Method for constructing genetic transformation system of Bacillus pasteurianus

Abstract

The present invention discloses a method for constructing a genetic transformation system of Bacillus pasteurianus, comprising the following steps: adding plasmid pBBR1MCS‑2 to an electroshock buffer containing competent cells of Bacillus pasteurianus, mixing, electroshock, adding a recovery medium, culturing for 2 to 3 hours, obtaining a bacterial solution, applying the bacterial solution to a strain selection medium for culturing, and using the colonies grown on the strain selection medium as transformants. The present invention uses lysozyme to partially enzymatically hydrolyze the cell wall of the Bacillus pasteurianus strain, and then electroshocks the obtained competent cells of Bacillus pasteurianus to obtain transformants. The present invention successfully uses the electroshock transformation method to transform the competent cells containing Can The plasmid pBBR1MCS‑2 carrying the gene was transformed into the Bacillus pasteurianus strain. After transfer, the target gene could still be detected in the transformant, indicating that the genetic transformation system was successfully constructed.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial genetic transformation, and specifically relates to a method for constructing a genetic transformation system of Bacillus pasteurianus. Background Art

[0002] Bacillus pasteurianus belongs to the genus Sporosarcina of the family Coccidae of the order Bacillus, also known as Sporosarcina pasteurianus, and is a non-pathogenic Gram-positive bacterium originating from soil. Bacillus pasteurianus is one of the most efficient microorganisms for biomineralization using urea degradation so far, and has been successfully applied in biosolidation. With the rapid development of bioinformatics and molecular genetics technology, genetic improvement of excellent strains with potential application value through genetic engineering technology has been favored. However, one of the key technical bottlenecks of genetic engineering modification is genetic transformation technology. The target strain can only be modified after the target gene to be modified is introduced into the target strain. Therefore, it is very important to explore and construct an efficient genetic transformation system for Bacillus pasteurianus.

[0003] Since Bacillus is a Gram-positive bacterium with a thick cell wall, the difficulty in transforming Bacillus and the low transformation efficiency have always attracted much attention. At present, although there are successful precedents for the genetic transformation method of Bacillus, except for the relatively mature transformation system of the model bacteria Bacillus subtilis, the genetic transformation systems of other Bacillus are still being tried and improved, and there has been no report on the genetic transformation method of Bacillus pasteurianus. Summary of the invention

[0004] In view of the shortcomings of the prior art, the object of the present invention is to provide a method for constructing a genetic transformation system of Bacillus pasteurianus.

[0005] The purpose of the present invention is achieved through the following technical solutions.

[0006] A method for constructing a genetic transformation system of Bacillus pasteurianus comprises the following steps:

[0007] Step (1), add a layer containing a density of 4×10 7 ~5×10 7Add 50-80 ng of plasmid pBBR1MCS-2 to 80-100 μL electroporation buffer of competent cells of Bacillus pasteurianus per μL, mix well, and then add to the electroporation cup that has been placed in an ice bath for 10-15 min. Place the electroporation cup in an ice bath for 10-15 min and perform electroporation using an electroporator: the electroporator parameters are voltage 2.9 kV, capacitance 25 μF, and resistance 200 Ω. Perform electroporation once, and the electroporation time is 4.5-4.8 ms. The electroporation buffer is a mixture of sorbitol, mannitol, glycerol, and water. The concentrations of sorbitol, mannitol, and glycerol in the electroporation buffer are 0.5 mol / L, 0.5 mol / L, and 10 g / L, respectively.

[0008] Step (2), taking a recovery medium and adding it to an electric shock cup, resuspending the competent cells of Bacillus pasteurianus, and shaking and culturing at 30°C and 180-220 rpm for 2-3 hours to obtain a bacterial solution, wherein the recovery medium is a mixture of 10 g / L (NH4)2SO4, 20 g / L yeast extract powder, 15.748 g / L Tris, 0.8 mol / L sorbitol and water;

[0009] Step (3), taking the bacterial liquid and applying it to the strain selection medium for cultivation, the colonies grown in the strain selection medium are transformants, wherein the method for obtaining the strain selection medium is: (NH4)2SO4, yeast extract powder, Tris, agar and water are mixed evenly to make (NH4)2SO4 10 g / L, yeast extract powder 20 g / L, Tris 15.748 g / L, agar 15 g / L, sterilize, and then add kanamycin solution to make the final concentration of kanamycin 100 μg / mL.

