Preparation method and transformation method of staphylococcus aureus electroporation competent cells

Abstract

The invention relates to a preparation method and a transformation method of staphylococcus aureus electroporation competent cells, and the method comprises the following steps: providing staphylococcus aureus thalli, and washing by using a glycerol-containing solution; resuspending the washed bacteria in a buffer solution containing lysozyme for incubation treatment; and resuspending the thalli by using an electroporation solution to obtain the staphylococcus aureus competent cells. The method disclosed by the invention has the beneficial effects that the competent cells are prepared by adopting lysozyme treatment, aiming at the characteristics of thick cell wall and complex cross-linking of staphylococcus aureus, the efficiency of exogenous DNA entering the cells is effectively improved by optimizing enzyme concentration and time, the conversion efficiency is greatly improved, and the preparation time and reagent consumption are reduced.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a method for preparing and transforming Staphylococcus aureus electroporation competent cells. Background Technology

[0002] Staphylococcus aureus is a Gram-positive opportunistic pathogen widely found in nature, posing a serious threat to clinical health. It can cause a range of diseases, including skin and mucous membrane infections, sepsis, bacteremia, and pneumonia. Its antibiotic resistance, pathogenicity, and persistent prevalence have become major challenges in global public health. Current research on the resistance and virulence mechanisms of Staphylococcus aureus, especially methicillin-resistant Staphylococcus aureus (MRSA), relies heavily on efficient and stable gene manipulation techniques.

[0003] In molecular biology research on Staphylococcus aureus, the introduction of exogenous genes is a crucial prerequisite for elucidating gene function and modifying genetically engineered strains. Currently, the main methods for gene introduction into Staphylococcus aureus include transduction, chemical transformation, and electroporation.

[0004] Transduction primarily utilizes bacteriophages carrying exogenous genes to infect Staphylococcus aureus and integrate the target gene into the Staphylococcus aureus genome. However, this method requires bacteriophages as vectors and is technically challenging. Chemical transformation, using methods such as CaCl2 to treat cells and make them receptive to exogenous genes, is a common method for transforming Gram-negative bacteria. However, due to the thicker cell wall of Staphylococcus aureus compared to Gram-negative bacteria, chemical transformation often struggles to introduce exogenous genes into the cells. Electroporation, on the other hand, uses a high-voltage pulse to momentarily electroporate Staphylococcus aureus, allowing the exogenous gene to enter the cell. Electroporation is currently the mainstream method for Staphylococcus aureus, offering simplicity, speed, and efficiency. However, its drawback is that electroporation efficiency is very low for some Staphylococcus aureus strains, especially methicillin-resistant Staphylococcus aureus (MRSA).

[0005] Electroporation requires the preparation of competent cells. For Staphylococcus aureus, the most common method is cryogenic washing. The core principle is to remove extracellular impurities and electrolytes under low temperature and hypotonic conditions, preventing cell rupture and arcing during electroporation. The procedure involves repeatedly washing the mid-logarithmic bacterial cells 3-5 times with pre-cooled ultrapure water or hypotonic buffer (e.g., 10% glycerol, 0.5M sucrose), maintaining the entire process on ice. Finally, the cells are resuspended and concentrated in hypotonic buffer to obtain competent cells. Other modified versions of this method exist, such as adding glycine during the mid-logarithmic growth phase to interfere with peptidoglycan synthesis in the cell wall, resulting in a thinner cell wall; or adding dithiothreitol (DTT) to reduce disulfide bonds on the cell membrane surface, decreasing membrane rigidity. All these modified methods are based on altering cell surface permeability. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing and transforming Staphylococcus aureus electroporation competent cells.

[0007] Firstly, a method for preparing Staphylococcus aureus electroporation competent cells is provided, including: Provide Staphylococcus aureus cells and wash with a glycerol-containing solution; The washed bacterial cells were resuspended in a buffer solution containing lysozyme for incubation. The bacterial cells were resuspended using an electroporation solution to obtain competent Staphylococcus aureus cells.

[0008] Preferably, the buffer solution containing lysozyme includes: Tris, sucrose, EDTA, NaCl, and lysozyme.

[0009] Preferably, the concentration of lysozyme is 15-30 μg / mL; Optionally, the incubation time is 10-30 minutes.

