CN108866043B - Pretreatment kit and method for bacterial plasmid extraction - Google Patents
Pretreatment kit and method for bacterial plasmid extraction Download PDFInfo
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Abstract
The invention relates to a pretreatment kit for bacterial plasmid extraction, which comprises the following components: the component A comprises: 100-200 mg/ml lysozyme; and B component: washing liquid; the component A is 100mg/ml lysozyme. The invention also discloses a pretreatment method for extracting the bacterial plasmid. The invention also provides a bacterial plasmid extraction kit and a method. The invention provides a pretreatment kit and a pretreatment method for extracting bacterial plasmids, which can effectively improve the extraction efficiency of the bacterial plasmids, particularly have very good extraction effect on lactobacillus plantarum, can overcome the problem that the existing method can not effectively extract gram-positive bacterial plasmids, and have wide market application prospect.
Description
Technical Field
The invention relates to a pretreatment kit and a method for bacterial plasmid extraction.
Background
Plasmids are nucleic acid molecules capable of self-replication in living cells. Plasmid-mediated horizontal gene transfer is considered to be the main driving force for bacterial diversity and adaptation. With the progress of research, plasmids have been widely used as tools for genetic research. The extraction of plasmid DNA is a key technology in molecular biology, and is an essential link in molecular technologies such as cloning, transfection and gene therapy. The research of bacteria finds that the new plasmid has very important function.
Gram-positive bacteria have high crosslinking degree and are difficult to crack due to peptidoglycan contained in the cell wall, so that the extraction of plasmids of the gram-positive bacteria is very difficult, and particularly, the extraction of one or more natural plasmids in a plurality of lactobacillus plantarum strains is more difficult. The prior literature reports that glass beads combined with short vortex time help to overcome the lysis difficulty, but the operation is complicated and the cost is high.
Disclosure of Invention
In order to solve the technical problems, the invention provides a pretreatment kit and a pretreatment method for bacterial plasmid extraction.
The invention relates to a pretreatment kit for bacterial plasmid extraction, which comprises the following components:
the component A comprises: 80-160mg/ml lysozyme;
and B component: washing liquid;
the component A is 100mg/ml lysozyme.
The washing solution is a 4-6% glucose solution or a solution I; the solution I is a Tris-HCl solution added with 10mmol/L EDTA, 50mmol/L glucose and 100 mu g/ml RNase A, wherein the concentration of the Tris-HCl solution is 25 mmol/L.
The concentration of the glucose solution was 5%.
The invention also provides a bacterial plasmid extraction kit, which comprises the following components:
(I) the pretreatment kit;
(II) plasmid extraction kit.
The component (II) is a kit for extracting plasmids by an alkaline lysis method.
The kit for extracting the plasmids by the alkaline lysis method comprises the following components:
solution I: adding Tris-HCl solution with 10mmol/L EDTA, 50mmol/L glucose and 100 mu g/ml RNase A, wherein the concentration of the Tris-HCl solution is 25 mmol/L;
solution II: adding a NaOH solution with the concentration of 250mmol/L of 1% (W/V) SDS;
solution III: the mixed solution of potassium acetate and acetic acid, wherein the concentration of the potassium acetate is 3mol/L, and the concentration of the acetic acid is 5 mol/L;
solution IV: each 1L of the solution contains 10mmol of Tris-HCl, 0.80L of ethanol and the balance of water (pH 7.5);
solution V: 10mmol/L Tris-HCl solution.
The invention also provides a pretreatment method for bacterial plasmid extraction, which adopts the pretreatment kit of any one of claims 1-4 for treatment and comprises the following steps: treating bacterial thallus with component A, and washing with component B.
The invention also provides a bacterial plasmid extraction method, which adopts the kit for extraction and comprises the following steps: pretreatment was carried out according to the above method, and extraction was carried out using a plasmid extraction kit.
The invention provides a pretreatment kit and a pretreatment method for extracting bacterial plasmids, which can effectively improve the extraction efficiency of the bacterial plasmids, particularly have very good extraction effect on lactobacillus plantarum, can overcome the problem that the existing method can not effectively extract gram-positive bacterial plasmids, and have wide market application prospect.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1: agarose gel electrophoresis images of the lactobacillus plantarum PC518 plasmid DNA were extracted using 7 protocols.
FIG. 2: the results of concentration determination of the plasmid DNA of Lactobacillus plantarum PC518 were extracted in 7 protocols.
FIG. 3: comparative figures of plasmid DNA obtained by our improved method and the plasmid isolation kit for gram-positive bacteria (Solarbio, Beijing, China). A. Concentration of plasmid DNA, B, plasmid DNA by agarose gel electrophoresis. Lanes 1 and 2: plasmid DNA of lactobacillus plantarum PC 518; lanes 3 and 4: plasmid DNA of lactobacillus plantarum 9L 15; lanes 5 and 6: plasmid DNA of Weissella cibaria M2; lanes 7 and 8: plasmid DNA of lactobacillus plantarum JS 193; lanes 9 and 10: plasmid DNA of Lactobacillus plantarum 410. Plasmid DNA was obtained from the Solambio gram-positive bacteria plasmid isolation kit (odd lanes) or our modified protocol (even lanes).
