or about 250 ml / min.

[0049] In certain embodiments, in step (a), the volume ratio of the lysate to the bacterial cell suspension is in the range of about 1:4 to about 1:1, about 1:3 to about 1:1, about 1:2 to about 1:1, about 1:4, about 1:3.5, about 1:3, about 1:2.5, about 1:2, about 1:1.5, or about 1:1.

[0050] In one embodiment, in step (a), the first mixture is incubated for about 1 minute to about 60 minutes, about 1 minute to about 45 minutes, or about 1 minute to about 30 minutes, about 1 minute to about 20 minutes, about 1 minute to about 15 minutes, about 1 minute to about 10 minutes, about 1 minute to about 8 minutes, about 1 minute to about 5 minutes, about 2 minutes to about 45 minutes, about 2 minutes to about 30 minutes, about 2 minutes to about 20 minutes, about 2 minutes to about 15 minutes, about 2 minutes to about 10 minutes, about 2 minutes to about 8 minutes, about 2 minutes to about 5 minutes, about 3 minutes to about 5 minutes, about 3 minutes to about 8 minutes, about 5 minutes to about 45 minutes, about 5 minutes to about 30 minutes, about 5 minutes to about 20 minutes, about 5 minutes to about 15 minutes, about 5 minutes to about 10 minutes, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, or about 8 minutes.

[0051] In certain embodiments, in step (a), the first mixture is shaken and / or incubated at a temperature in the range of about 20°C to about 25°C or at ambient temperature. The hydrolysis may be carried out at a temperature in the range of about 1°C to about 37°C, about 2°C to about 37°C, about 10°C to about 15°C, about 15°C to about 20°C, about 20°C to about 25°C, about 25°C to about 30°C, about 30°C to about 35°C, about 4°C to about 35°C, about 10°C to about 37°C, about 15°C to about 37°C, about 20°C to about 37°C, about 25°C to about 37°C, about 2°C to about 8°C, about 4°C to about 8°C, about 2°C to about 4°C, about 6°C to about 8°C, about 4°C to about 10°C, about 4°C to about 15°C, about 4°C to about 20°C, about 4°C to about 25°C, about 4°C to about 30°C, about 4°C to about 35°C, about 4°C to about 37°C, about 4°C, about 25°C, or about 37°C. The temperature may be in a range between the temperatures of any two integer values selected from about 1°C to about 37°C. The temperature may be the temperature of any one integer value selected from those including about 1°C to about 37°C or about 15°C to about 35°C. A temperature from room temperature (ambient temperature) to about 37°C can be used. The temperature may be any temperature including between room temperature and about 37°C. A temperature of about 20°C to about 35°C may be used. The temperature may be any temperature including about 20°C to about 25°C. The temperature may be about 25°C.

[0052] In certain embodiments, in step (b), the volume ratio of the buffer to the preliminary bacterial cell lysate is in the range of about 1:4 to about 1:1, about 1:3 to about 1:1, about 1:2 to about 1:1, about 1:4, about 1:3.5, about 1:3, about 1:2.5, about 1:2, about 1:1.5, or about 1:1.

[0053] In one embodiment, in step (b), the second mixture is incubated for about 1 minute to about 60 minutes, about 1 minute to about 45 minutes, or about 1 minute to about 30 minutes, about 1 minute to about 20 minutes, about 1 minute to about 15 minutes, about 1 minute to about 10 minutes, about 1 minute to about 8 minutes, about 1 minute to about 5 minutes, about 2 minutes to about 45 minutes, about 2 minutes to about 30 minutes, about 2 minutes to about 20 minutes, about 2 minutes to about 15 minutes, about 2 minutes to about 10 minutes, about 2 minutes to about 8 minutes, about 2 minutes to about 5 minutes, about 3 minutes to about 5 minutes, about 3 minutes to about 8 minutes, about 5 minutes to about 45 minutes, about 5 minutes to about 30 minutes, about 5 minutes to about 20 minutes, about 5 minutes to about 15 minutes, about 5 minutes to about 10 minutes, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes or about 10 minutes.