[0010] Preferably, the electroporation solution comprises sucrose and glycerol; Optionally, the Staphylococcus aureus cells are in the late stage of exponential growth; Optionally, the OD of the Staphylococcus aureus cells 600 =0.6-0.7; Optionally, the Staphylococcus aureus cells are centrifuged, the supernatant is discarded, and then resuspended in a glycerol-containing solution, centrifuged again, and the supernatant is discarded to obtain the washed cells.

[0011] Preferably, the Staphylococcus aureus is methicillin-resistant Staphylococcus aureus.

[0012] In a second aspect, Staphylococcus aureus electroporation competent cells prepared by any of the methods described in the first aspect are provided.

[0013] Thirdly, a method for transforming Staphylococcus aureus electroporation competent cells as described in the second aspect is provided, including: Mix Staphylococcus aureus electroporation competent cells with the DNA to be transformed and incubate on ice; The mixture was added to a pre-cooled electroporation cuvette and subjected to electroporation. After electric shock, resuscitation medium was added for resuscitation culture. The revived bacterial culture was spread onto a selective culture medium for incubation to obtain Staphylococcus aureus transformed cells.

[0014] Preferably, the Staphylococcus aureus transformed cells contain exogenous DNA; Optionally, electroporation can be performed under 2.0-3.0 kV conditions; Optionally, the resuscitation medium contains sucrose; Optionally, the resuscitation medium includes tryptone soybean broth (TSB) medium; Optionally, the revived bacterial culture can be centrifuged, the supernatant discarded, and spread onto a selective medium for further culture. Optionally, when centrifuging the revived bacterial culture, centrifuge at 4000-5000 rpm for 3-5 minutes.

[0015] Fourthly, a kit comprising Staphylococcus aureus electroporation competent cells as described in the second aspect is provided.

[0016] Fifthly, a special buffer solution for preparing Staphylococcus aureus electroporation competent cells as described in the second aspect is provided, comprising Tris, sucrose, EDTA, NaCl, and lysozyme.

[0017] The beneficial effects of this invention are: This invention uses lysozyme treatment to prepare competent cells. Targeting the characteristics of Staphylococcus aureus cell walls with thick walls and complex cross-linking, the method effectively improves the efficiency of exogenous DNA entering cells by optimizing enzyme concentration and time, greatly increasing transformation efficiency, reducing preparation time and reagent consumption. Moreover, this method is applicable to a variety of strains, including methicillin-resistant Staphylococcus aureus, solving the problem of the difficulty of electroporation of drug-resistant strains in traditional methods. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the lysozyme-based competent cell preparation and electroporation process provided by the present invention; Figure 2A schematic diagram showing the number of single colonies of methicillin-resistant Staphylococcus aureus Mw2 competent cells after electroporation prepared by conventional methods; Figure 3 A schematic diagram showing the number of single colonies of methicillin-resistant Staphylococcus aureus Mw2 competent cells after electroporation prepared by the lysozyme method provided in this invention. Figure 4 The image shows the nucleic acid electrophoresis diagram of the plasmid used in this invention to identify Staphylococcus aureus lysozyme competent electroporated single clones by PCR using specific primers on the transformed plasmid; Figure 5 This is a schematic diagram illustrating the electroconversion efficiency ratios of lysozyme competent cells and sucrose competent cells provided by the present invention. Detailed Implementation

[0019] The present invention will be further described below with reference to embodiments. The description of the embodiments below is only for the purpose of helping to understand the present invention. It should be noted that those skilled in the art can make several modifications to the present invention without departing from the principle of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

[0020] Example 1: Staphylococcus aureus is a Gram-positive bacterium with a thicker cell wall and a higher degree of cross-linking than Gram-negative bacteria. Different Staphylococcus aureus strains have different gene expression levels, cell wall and cell membrane thickness and composition, and different sensitivities to electrical pulses. These differences lead to the following disadvantages in conventional competent cell preparation methods: (1) low electroporation efficiency; (2) poor strain adaptability, making it difficult to electroporate some complex drug-resistant strains; (3) large reagent consumption, such as the need to consume several hundred milliliters of sucrose solution to prepare one competent cell using conventional methods.

[0021] To address the problems of the prior art, Embodiment 1 of this application provides a method for preparing electroporation competent cells using lysozyme treatment, which can reduce the amount of reagents used in the preparation of competent cells, reduce the frequency of reagent preparation, and improve the electroporation efficiency for Staphylococcus aureus, especially methicillin-resistant Staphylococcus aureus.

[0022] like Figure 1 As shown, the method for preparing Staphylococcus aureus electroporation competent cells treated with lysozyme provided in this application includes: Step 1: Cultivate Staphylococcus aureus.