Detailed Description
Example 1 pretreatment kit and method for bacterial plasmid extraction of the present invention
1. Kit composition
The component A comprises: 100mg/ml lysozyme
And B component: 5% (meaning 5g/100ml) glucose solution
2. Application method
Adding 100mg/ml lysozyme into bacteria, incubating for 10 minutes at 37 ℃, and centrifuging the suspension for 1 minute at the room temperature at 11000 Xg after lysozyme treatment; two more washes were performed, the procedure was as follows: the pellet was gently suspended in 500. mu.l of 5% glucose solution, centrifuged at 11000 Xg for 1 minute, and the supernatant was discarded.
Example 2 pretreatment kit and method for bacterial plasmid extraction
1. Kit composition
The component A comprises: 120mg/ml lysozyme
And B component: 4% glucose solution
2. Application method
Adding 150mg/ml lysozyme into bacteria, incubating for 10 minutes at 37 ℃, and centrifuging the suspension for 1 minute at the room temperature at 11000 Xg after lysozyme treatment; two more washes were performed, the procedure was as follows: the pellet was gently suspended in 500. mu.l of 5% glucose solution, centrifuged at 11000 Xg for 1 minute, and the supernatant was discarded.
Example 3 pretreatment kit and method for bacterial plasmid extraction of the present invention
1. Kit composition
The component A comprises: 160mg/ml lysozyme
And B component: 5% glucose solution
2. Application method
Adding 200mg/ml lysozyme into bacteria, incubating for 10 minutes at 37 ℃, and centrifuging the suspension for 1 minute at the room temperature at 11000 Xg after lysozyme treatment; two more washes were performed, the procedure was as follows: the pellet was gently suspended in 500. mu.l of 5% glucose solution, centrifuged at 11000 Xg for 1 minute, and the supernatant was discarded.
Example 4 pretreatment kit and method for bacterial plasmid extraction of the present invention
1. Kit composition
The component A comprises: 80mg/ml lysozyme
And B component: 25mmol/L Tris-HCl (pH8.0), 10mmol/L EDTA, 50mmol/L glucose, 100. mu.g/ml RNase A
2. Application method
Adding 100mg/ml lysozyme into bacteria, incubating for 10 minutes at 37 ℃, and centrifuging the suspension for 1 minute at the room temperature at 11000 Xg after lysozyme treatment; two more washes were performed, the procedure was as follows: the precipitate was gently suspended in 500. mu.l of solution I, centrifuged at 11000 Xg for 1 minute, and the supernatant was discarded.
Example 5 bacterial plasmid extraction kit and method of the invention
1. Kit composition
(1) Pretreatment kit
Any one of the kits described in examples 1 to 4;
(2) lysis kit
Solution I: 25mmol/L Tris-HCl (pH8.0), 10mmol/L EDTA, 50mmol/L glucose, 100. mu.g/ml RNase A
Solution II: 250mmol/L NaOH, 1% (W/V) SDS (sodium dodecyl sulfate);
solution III: 3mol/L potassium acetate and 5mol/L acetic acid;
solution IV: Tris-HCl pH 7.510 mmol/L, 80% ethanol;
solution V: 10mmol/L Tris-HCl pH 7.5.
2. Application method
(1) Pretreatment
Treating the above-mentioned sample with any one of the kits described in examples 1 to 4 by the corresponding method described in examples 1 to 4;
(2) cracking
Mu.l of solution II (component: 250mmol/L NaOH, 1% (W/V) SDS (sodium dodecyl sulfate)) was added to 250. mu.l of the cell resuspended in solution I, and the cell was gently inverted 6 to 8 times to lyse the cell sufficiently.
② adding 350 mul solution III (components: 3mol/L potassium acetate, 5mol/L acetic acid) into a centrifuge tube, immediately and gently turning up and down for 6-8 times, and fully mixing, wherein white flocculent precipitate appears. Centrifuge at 13,400 Xg for 10 min.
Thirdly, transferring the supernatant into an adsorption column (the adsorption column is put into a collecting pipe), and paying attention to prevent the precipitate from being sucked out as much as possible. Centrifuging at 13,400 Xg for 30-60sec, discarding the waste liquid in the collection tube, and placing the adsorption column in the collection tube.
Fourthly, 500 mul of solution IV (component: Tris-HCl with pH of 7.510 mmol/L and 80% ethanol) is added into the adsorption column, the mixture is centrifuged for 30 to 60sec at 13,400 Xg, waste liquid in the collection tube is poured out, and the adsorption column is replaced into the collection tube again.