[0054] In certain embodiments, in step (b), the second mixture is shaken and / or incubated at a temperature in the range of about 20°C to about 25°C, or at ambient temperature. The hydrolysis may be carried out at a temperature in the range of about 1°C to about 37°C, about 2°C to about 37°C, about 10°C to about 15°C, about 15°C to about 20°C, about 20°C to about 25°C, about 25°C to about 30°C, about 30°C to about 35°C, about 4°C to about 35°C, about 10°C to about 37°C, about 15°C to about 37°C, about 20°C to about 37°C, about 25°C to about 37°C, about 2°C to about 8°C, about 4°C to about 8°C, about 2°C to about 4°C, about 6°C to about 8°C, about 4°C to about 10°C, about 4°C to about 15°C, about 4°C to about 20°C, about 4°C to about 25°C, about 4°C to about 30°C, about 4°C to about 35°C, about 4°C to about 37°C, about 4°C, about 25°C, or about 37°C. The temperature may be in a range between the temperatures of any two integer values selected from about 1°C to about 37°C. The temperature may be the temperature of any one integer value selected from those including about 1°C to about 37°C, or about 15°C to about 35°C. A temperature from room temperature (ambient temperature) to about 37°C can be used. The temperature may be any temperature including between room temperature and about 37°C. A temperature of about 20°C to about 35°C may be used. The temperature may be any temperature including about 20°C to about 25°C. The temperature may be about 25°C.

[0055] The buffer can be an acetate buffer or a citrate buffer. The acetate buffer can be a potassium acetate buffer and / or a sodium acetate buffer. In certain embodiments, the buffer is a potassium acetate buffer having a concentration in the range of about 1 M (mol / L) to about 5 M, about 1 M to about 4 M, about 1 M to about 3 M, about 1 M to about 2 M, about 2 M to about 5 M, about 2 M to about 4 M, about 2 M to about 3 M, about 3 M to about 5 M, about 3 M to about 4 M, about 1 M, about 1.5 M, about 2 M, about 2.5 M, about 3 M, about 3.5 M, about 4 M, about 4.5 M, or about 5 M.

[0056] The buffer can have a pH in the range of about pH 3 to about pH 7, about pH 3 to about pH 6.5, about pH 3 to about pH 6, about pH 3 to about pH 5.5, about pH 3 to about pH 5, about pH 3 to about pH 4, about pH 4 to about pH 7, about pH 4 to about pH 6.5, about pH 4 to about pH 6, about pH 4 to about pH 5.5, about pH 4 to about pH 5, about pH 5 to about pH 7, about pH 5 to about pH 6.5, about pH 5 to about pH 6, about pH 5 to about pH 5.5, about pH 5.5 to about pH 6, about pH 4, about pH 4.5, about pH 5, about pH 5.5, or about pH 6.

[0057] In one embodiment, in step (b), the buffer comprises an acetate buffer (such as a potassium acetate buffer).

[0058] In one embodiment, in step (b), while shaking the preliminary bacterial cell lysate, a buffer solution is added to the preliminary bacterial cell lysate at a flow rate in the range of about 50 ml / min to about 400 ml / min, about 50 ml / min to about 350 ml / min, about 50 ml / min to about 300 ml / min, about 50 ml / min to about 250 ml / min, about 50 ml / min to about 200 ml / min, about 100 ml / min to about 400 ml / min, about 100 ml / min to about 350 ml / min, about 100 ml / min to about 300 ml / min, about 100 ml / min to about 250 ml / min, about 100 ml / min to about 200 ml / min, about 150 ml / min to about 400 ml / min, about 150 ml / min to about 350 ml / min, about 150 ml / min to about 300 ml / min, about 150 ml / min to about 250 ml / min, about 150 ml / min to about 200 ml / min, about 200 ml / min to about 400 ml / min, about 200 ml / min to about 350 ml / min, about 200 ml / min to about 300 ml / min, about 200 ml / min to about 250 ml / min, about 150 ml / min, about 200 ml / min or about 250 ml / min.