[0010] In the step (1), the Bacillus pasteurianus competent cells are Bacillus pasteurianus ATCC 11859 competent cells.

[0011] In the step (1), the method for obtaining the competent cells of Bacillus pasteurianus comprises the following steps:

[0012] S1, inoculate Bacillus pasteurianus ATCC 11859 into YE liquid medium and culture at 30 °C and 180-220 rpm until OD 600 The value was set to 2.0-2.2 to activate Bacillus pasteurianus ATCC 11859 and obtain the activated bacterial solution. The activated bacterial solution was inoculated into SYE liquid medium at a 5% inoculum amount and cultured at 30°C and 180-220 rpm until the OD 600The value is 1.4-1.8, and the culture solution is obtained, wherein the SYE liquid culture medium includes 10 g / L (NH4)2SO4, 20 g / L yeast extract powder, 15.748 g / L Tris, 0.3 mol / L sorbitol and water; the YE liquid culture medium includes 10 g / L (NH4)2SO4, 20 g / L yeast extract powder, 15.748 g / L Tris and water;

[0013] S2, adding lysozyme solution to the culture medium so that the final concentration of lysozyme is 10 μg / mL, shaking and culturing at 30°C and 180-220 rpm for 30 min to enzymatically hydrolyze part of the cell wall of Bacillus pasteurianus ATCC 11859, collecting the bacterial solution after enzymatic hydrolysis of part of the cell wall in a sterile centrifuge tube after ice bath, ice bathing for 10-15 min to stop bacterial growth, centrifuging at 4°C after ice bathing, discarding the supernatant, and collecting the bacterial bodies;

[0014] S3, wash the bacteria 4-5 times repeatedly, add 80-100 μL of electroporation buffer to resuspend, and obtain Bacillus pasteurianus competent cells in electroporation buffer.

[0015] In S3, washing the bacteria each time includes: adding an electroporation buffer that has been pre-bathed on ice to the bacteria, resuspending by pipetting, centrifuging the resuspended bacteria at 4°C, and collecting the bacteria.

[0016] The method for obtaining a kanamycin solution comprises: dispersing kanamycin in water to make the concentration of kanamycin be 100 mg / mL, filtering and sterilizing to obtain a kanamycin solution.

[0017] The method for obtaining the lysozyme solution comprises: dispersing the lysozyme in water so that the concentration of the lysozyme is 10 mg / mL, filtering and sterilizing, and obtaining the lysozyme solution.

[0018] In the method for obtaining the strain selection medium in step (3), the pH of the strain selection medium is a natural value.

[0019] Beneficial effects brought by the technical solution of the present invention:

[0020] Compared with the prior art, the present invention uses lysozyme to partially enzymatically hydrolyze the cell wall of Bacillus pasteurianus ATCC 11859, and then electroporates the obtained competent cells of Bacillus pasteurianus to obtain transformants. The present invention successfully uses electroporation to transform the KanThe plasmid pBBR1MCS-2 carrying the gene was transformed into Bacillus pasteurianus ATCC 11859. After the transformation, the target gene could still be detected in the transformant, indicating that the genetic transformation system was successfully constructed. The genetic transformation system can be used for the genetic manipulation and function of genes in Bacillus pasteurianus ATCC 11859. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The plasmid of Bacillus pasteurianus ATCC 11859 was amplified by PCR. Kan Gene products and plasmids of transformants were amplified by PCR Kan The gene product and plasmid pBBR1MCS-2 were amplified by PCR Kan PCR amplification of gene products and water Kan Electropherogram of gene products. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments.