[0023] For example, a single colony of Staphylococcus aureus was picked from trypto-soy agar (TSA) solid medium and placed into a 12 ml shake tube containing 3 ml of trypto-soy liquid medium (TSB). The tube was then placed on a shaker and cultured overnight for 16 hours at 37°C and 220 rpm until the plateau phase. Subsequently, the overnight saturated culture of Staphylococcus aureus was diluted 1:100 and transferred to 50 ml of trypto-soy liquid medium (TSB). The tube was then cultured on a shaker at 37°C and 220 rpm until the late exponential growth phase (OD). 600 =0.6-0.7. Among them, Staphylococcus aureus is selected from methicillin-resistant Staphylococcus aureus, antibiotic-sensitive Staphylococcus aureus (such as Newman), and drug-resistant Staphylococcus aureus MRSA (such as Mw2), etc.

[0024] Step 2: Collect the bacterial cells and resuspend them using a glycerol-containing solution.

[0025] For example, after centrifuging the bacterial culture at 4°C and 7000 rpm for 5 min, the supernatant was discarded, and the cell pellet was resuspended in 1 mL of ice-cold 10% sterile glycerol and transferred to an Eppendorf tube.

[0026] Step 3: Resuspend the washed bacterial cells in a buffer solution containing lysozyme for incubation.

[0027] For example, after centrifugation at 7000 rpm for 5 min at 4 °C, the supernatant was discarded, and the cell pellet was resuspended in 500 μl of lysozyme buffer and incubated in a water bath at 37 °C for 20 min. The lysozyme buffer contains 10 mM Tris (pH 8.0), 20% sucrose, 10 mM EDTA, 50 mM NaCl, and 30 μg / mL lysozyme. The concentrations of sucrose, glycerol, Tris, EDTA, and NaCl can be adjusted appropriately.

[0028] This application is the first to propose a novel method for preparing Staphylococcus aureus electroporation competent cells using lysozyme. Lysozyme is a natural enzyme widely found in living organisms (such as tears, saliva, egg white, etc.), which can destroy peptidoglycan in bacterial cell walls, leading to bacterial lysis. Its known uses in daily life, industry, and medical health are mainly: (1) as a natural antibacterial agent, inhibiting the growth and contamination of Gram-positive bacteria, thereby achieving antibacterial, anti-inflammatory, and antiseptic effects; (2) in the field of biological research, it is often used to destroy bacteria or certain eukaryotic cells when extracting cell contents. All of the above functions are based on the purpose of destroying bacterial cells and lysing cells. However, this invention proposes an unconventional new approach based on the working principle of lysozyme, using lysozyme to treat Staphylococcus aureus to prepare competent cells. This method requires strict control and optimization of the lysozyme dosage and treatment time. Overtreatment can severely inhibit the growth of Staphylococcus aureus, while undertreatment can affect the dissolution of the Staphylococcus aureus cell wall, ultimately leading to poor electroporation results in competent cells. This invention provides a feasible solution for the application of lysozyme in the preparation and electroporation of competent Staphylococcus aureus cells by proposing an optimized lysozyme dosage, treatment time scheme, and a suitable buffer system. This method solves the problem of thick cell walls and complex peptidoglycan cross-linking in Gram-positive bacteria, and improves the efficiency of exogenous DNA electroporation into Staphylococcus aureus cells.

[0029] Specifically, in step 3, this application uses lysozyme treatment to loosen the cell walls of Staphylococcus aureus, followed by washing with an electroporation solution to maintain osmotic balance inside and outside the cells, preventing cell rupture and improving electroporation efficiency. First, it has a wider applicability to Staphylococcus aureus strains, suitable for Staphylococcus aureus with different cell wall thicknesses. Second, it shortens the preparation process of Staphylococcus aureus electroporation competent cells. Traditional methods typically require 2-3 hours to prepare Staphylococcus aureus competent cells, while the method of this invention can complete the preparation within 1 hour. Third, it reduces buffer consumption. Existing technologies typically require tens of ml of buffer per wash, while this invention only requires 1 ml of buffer per resuspension wash, significantly reducing buffer consumption and saving not only the frequency and time of buffer preparation but also reagent costs. Fourth, compared with traditional methods (taking the 0.5M sucrose washing method as an example), the electroporation efficiency of Staphylococcus aureus competent cells prepared by the lysozyme method in this invention is greatly improved.

[0030] Step 4: Resuspend the bacterial cells in electroporation solution to obtain competent Staphylococcus aureus cells.