[0059] In one embodiment, the bacterial cell suspension contains a buffer solution.

[0060] In one embodiment, the bacterial cell suspension is prepared by suspending bacterial cells in a suspension buffer and shaking the suspended bacterial cells to obtain the bacterial cell suspension.

[0061] The suspension buffer can be Tris buffer, TAPS buffer, Bicine buffer, Tricine buffer, TAPSO buffer, HEPES buffer, TES buffer, MOPS buffer or PIPES buffer.

[0062] The suspension buffer can have a pH in the range of about pH 6 to about pH 9, about pH 6.5 to about pH 9, about pH 7 to about pH 9, about pH 7.5 to about pH 9, about pH 8 to about pH 9, about pH 8.5 to about pH 9, about pH 6 to about pH 8, about pH 6.5 to about pH 8, about pH 7 to about pH 8, about pH 7.5 to about pH 8, about pH 6 to about pH 8.5, about pH 7 to about pH 8.5, about pH 8 to about pH 8.5, about pH 7, about pH 7.5, about pH 8, about pH 8.5, or about pH 9.

[0063] In one embodiment, the suspension buffer contains Tris buffer.

[0064] In one embodiment, the suspended bacterial cells are shaken at a rotational speed in the range of about 10 rpm to about 30 rpm, about 10 rpm to about 20 rpm, about 10 rpm to about 25 rpm, about 10 rpm to about 15 rpm, about 15 rpm to about 30 rpm, about 15 rpm to about 25 rpm, about 15 rpm to about 20 rpm, about 20 rpm to about 30 rpm, about 20 rpm to about 25 rpm, about 25 rpm to about 30 rpm, about 10 rpm, about 15 rpm, about 20 rpm, about 25 rpm, or about 30 rpm.

[0065] In one embodiment, the suspended bacterial cells are shaken for about 1 minute to about 60 minutes, about 1 minute to about 45 minutes, about 1 minute to about 30 minutes, about 1 minute to about 20 minutes, about 1 minute to about 10 minutes, about 1 minute to about 5 minutes, about 1 minute to about 4 minutes, about 1 minute to about 3 minutes, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, or about 5 minutes.

[0066] In certain embodiments, a first oscillator is used in step (a), a second oscillator is used in step (b), and a third oscillator is used when preparing the bacterial cell suspension.

[0067] In one embodiment, the first oscillator, the second oscillator, and the third oscillator are the same oscillator or different oscillators.

[0068] In one embodiment, the first oscillator is a 3D (three-dimensional) or 2D oscillator, and / or the second oscillator is a 3D or 2D oscillator.

[0069] In one embodiment, the third oscillator is a 3D or 2D oscillator.

[0070] In one embodiment, the first oscillator is a back-and-forth shaker, a left-and-right shaker, and / or a waveform shaker.

[0071] In one embodiment, the second oscillator is a front-back oscillating oscillator, a left-right oscillating oscillator, and / or a waveform oscillating oscillator.

[0072] In one embodiment, the third oscillator is a front-back oscillating oscillator, a left-right oscillating oscillator, and / or a waveform oscillating oscillator.

[0073] In one embodiment, the lysate contains a NaOH / SDS lysate.

[0074] In one embodiment, the lysate contains NaOH and / or SDS.

[0075] In one embodiment, the first oscillator includes a first tray.

[0076] In one embodiment, the second oscillator includes a second tray.

[0077] In one embodiment, the third oscillator includes a third tray.

[0078] In one embodiment, in step (a), the first mixture is placed on the first tray for oscillation, and the first tray forms an angle of about 10 to about 20 degrees with respect to the horizontal plane.

[0079] In one embodiment, the bacterial cell suspension is placed in a liquid dispensing bag.

[0080] In one embodiment, in step (b), the preliminary bacterial cell lysate is placed on the second tray for oscillation, and the second tray forms an angle of about 10 to about 20 degrees with respect to the horizontal plane.