[0023] Strain: Bacillus pasteurianus ATCC 11859;

[0024] Plasmid pBBR1MCS-2 contains Kan Gene, the DNA of plasmid pBBR1MCS-2 is shown in SEQ ID NO.1, Kan The DNA of the gene is shown as SEQ ID NO.2.

[0025] YE liquid medium includes: 10 g / L (NH4)2SO4, 20 g / L yeast extract powder, 15.748 g / L Tris and water, natural pH, sterilized at 121℃ for 20 min before use;

[0026] YE solid medium includes: 10 g / L (NH4)2SO4, 20 g / L yeast extract powder, 15.748 g / L Tris, 15 g / L agar and water, natural pH, sterilized at 121 °C for 20 min before use;

[0027] SYE liquid medium includes: 10 g / L (NH4)2SO4, 20 g / L yeast extract powder, 15.748 g / L Tris, 0.3 mol / L sorbitol and water, natural pH, sterilized at 121 °C for 20 min before use;

[0028] Recovery medium: 10 g / L (NH4)2SO4, 20 g / L yeast extract powder, 15.748 g / L Tris, 0.8 mol / L sorbitol and water, natural pH, sterilized at 121 °C for 20 min before use;

[0029] The method for obtaining the strain selection medium is to mix (NH4)2SO4, yeast extract powder, Tris, agar and water evenly to make (NH4)2SO4 10 g / L, yeast extract powder 20 g / L, Tris 15.748 g / L, agar 15 g / L, sterilize at 121°C for 20 min, and then add kanamycin solution to make the final concentration of kanamycin 100 μg / mL, and the pH is natural value.

[0030] Kanamycin solution: Disperse kanamycin in water to obtain a kanamycin concentration of 100 mg / mL, and filter the solution through a 0.22 μm filter to obtain a kanamycin solution.

[0031] Lysozyme solution: Disperse lysozyme in water to a concentration of 10 mg / mL, and sterilize by filtration using a 0.22 μm filter to obtain a lysozyme solution;

[0032] Electroporation buffer: Sorbitol, mannitol and glycerol were dispersed in ultrapure water and sterilized at 121°C for 20 min to obtain an electroporation buffer, wherein the pH of the electroporation buffer was a natural value, the concentration of sorbitol was 0.5 mol / L, the concentration of mannitol was 0.5 mol / L, and the concentration of glycerol was 10 g / L in the electroporation buffer;

[0033] CaCl2 mixed solution: 15% (v / v) glycerol is dispersed in a CaCl2 aqueous solution, wherein the CaCl2 aqueous solution is a mixture of CaCl2 and water, and the concentration of CaCl2 in the CaCl2 aqueous solution is 0.1 mol / L.

[0034] Example 1

[0035] The method for obtaining competent cells of Bacillus pasteurianus comprises the following steps:

[0036] S1, inoculate Bacillus pasteurianus ATCC 11859 stored at -20 ℃ into YE liquid medium, and culture at 30 ℃ and 200 rpm for 48 h until OD 600 The value was 2.1±0.1 to activate Bacillus pasteurianus ATCC 11859 to obtain an activated bacterial solution, which was inoculated into SYE liquid medium at a 5% inoculum amount and cultured at 30°C and 200rpm with shaking until OD 600 The value was 1.6±0.2, and the culture solution was obtained.

[0037] S2, add lysozyme solution to the culture medium to make the final concentration of lysozyme 10 μg / mL, and culture with shaking at 30°C and 200 rpm for 30 min to enzymatically hydrolyze part of the cell wall of Bacillus pasteurianus ATCC 11859. Collect the bacterial solution after enzymatic hydrolysis of part of the cell wall in a sterile centrifuge tube after ice bath (2 mL / tube), and ice bathe for 10 min to stop bacterial growth. After ice bath, centrifuge at 4°C and 12000 rpm for 3 min, discard the supernatant, and try to absorb the residual liquid to collect the bacterial bodies.

[0038] S3, repeat washing the bacteria 4 times, add 100 μL of electroporation buffer to resuspend, and obtain Bacillus pasteurianus competent cells in the electroporation buffer, wherein each washing of the bacteria includes: adding 1 mL of electroporation buffer pre-bathed on ice to the bacteria, gently pipetting back and forth to resuspend the bacteria, and centrifuging the resuspended bacteria at 4 ° C and 12000 rpm for 3 min to collect the bacteria.