Fifthly, 50 mul solution V (component: 10mmol/L Tris-HCl pH7.5) is added into the adsorption column, and centrifugation is carried out for 1min at 13,400 Xg, thus obtaining plasmid solution.
The advantageous effects of the present invention are described below by way of test examples.
Test example 1 method for extracting bacterial plasmid of the present invention
1 Experimental strains
Lactobacillus plantarum PC518, 410, 9L15 and JS193 strains isolated from Chinese sauerkraut. Weissella cibaria M2 strain was isolated from the gut of pandas. The strains were cultured in MRS medium at 37 ℃ for 20 hours under static anaerobic conditions.
Lactobacillus plantarum PC518, 410, 9L15 and JS193 strains, Weissella sinus (Weissella cibaria) M2 strains, all clinically isolated strains, were identified as Lactobacillus plantarum and Weissella sinus (Weissella cibaria).
2 conditional screening
2.1 screening method
2.1.1 Lysozyme treatment and removal
There were 7 protocols in the study. 8 ml of culture broth (Lactobacillus plantarum PC518) were harvested in sterile Eppordf tubes, centrifuged at 11000 Xg for 1min and the pellet was used for plasmid extraction. The lysozyme treatment and removal protocol is shown in table 1.
TABLE 1.7 Lysozyme treatment and removal protocol for Lactobacillus plantarum PC518 bacteria
a: adding lysozyme with the final concentration of 100mg/ml into the solution I;b: adding lysozyme with the final concentration of 10mg/ml into the solution I;c: preparing in sterile water;d250. mu.l of solution I ingredient.
To 250. mu.l of solution I (solution I: 25mmol/L Tris-HCl (pH8.0), 10mmol/L EDTA, 50mmol/L glucose, 100. mu.g/ml RNase A) was added 100mg/ml or 10mg/ml lysozyme, followed by incubation at 37 ℃ or 50 ℃ for 10 minutes or 30 minutes, and after the lysozyme treatment, the suspension was centrifuged at 11000 Xg for 1 minute at room temperature. Finally, to completely remove lysozyme, two washes were performed: the pellet was gently suspended in 500. mu.l of 5% glucose solution or solution I, centrifuged at 11000 Xg for 1min, and the supernatant was discarded.
2.1.2 plasmid extraction
Mu.l of solution II (component: 250mmol/L NaOH, 1% (W/V) SDS (sodium dodecyl sulfate)) was added to 250. mu.l of the cell resuspended in solution I, and the cell was gently inverted 6 to 8 times to lyse the cell sufficiently.
② adding 350 mul solution III (components: 3mol/L potassium acetate, 5mol/L acetic acid) into a centrifuge tube, immediately and gently turning up and down for 6-8 times, and fully mixing, wherein white flocculent precipitate appears. Centrifuge at 13,400 Xg for 10 min.
Thirdly, transferring the supernatant into an adsorption column (the adsorption column is put into a collecting pipe), and paying attention to prevent the precipitate from being sucked out as much as possible. Centrifuging at 13,400 Xg for 30-60sec, discarding the waste liquid in the collection tube, and placing the adsorption column in the collection tube.
Fourthly, 500 mul of solution IV (component: Tris-HCl with pH of 7.510 mmol/L and 80% ethanol) is added into the adsorption column, the mixture is centrifuged for 30 to 60sec at 13,400 Xg, waste liquid in the collection tube is poured out, and the adsorption column is replaced into the collection tube again.
Fifthly, 50 mul solution V (component: 10mmol/L Tris-HCl pH7.5) is added into the adsorption column, and centrifugation is carried out for 1min at 13,400 Xg, thus obtaining plasmid solution.
2.1.3 plasmid assay
Plasmid DNA was evaluated by agarose gel electrophoresis using a horizontal electrophoresis system (Bio-Rad USA). The number and intensity of bands represent the quality and yield of the extracted plasmid DNA. In addition, plasmid DNA was quantified by a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA) at 260 and 280 nm. The ratio of A260/A280 of the high-quality DNA is 1.8-2.0.
2.2 screening results
The agarose gel electrophoresis results show that the quality and yield of plasmid DNA extracted by the P1, P2 and P3 protocols are higher than those of the P4, P5, P6 and P7 protocols. It is clear that 8-9 recognizable bands were present in the lanes of the P1, P2 and P3 protocols, that only 3-5 weak bands were present in the lanes of the P4, P5 and P6 protocols, and that the bands were diffuse in the lanes of the P7 protocol. In addition, in the lanes of the P1, P2, and P3 schemes, the band of the lane of the P2 scheme was the brightest, and the band of the lane of the P1 scheme was the weakest (fig. 1).