[0081] In one embodiment, the preliminary bacterial cell lysate is placed in a liquid dispensing bag.

[0082] In one embodiment, the bacterial cells are engineered bacterial cells containing at least one plasmid.

[0083] In certain embodiments, the bacterial cell is Escherichia coli bacteria.

[0084] In one embodiment, the bacterial cell is Escherichia coli Top10.

[0085] In one embodiment, the plasmid is the P1 plasmid.

[0086] The present disclosure also provides a method for preparing a plasmid. The method can include the following steps: (a) lysing the bacterial cells using the method for lysing bacterial cells to obtain a bacterial cell lysate, wherein the bacterial cells contain a plasmid, and (b) isolating the plasmid from the bacterial cell lysate.

[0087] The bacterial cells can be any suitable bacterial cells. The plasmid can be any suitable type of plasmid.

[0088] The present invention provides a method for lysing bacterial cells, the method including the following steps: (1) placing the bacterial cell solution into a first shaker for shaking, then adding the lysate to the bacterial solution while shaking, shaking, and mixing, and then allowing to stand to obtain a primary bacterial cell lysate, wherein the rotation speed of the first shaker is from 10 to 30 rpm and the mixing time is from 1 to 10 minutes, (2) placing the primary bacterial cell lysate into a second shaker for shaking, then adding the buffer to the primary bacterial cell lysate while shaking, shaking, and mixing, and then allowing to stand to obtain a bacterial cell lysate, wherein the rotation speed of the second shaker is from 10 to 70 rpm and the mixing time is from 1 to 10 minutes.

[0089] The bacterial cells can be engineered bacterial cells modified by a plasmid.

[0090] The plasmid may include a P1 plasmid.

[0091] The bacterial cells may include Top10 Escherichia coli bacteria.

[0092] In one embodiment of the present disclosure, in step (1), the rotation speed of the first oscillator is 10 to 20 rpm.

[0093] In one embodiment of the present disclosure, in step (1), the standing time is 1 to 60 minutes, 1 to 8 minutes, or 1 to 5 minutes.

[0094] In one embodiment of the present disclosure, in step (1), the mixing time is 1 to 8 minutes, or 3 to 8 minutes.

[0095] In one embodiment of the present disclosure, in step (1), the bacterial cell solution includes a bacterial cell buffer.

[0096] Typically, in step (1), the bacterial cell solution is prepared by the following method: (1-1) After mixing the bacterial cells and the buffer, shake and mix with a third oscillator to obtain a bacterial cell solution.

[0097] In one embodiment, in step (1-1), the buffer includes a Tris buffer.

[0098] In one embodiment, in step (1-1), the rotation speed of the third oscillator is 10 to 70 rpm, or 10 to 20 rpm.

[0099] In one embodiment, in step (1-1), the mixing time is 1 to 5 minutes.

[0100] In one embodiment of the present disclosure, in step (1), the lysate is added to the primary bacterial cell lysate while shaking at a flow rate of 50 to 400 ml / min, 50 to 300 ml / min, 50 to 200 ml / min, or 150 to 200 ml / min.

[0101] In the method of the present disclosure, the first oscillator, the second oscillator, and the third oscillator are the same oscillator or different oscillators.

[0102] Typically, the first oscillator is a 3D or 2D oscillator.

[0103] Typically, the second oscillator is a 3D or 2D oscillator.

[0104] Typically, the third oscillator is a 3D or 2D oscillator.

[0105] Typically, the first oscillator is a front - back oscillating oscillator, a left - right oscillating oscillator, and / or a waveform oscillating oscillator.

[0106] Typically, the second oscillator is a front - back oscillating oscillator, a left - right oscillating oscillator, and / or a waveform oscillating oscillator.

[0107] Typically, the third oscillator is a front - back oscillating oscillator, a left - right oscillating oscillator, and / or a waveform oscillating oscillator.

[0108] In one embodiment of the present disclosure, in step (1), the lysate contains a NaOH / SDS lysate.

[0109] In one embodiment, in step (1), the lysate contains a NaOH / SDS component.