[0039] Example 2

[0040] A method for constructing a genetic transformation system of Bacillus pasteurianus comprises the following steps:

[0041] Step (1), adding the competent cells of Bacillus pasteurianus in Example 1 (the competent cell density of Bacillus pasteurianus is 5×10 7 50 ng of plasmid pBBR1MCS-2 was added to 100 μL of electroporation buffer (100 μL / μL), and gently mixed with a pipette. Then, the mixture was added to the electroporation cup that had been placed in an ice bath for 10 min. The electroporation cup was placed in an ice bath for 10 min. After the water on the outer wall of the electroporation cup after the ice bath was wiped dry with lens paper, the electroporation was performed using an electroporator: the electroporator parameters were 2.9 kV voltage, 25 μF capacitance, and 200 Ω resistance. The electroporation was performed once, and the duration of one electroporation was 4.7 ms.

[0042] Step (2), immediately after the electroporation, use a pipette to take 1 mL of recovery medium and add it to the electroporation cup to resuspend the competent cells of Bacillus pasteurianus, then transfer them to a centrifuge tube and culture them at 30°C and 200 rpm for 2 h to obtain a bacterial solution;

[0043] Step (3): Spread 100 μL of bacterial solution onto the strain selection medium and culture for 3 days. The colonies grown on the strain selection medium are transformants.

[0044] Comparative Examples 1 to 3

[0045] A method for constructing a genetic transformation system of Bacillus pasteurianus is basically the same as the construction method of Example 2, except that the voltage in step (1) is W kV, and the W in the genetic transformation system of Bacillus pasteurianus of Comparative Examples 1 to 3 and the colony growth in the strain selection medium are shown in Table 1.

[0046] Table 1

[0047] Comparative Example W Colony growth Comparative Example 1 2.1 kV No colonies grown Comparative Example 2 2.5 kV No colonies grown Comparative Example 3 3 kV No colonies grown

[0048] Comparative Examples 4 to 6

[0049] A method for verifying Bacillus pasteurianus competent cells comprises the following steps:

[0050] Step (1), containing the competent cells of Bacillus pasteurianus in Example 1 (the competent cell density of Bacillus pasteurianus is 5×10 7 100 μL of shock buffer (100 μL / μL) was added to the shock cup which had been placed in an ice bath for 10 min. The shock cup was placed in an ice bath for 10 min. The water on the outer wall of the shock cup after the ice bath was wiped dry with lens paper, and then the shock was performed using an electroporator: the parameters of the electroporator were voltage Y kV, capacitance 25 μF, and resistance 200 Ω, and the shock was performed once, and the shock time was 4.7 ms.

[0051] Step (2), immediately after the electroporation, use a pipette to take 1 mL of recovery medium and add it to the electroporation cup to resuspend the competent cells of Bacillus pasteurianus, then transfer them to a centrifuge tube and culture them at 30°C and 200 rpm for 2 h to obtain a bacterial solution;

[0052] Step (3): Spread 100 μL of bacterial solution onto YE solid medium and culture for 3 days.

[0053] The colony growth conditions in the Y and YE solid culture media in the competent cells of Bacillus pasteurianus of Comparative Examples 4 to 6 are shown in Table 2.

[0054] Table 2

[0055] Comparative Example Y Colony growth Comparative Example 4 2.1 kV Normal growth Comparative Example 5 2.5 kV Normal growth Comparative Example 6 3 kV No colonies grown

[0056] Combined with Comparative Examples 1 and 2, the colonies in the YE solid culture medium of Comparative Examples 4 and 5 grew normally, indicating that the voltage during electric shock was too low (2.1 kV and 2.5 kV), resulting in the failure of plasmid pBBR1MCS-2 to enter the competent cells of Bacillus pasteurianus. Under the condition of 3 kV voltage, the bacterial liquid after electric shock without the plasmid pBBR1MCS-2 was coated on the YE solid culture medium and could not grow, indicating that the voltage during electric shock was too high, resulting in the death of the competent cells of Bacillus pasteurianus.