[0031] For example, after centrifugation at 7000 rpm for 5 min at 4°C, the supernatant was discarded, and the cell pellet was resuspended in 1 ml of electroporation solution. This step was repeated 3-4 times, and the cells were finally resuspended in 1 ml of electroporation solution, which consisted of 0.5 M sucrose and 10% glycerol by volume.

[0032] In the above embodiments, the preparation of competent cells must be carried out entirely on ice or at a low temperature of 0-4°C.

[0033] Example 2: Based on Example 1, Example 2 of this application provides another method for preparing Staphylococcus aureus electroporation competent cells, including: Step 1: Cultivate Staphylococcus aureus.

[0034] Step 2: Collect the bacterial cells and resuspend them using a glycerol-containing solution.

[0035] Step 3: Resuspend the washed bacterial cells in a buffer solution containing lysozyme for incubation.

[0036] In step 3, this application uses the preferred conditions of 30 μg / ml lysozyme for 20 min. Without departing from the technical solution of this invention, reasonable adjustments are made to the lysozyme concentration and treatment time, such as: 30 μg / ml lysozyme for 10 min; 30 μg / ml lysozyme for 30 min; 15 μg / ml lysozyme for 30 min; 15 μg / ml lysozyme for 20 min.

[0037] In addition, lysozyme is derived from egg white; lysozyme from other mammalian or microbial sources can be used as a substitute.

[0038] Step 4: Resuspend the bacterial cells in electroporation solution to obtain competent Staphylococcus aureus cells.

[0039] Step 5: Aliquot and preserve the competent Staphylococcus aureus cells.

[0040] For example, Staphylococcus aureus competent cells resuspended in electroporation solution were rapidly aliquoted into pre-chilled 1.5 ml ep tubes, 80 μl per tube, and then quickly transferred to an ultra-low temperature freezer at -80°C for storage until use.

[0041] It should be noted that the parts in this embodiment that are the same as or similar to those in Embodiment 1 can be referred to each other, and will not be repeated in this application.

[0042] Example 3: Based on Example 2, Example 3 of this application provides a method for transforming Staphylococcus aureus electroporation competent cells, including: Step 1: Mix Staphylococcus aureus electroporation competent cells with the DNA to be transformed and incubate on ice.

[0043] For example, place competent cells on ice for 15 minutes to allow them to thaw naturally. Add 1-8 μg of target DNA using a pipette tip, gently stirring to mix the competent cells and DNA evenly; do not vigorously pipette. Incubate the DNA and competent cell mixture on ice for 30 minutes, while pre-cooling a sterile 0.2 cm gap electroporation cuvette (Bio-Rad) on ice.

[0044] Step 2: Add the mixture to a pre-cooled electroporation cuvette and perform electroporation.

[0045] For example, the mixture of DNA and competent cells was added to a pre-cooled electroporation cuvette. The water on the outside glass and electrode plates of the electroporation cuvette was quickly wiped dry with a paper towel, and then immediately placed in an electroporator for electroporation at 25 μF, 400 ohms and 3.0 kV.

[0046] Step 3: After electric shock, add resuscitation medium and carry out resuscitation culture.

[0047] Staphylococcus aureus cells treated with lysozyme and then electroporated require a 5-hour recovery period. The actual recovery speed can be adjusted between 120-200 rpm, and the recovery time between 2-5 hours. For example, immediately after electroporation, 400 μL of STSB (TSB containing 0.5 M sucrose) is added to the electroporation cuvette, gently pipetted, transferred to a sterile EP tube, and placed in a shaker at 37°C and 150 rpm for 5 hours. The electroporation recovery medium is STSB tryptone-soybean liquid medium containing 20% ​​sucrose. After lysozyme treatment and electroporation, the surface of Staphylococcus aureus cells is fragile. 20% sucrose effectively maintains the osmotic pressure inside and outside the Staphylococcus aureus cells, preventing cell rupture and death, and thus improving the survival rate of Staphylococcus aureus after electroporation.

[0048] Furthermore, the electroporation voltage, taking 3000V as an example, can be optimized within the range of 2000-3000V.

[0049] Step 4: Spread the revived bacterial solution onto a selective culture medium for culture to obtain Staphylococcus aureus transformed cells.