[0110] In one embodiment of the present disclosure, the first oscillator includes a first tray.

[0111] In one embodiment of the present disclosure, the second oscillator includes a second tray.

[0112] In one embodiment of the present disclosure, the third oscillator includes a third tray.

[0113] In one embodiment, in step (1), the bacterial cell solution is placed in the first tray for oscillation.

[0114] In one embodiment, the first tray has an angle of 10 to 20 degrees with respect to the horizontal plane.

[0115] In one embodiment, the bacterial cell lysate is placed in a liquid dispensing bag.

[0116] In one embodiment, in step (2), the primary bacterial cell lysate is placed in the second tray for shaking.

[0117] In one embodiment, the second tray has an angle of 10 to 20 degrees with respect to the horizontal plane.

[0118] In one embodiment, the primary bacterial cell lysate is placed in a liquid dispensing bag.

[0119] In one embodiment, in step (2), the standing time is 1 to 60 minutes.

[0120] In one embodiment, in step (2), the buffer solution includes an acetate (such as potassium acetate) buffer solution.

[0121] The buffer solution is added to the primary bacterial cell lysate while shaking at a flow rate of 50 to 400 ml / min.

[0122] In one embodiment of the present disclosure, in step (2), the lysis buffer solution is added into the primary bacterial cell lysate while shaking at a flow rate of 50 to 400 ml / min, 50 to 300 ml / min, 50 to 200 ml / min, or 150 to 200 ml / min.

[0123] In one embodiment of the present disclosure, in step (2), the rotation speed of the second shaker is 30 to 55 rpm, or 40 to 50 rpm.

[0124] In one embodiment of the present disclosure, in step (2), the mixing time is 3 to 8 minutes.

[0125] In one embodiment of the present disclosure, in step (2), the standing time is 1 to 60 minutes, or 3 to 8 minutes.

[0126] The present disclosure also provides a method for preparing a plasmid, the method comprising the following steps: (a) lysing the engineered bacterial cells modified by the plasmid by the method for lysing bacterial cells according to claim 1 to obtain a bacterial cell lysate; (b) isolating the plasmid from the bacterial cell lysate.

[0127] This method provides the following main advantages.

[0128] 1. The method for lysing bacterial cells of the present disclosure has simple operations, saves time and labor, is convenient, and is suitable for industrial production.

[0129] 2. Compared with the conventional manual lysis method or stirring lysis method, the method for lysing bacterial cells of the present disclosure can significantly improve the sufficient mixing of bacterial cells and the lysing solution, thereby releasing the plasmid from the bacterial cells, increasing the yield of the plasmid, and increasing the concentration of the plasmid and the content of supercoils.

[0130] 3. The method for lysing bacterial cells of the present disclosure can overcome the influence of human factors and can be stably scaled up.

[0131] The present disclosure will be further described below in connection with specific embodiments. It should be understood that these embodiments are used only to illustrate the present disclosure and are not used to limit the scope of the present disclosure. In the following examples, experimental methods not indicated for specific conditions generally follow conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0132] [Example 1] 1. Culturing and collecting bacteria Reagents and materials: LB medium; Top10 Escherichia coli; 2L conical flask; kanamycin or ampicillin concentrate; pipette; 500ml centrifuge bottle.

[0133] Equipment: constant temperature shaker; high-speed centrifuge; AKTAflux 6; peristaltic pump.

[0134] Inoculation and culture: A Top10 Escherichia coli starter culture (containing the P1 plasmid with a size of about 2000bp) was added to LB medium at a volume ratio of 1:1000. Kanamycin or ampicillin concentrate was added at a volume ratio of 1:500 (concentrate:LB medium). The final concentration of kanamycin or ampicillin was 100 ng / ml - 200 ng / ml. The bacteria were grown at 37°C for 10 to 30 hours while shaking (260 rpm). The bacterial cells were collected by centrifugation or ultrafiltration.