[0057] Comparative Example 7

[0058] The method for obtaining competent cells of Bacillus pasteurianus comprises the following steps:

[0059] Step (1), inoculate Bacillus pasteurianus ATCC 11859 stored at -20°C into YE liquid culture medium, and culture at 30°C and 200 rpm for 48 h until OD 600 The value was 2.1±0.1 to activate Bacillus pasteurianus ATCC 11859 to obtain an activated bacterial solution, which was inoculated into YE liquid medium at a 5% inoculation rate and cultured at 30°C and 200rpm until OD 600 The value was 0.4±0.1, and the culture solution was obtained.

[0060] Step (2), take 2 mL of culture medium and place it in a centrifuge tube, seal the centrifuge tube and place it on ice for 20 min, centrifuge it at 4 °C and 12000 rpm for 3 min, discard the supernatant, and try to absorb the remaining liquid to obtain the bacteria. Add 1 mL of 0.1 mol / LCaCl2 aqueous solution that has been placed on ice for 30 min to the centrifuge tube, gently shake the centrifuge tube with a vortex oscillator to disperse the bacteria as much as possible, continue to place it on ice for 30 min, centrifuge it at 4 °C and 12000 rpm for 3 min, and discard the supernatant.

[0061] Step (3), add 120 μL of the CaCl2 mixed solution that has been pre-bathed on ice to the centrifuged bacteria, gently blow the bacterial solution with a pipette and mix it, and obtain Bacillus pasteurianus competent cells in the CaCl2 mixed solution.

[0062] Comparative Example 8

[0063] A method for constructing a genetic transformation system of Bacillus pasteurianus comprises the following steps:

[0064] Step (1), adding competent cells of Bacillus pasteurianus in Comparative Example 7 (cell density of 5×10 4 Add 50 ng of plasmid pBBR1MCS-2 to 100 μL CaCl2 mixed solution (100 μL / μL), gently rotate the centrifuge tube by hand to mix thoroughly to obtain bacterial cells, and place the centrifuge tube containing the bacterial cells in an ice bath for 30 min.

[0065] Step (2): After the ice bath, place the centrifuge tube containing the bacteria in a 42°C hot water bath for heat shock for 90 seconds, then immediately remove from the ice bath for 2 minutes to allow the bacteria to cool down rapidly. After the ice bath, add 180 μL YE liquid culture medium and warm the tube in a 30°C constant temperature water bath for 45 minutes to allow the bacteria to fully recover.

[0066] Step (3), take 100 μL of the incubated bacteria and spread it on the strain selection medium and culture it for 3 days. No colonies grew on the strain selection medium.

[0067] The calcium chloride method is often used to obtain competent Escherichia coli cells. Comparative Examples 7 and 8 show that the calcium chloride method is not suitable for obtaining competent Bacillus pasteurianus cells.

[0068] PCR identification:

[0069] (1) Plasmid extraction method: Pick a single colony and inoculate it into YE liquid culture medium, shake and culture it at 30℃ and 200 rpm for 24 h, add lysozyme solution to make the final concentration of lysozyme 50 μg / mL, and react at 30℃ and 200 rpm for 30 min. Use TIANGEN rapid plasmid extraction kit (model DP105) to extract plasmid (see the instructions of TIANGEN rapid plasmid extraction kit for specific operation procedures).

[0070] A single colony of the transformant obtained in step (3) of Example 2 was picked and operated according to the "Plasmid Extraction Method" to obtain the plasmid of the transformant. A single colony of Bacillus pasteurianus ATCC 11859 was picked and operated according to the "Plasmid Extraction Method" to obtain the plasmid of Bacillus pasteurianus ATCC 11859.

[0071] (2) PCR amplification of target gene: The plasmid of Bacillus pasteurianus ATCC 11859, the plasmid of transformant, the plasmid pBBR1MCS-2 and water were used as templates respectively. Kan Design forward primers based on conserved sequences Kan- F and reverse primer Kan- R, PCR amplification was performed, and the length of the amplified fragment was approximately 283 bp.