[0050] For example, after resuscitating Staphylococcus aureus, centrifuge at 5000 rpm for 3 min, remove excess supernatant, retain 100 μl, resuspend the bacterial pellet by pipetting, and spread it all onto selective tryptone soy agar plates (TSA). Incubate overnight at 37°C (16-24 h). The next day, count the colonies on the plates and calculate the electroporation titer. If necessary, pick colonies to extract genomic DNA or plasmids, and confirm successful DNA transformation into Staphylococcus aureus cells by polymerase chain reaction (PCR). In this example, the existing plasmid pWWW412-A gene was used for electroporation. After transformation, single clones were picked and cultured overnight in tryptophan soybean liquid medium (TSB) for 16 hours. The plasmid was then extracted using a plasmid extraction kit, and polymerase chain reaction (PCR) was performed using specific primers on plasmid pWWW412. Nucleic acid electrophoresis was then performed; the appearance of the target band (2660 bp) of DNA confirmed successful transformation of the plasmid into Staphylococcus aureus. The electrophoresis results are shown below. Figure 4 As shown, the results demonstrate that the plasmid was successfully electroporated into Staphylococcus aureus.

[0051] In addition, such as Figure 5 As shown, compared with the traditional method (taking the 0.5M sucrose washing method as an example), the preparation time of competent Staphylococcus aureus electroporation competent cells prepared by the lysozyme method is shortened by 2 hours, and the conversion efficiency is increased by 12 times, which greatly improves the conversion efficiency.

[0052] It should be noted that the transformation method provided in this embodiment is the corresponding method for Staphylococcus aureus electroporation competent cells provided in Example 2. Therefore, the parts in this embodiment that are the same as or similar to those in Example 2 can be referred to each other and will not be repeated in this application.

Claims

1. A method for preparing Staphylococcus aureus electroporation competent cells, characterized in that, include: Provide Staphylococcus aureus cells and wash with a glycerol-containing solution; The washed bacterial cells were resuspended in a buffer solution containing lysozyme for incubation. The bacterial cells were resuspended using an electroporation solution to obtain competent Staphylococcus aureus cells.

2. The method for preparing Staphylococcus aureus electroporation competent cells according to claim 1, characterized in that, The buffer solution containing lysozyme includes: Tris, sucrose, EDTA, NaCl, and lysozyme.

3. The method for preparing Staphylococcus aureus electroporation competent cells according to claim 1, characterized in that, The concentration of lysozyme in the buffer solution is 15-30 μg / mL; Optionally, the incubation time is 10-30 minutes.

4. The method for preparing Staphylococcus aureus electroporation competent cells according to claim 1, characterized in that, The electroporation solution contains sucrose and glycerol; Optionally, the Staphylococcus aureus cells are in the late stage of exponential growth; Optionally, the OD of the Staphylococcus aureus cells 600 =0.6-0.7; Optionally, the Staphylococcus aureus cells are centrifuged, the supernatant is discarded, and then resuspended in a glycerol-containing solution, centrifuged again, and the supernatant is discarded to obtain the washed cells.

5. The method for preparing Staphylococcus aureus electroporation competent cells according to claim 1, characterized in that, The Staphylococcus aureus mentioned is methicillin-resistant Staphylococcus aureus.

6. Staphylococcus aureus electroporation competent cells prepared by the method according to any one of claims 1 to 5.

7. A method for transforming Staphylococcus aureus electroporation competent cells as described in claim 6, characterized in that, include: Mix Staphylococcus aureus electroporation competent cells with the DNA to be transformed and incubate on ice; The mixture is added to a pre-cooled electroporation container and subjected to electric shock. After electric shock, resuscitation medium was added for resuscitation culture. The revived bacterial culture was spread onto a selective culture medium and cultured to obtain Staphylococcus aureus transformed cells.

8. The method for transforming Staphylococcus aureus electroporation competent cells according to claim 7, characterized in that, The Staphylococcus aureus transformed cells contain exogenous DNA; Optionally, electroporation can be performed under 2.0-3.0 kV conditions; Optionally, the resuscitation medium contains sucrose; Optionally, the resuscitation medium includes tryptone soybean broth (TSB) medium; Optionally, the revived bacterial culture can be centrifuged, the supernatant discarded, and spread onto a selective medium for further culture. Optionally, when centrifuging the revived bacterial culture, centrifuge at 4000-5000 rpm for 3-5 minutes.

9. A kit comprising Staphylococcus aureus electroporation competent cells as described in claim 6.

10. A specific buffer solution for preparing Staphylococcus aureus electroporation competent cells as described in claim 6, characterized in that, It contains Tris, sucrose, EDTA, NaCl, and lysozyme.

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