[0135] 2. Bacterial cell lysis Reagents and materials: Tris buffer (50 mM Tris-Cl, 10 mM EDTA, 50 mM glucose, pH 8.0); NaOH / SDS lysate (1% SDS (w / v) / 200 mM NaOH); potassium acetate buffer (3.0 M potassium acetate, pH 5.0); undulating liquid dispensing bag; pipette; and centrifuge tube.

[0136] Equipment: 3D shaker (SCILOGEX) (capable of providing undulating shaking); and peristaltic pump.

[0137] 2.1 Addition of Tris buffer: 75 ml of Tris buffer was added to each centrifuge bottle containing bacterial cells collected from 400 mL - 500 mL of bacterial culture. The bacterial cells were resuspended and mixed with the Tris buffer.

[0138] In the manual lysis method, the bacterial cell suspension in one centrifuge bottle was used to manually lyse the bacterial cells.

[0139] In the oscillator lysis method, a peristaltic pump was used to pump 1-2 liters of bacterial cell suspension into a 5L liquid dispensing bag. Next, the liquid dispensing bag was placed on the tray of a 3D oscillator. Then, the bacterial cells in the liquid dispensing bag were incubated while shaking at 15 rpm for about 3 minutes.

[0140] 2.2 Addition of NaOH / SDS lysate: Manual lysis method: 75 ml of NaOH / SDS lysate was added to the centrifuge bottle selected for the manual lysis method. Then, the bottle was gently inverted 3 times for mixing and then incubated without shaking for 1 minute.

[0141] Oscillator lysis method: The rotation speed of the 3D oscillator was set to 15 rpm (the angle between the tray of the 3D oscillator and the horizontal plane was 15°). While shaking the liquid dispensing bag, a peristaltic pump was used to pump 1-2 liters of NaOH / SDS lysate into the 5L liquid dispensing bag (containing 1-2 liters of bacterial cell suspension) at a flow rate of 200 ml / min. After mixing for 5 minutes, the oscillator was stopped and the sample was incubated without shaking for 2 minutes.

[0142] 2.3 Addition of potassium acetate buffer: Manual lysis method: 75 ml of potassium acetate buffer was added to the centrifuge bottle selected for the manual lysis method. Then, the bottle was gently inverted 6 times for mixing. A 1 ml sample of the lysate was taken and added to a 1.5 ml centrifuge tube for subsequent tests.

[0143] Oscillator lysis method: The rotation speed of the 3D oscillator was set to 45 rpm. While shaking the liquid dispensing bag, a peristaltic pump was used to pump 1-2 liters of potassium acetate buffer into the liquid dispensing bag at a flow rate of 200 ml / min. After mixing for 5 minutes, the oscillator was stopped and the sample was incubated without shaking for 5 minutes. Then, a 1 ml sample of the lysate was taken and added to a 1.5 ml centrifuge tube.

[0144] 3. Experimental results 1. Determination of plasmid concentration Apparatus: Ultra-high sensitivity ultraviolet spectrophotometer Samples of the above two groups of lysis products were tested for plasmid concentration using an ultra-high sensitivity UV spectrophotometer. 2 μl was taken each time for detection, and the tests for each group were repeated 3 times. The results are shown in Table 1 below.

[0145]

Table 1

[0146] As can be seen from Table 1, the average plasmid concentration of the lysis products obtained in the oscillator lysis method group ("system") was 679.5 ng / μl, while the average plasmid concentration of the lysis products obtained in the manual lysis method group ("manual") was 597.4 ng / μl. Therefore, lysis by the oscillator method enables the complete mixing of bacterial cells with the lysate as compared to lysis by manual operation. As a result, plasmids are released more sufficiently from bacterial cells, and a higher plasmid yield can be obtained.

[0147] 2. DNA Gel Electrophoresis Detection and Plasmid Supercoil Content Analysis Equipment: Digital display electrophoresis apparatus, gel imager.

[0148] According to the results of plasmid concentration, 1000 ng - 1100 ng of lysis products in each of the two groups were taken for DNA gel electrophoresis detection (120 V, 30 minutes). After electrophoresis, analysis was performed using a gel imager. The results of DNA gel electrophoresis are shown in Figure 1.