The A260/A280 ratio of plasmid DNA extracted by the 7 schemes is 1.8-2.0. The concentrations of plasmid DNA were higher for the P1, P2, and P3 protocols than for the plasmids extracted from the P4, P5, and P6 protocols. Except for the plasmids extracted by the P7 protocol, the P2 protocol extracted the highest concentration of plasmid and the P5 protocol extracted the lowest concentration of plasmid DNA (FIG. 2). The results of the spectrophotometer measurement and the agarose gel electrophoresis result were substantially the same.
When the lysozyme treatment concentration is higher and lysozyme in alkaline lysis step before is removed, plasmid DNA yield and quality is significantly increased, for example using 100mg/ml lysozyme and increasing lysozyme removal step of P1, P2 and P3 scheme; if the lysozyme treatment concentration is merely increased as in the case of the P5 protocol, no good results are obtained.
The plasmid DNA of P1, P2, P3, P4, P5 and P6 were all superior in quality to the plasmid DNA extracted by the 50 ℃ P7 protocol under lysozyme treatment at 37 ℃ (FIG. 1). It is presumed that the increase in temperature leads to a crisis in survival of cells and self-exhaustion. The only difference between the P1 and P2 protocols was the duration of lysozyme treatment, which indicated that the extension of lysozyme treatment was detrimental to plasmid extraction. Lysozyme digestion times of more than 20 minutes at 37 ℃ can lead to loss of plasmid (Cataloluk, 2003). Long lysozyme treatment allows endogenous nucleases to become active (Ledeboer et al, 1976).
The washing solution containing 5% glucose can regulate membrane permeability and control release of intracellular contents through osmotic pressure equilibrium. Better results were obtained with the P2 protocol using a wash solution containing 5% glucose than with the P3 protocol using a wash solution without 5% glucose.
Therefore, after the lysozyme treatment of 100mg/ml, the lysozyme is removed by washing with 5% glucose or solution I, then the alkaline lysis treatment is carried out to extract plasmids, the concentration of the extracted plasmid DNA is high, and the purpose of effective extraction can be realized, wherein the preferred lysozyme treatment mode of the invention is a mode of P2: to 250. mu.l of solution I, 100mg/ml were added and incubated at 37 ℃ for 10 minutes, after the lysozyme treatment, the suspension was centrifuged at 11000 Xg for 1 minute at room temperature and washed twice in order to completely remove the lysozyme: the pellet was gently suspended in 500. mu.l of 5% glucose solution, centrifuged at 11000 Xg for 1 minute, and the supernatant was discarded.
Comparison of commercial kits
3.1 method
Plasmid DNA of Lactobacillus plantarum PC518, 410, 9L15 and JS193 strains and Weissella civorans M2 strains were extracted using a gram-positive bacteria plasmid isolation kit (Solarbio, Beijing, China) and the preferred method of the present invention (after treatment in section 2.1.1, section P2, then in section 2.1.2), respectively. The plasmid DNA was determined by agarose gel electrophoresis and spectrophotometer.
3.2 results
Compared with a gram-positive bacterium plasmid separation kit (Solarbio, beijing, china), the improved method has higher plasmid extraction efficiency. When we used the modified method, the bands on the gel were more bright, FIG. 3B), and the plasmid concentration was higher (FIG. 3A). Compared with the commercial kit, the plasmid concentration of the test strain is respectively increased by 10.6, 9.5, 1.5, 6 and 5.6 times.
Experimental results show that the method can effectively improve the extraction efficiency of the bacterial plasmids, wherein the extraction effect on the plant lactobacillus is better.
In conclusion, the invention obviously improves the plasmid extraction efficiency through a specific pretreatment mode, and has good application prospect.
Claims (5)
1. A pretreatment method for bacterial plasmid extraction is characterized in that: adopting a pretreatment kit for treatment, and comprising the following steps: treating bacterial thallus with component A, and washing with component B; the pretreatment kit comprises the following components:
the component A comprises: 80-160mg/ml lysozyme;
and B component: washing liquid; the washing solution is a 4-6% w/v glucose solution or a solution I; the solution I is a Tris-HCl solution added with 10mmol/L EDTA, 50mmol/L glucose and 100 mu g/ml RNase A, wherein the concentration of the Tris-HCl solution is 25 mmol/L; when in use: the bacterial cells were treated with component A and then washed with component B.
2. The pretreatment method according to claim 1, wherein: the component A is 100mg/ml lysozyme.
3. The pretreatment method according to claim 1, wherein: the concentration of the glucose solution was 5%.
4. A bacterial plasmid extraction method is characterized in that: pretreatment is carried out according to the method of any one of claims 1 to 3, followed by extraction using a plasmid extraction kit.
5. The extraction process according to claim 4, characterized in that: the plasmid extraction kit is a kit for extracting plasmids by an alkaline lysis method.
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