[0149] The results of DNA gel electrophoresis (Figure 1) show that the lysis products obtained in the oscillator lysis method group (system lysis) and the manual lysis method group contain plasmid electrophoresis bands at the same position. This confirms that the plasmids in bacterial cells did not change due to different lysis methods. This method obtained correct lysis products that were consistent with those obtained by the manual method.

[0150] The bands in the DNA gel electrophoresis of FIG. 1 were further analyzed as shown in FIGS. 2 and 3.

[0151] When analyzing the supercoil content of the lysates, it was suggested that the supercoiled plasmid content of the oscillator lysis method group (lane 2) was 78%, while that of the manual lysis method group (lane 3) was 76.5%. By the oscillator operation method, the bacterial cells could be completely mixed with the lysate. The plasmid isolated using the oscillator lysis method had a higher supercoil content than the manual lysis method group.

[0152] Conclusion When using the same lysis system (e.g., the same lysate, etc.), the lysis method using an oscillator has several advantages compared to the conventional manual method. Lysis can be controlled and quantified, is efficient, and produces a higher concentration of plasmid with a higher supercoil content.

[0153] The scope of the present disclosure is not limited by what has been specifically shown and described above. Those skilled in the art will recognize that suitable alternatives exist for the illustrated examples of materials, configurations, structures, and dimensions. A number of references, including patents and various publications, have been cited and considered in the description of the present invention. The citation and consideration of such references are presented solely to clarify the description of the present invention and do not admit that any reference is prior art to the present disclosure described herein. All references cited and discussed in this specification are hereby incorporated by reference in their entirety. Variations, modifications, and other embodiments of what is described herein will occur to those skilled in the art without departing from the spirit and scope of the present invention. Specific embodiments of the present disclosure have been shown and described, but it will be apparent to those skilled in the art that changes and modifications can be made without departing from the spirit and scope of the present invention. The matters described in the foregoing description and the accompanying drawings are provided by way of example only and not as limitations.

Claims

1. A method for lysing bacterial cells, comprising: (a) adding a lysing solution to a bacterial cell suspension to obtain a first mixture, shaking the first mixture at a rotational speed in the range of 10 rpm to 30 rpm for 1 minute to 10 minutes, and incubating the first mixture to obtain a preliminary bacterial cell lysate; and (b) adding a buffer solution to the preliminary bacterial cell lysate to obtain a second mixture, shaking the second mixture at a rotational speed in the range of 10 rpm to 70 rpm for 1 minute to 10 minutes, and incubating the second mixture to obtain a bacterial cell lysate. A method comprising the above steps.

2. The method according to claim 1, wherein in step (a), the first mixture is shaken at a rotational speed in the range of 10 rpm to 20 rpm.

3. The method according to claim 1, wherein in step (a), the first mixture is shaken for 1 minute to 8 minutes.

4. The method according to claim 1, wherein in step (a), the first mixture is shaken for 3 minutes to 8 minutes.

5. The method according to claim 1, wherein in step (b), the second mixture is shaken at a rotational speed in the range of 30 rpm to 55 rpm.

6. The method according to claim 1, wherein in step (b), the second mixture is shaken at a rotational speed in the range of 40 rpm to 50 rpm.

7. The method according to claim 1, wherein in step (b), the second mixture is shaken for 3 minutes to 8 minutes.

8. The method according to claim 1, wherein the lysing solution is an alkaline lysis buffer.

9. The method according to claim 8, wherein the alkaline lysis buffer contains 1% SDS (w / v) and 200 mM NaOH.

10. The method according to claim 1, wherein the buffer solution is an acetate buffer.

11. The method according to claim 10, wherein the acetate buffer is a potassium acetate buffer.

12. A method for preparing a plasmid, comprising: (a) lysing bacterial cells by the method according to claim 1 to obtain a bacterial cell lysate; and (b) isolating the plasmid from the bacterial cell lysate. A method comprising the above steps.

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