[0072] Primers (5'-3'):

[0073] The forward primer is shown as SEQ ID NO.3.

[0074] The reverse primer is shown as SEQ ID NO.4.

[0075] PCR amplification: The PCR system is shown in Table 3. The PCR reaction program is shown in Table 4. After PCR amplification, the PCR amplification products of the four templates are: The plasmid of Bacillus pasteurianus ATCC 11859 was amplified by PCR Kan Gene products and plasmids of transformants were amplified by PCR Kan The gene product and plasmid pBBR1MCS-2 were amplified by PCR Kan PCR amplification of gene products and water Kan Gene product.

[0076] The 500 bp marker and the PCR amplification products of each template were detected by 3.5% agarose gel electrophoresis, and the UV gel imaging system was used to observe and photograph the amplified products. The 500 bp marker was used as a molecular weight standard and reference. The plasmid pBBR1MCS-2 was amplified by PCR. Kan The gene product was used as a positive control and water was amplified by PCR Kan The gene product was used as a negative control, and the photographic results were as follows Figure 1 As shown, Figure 1 Lane M: 500 bp marker band, Lane 1: plasmid of Bacillus pasteurianus ATCC 11859 amplified by PCR Kan Bands of gene products, Lane 2: Plasmid of transformant amplified by PCR Kan Band of gene product, lane 3: plasmid pBBR1MCS-2 amplified by PCR Kan Bands of gene products, lane 4: water amplified by PCR Kan Bands of gene products.

[0077] Figure 1 The primers in lane 1 of the experiment had non-specific binding to Bacillus pasteurianus ATCC 11859, but the band size was consistent with the target gene ( Kan gene), and the plasmid of the transformant was amplified by PCR Kan The size of the gene product is related to the plasmid pBBR1MCS-2 used for PCR amplification Kan The size of the gene products was the same, and by comparing with the marker, it was determined that the size of the plasmid fragment was approximately 283 base pairs. This result showed that Kan The gene was successfully transferred into the electroporated Bacillus pasteurianus ATCC 11859. Under the same conditions, water was used as a template for PCR amplification. Kan There were no bands of gene products in the lanes, indicating that the primers, enzymes and water used were free of contamination, indicating that the genetic transformation system was successfully constructed.

[0078] Table 3

[0079] Reagents required for PCR system volume template 1 μL Primer (-F) 0.5 μL Primer (-R) 0.5 μL 2×Taq 10 μL <![CDATA[Double-distilled water (ddH2O)]]> 8 μL

[0080] Table 4

[0081] PCR reaction procedure temperature time Pre-denaturation 94 ℃ 3 min transsexual 94 ℃ 3 min annealing 61 ℃ 30 sec extend 72 ℃ 1 min Rear extension 72 ℃ 5 min

[0082] When the plasmid of the transformant is used as a template, the plasmid of the transformant is amplified by PCR Kan The reverse sequence of the gene product sequencing result is shown in SEQ ID NO.5. The plasmid of the transformant was amplified by PCR. KanThe forward sequence of the gene product sequencing result is shown as SEQ ID NO.6.

[0083] The present invention is described above by way of example. It should be noted that, without departing from the core of the present invention, any simple deformation, modification or other equivalent replacement that can be made by those skilled in the art without inventive effort falls within the protection scope of the present invention.

Claims

1. A method for constructing a genetic transformation system of Bacillus pasteurianus, characterized in that: The following steps are involved: Step (1), add a layer containing a density of 4×10 7 ~5×10 7 Add 50-80 ng of plasmid pBBR1MCS-2 to 80-100 μL electroporation buffer of competent cells of Bacillus pasteurianus per μL, mix well, and then add to the electroporation cup that has been placed in an ice bath for 10-15 min. Place the electroporation cup in an ice bath for 10-15 min and perform electroporation using an electroporator: the electroporator parameters are voltage 2.9 kV, capacitance 25 μF, and resistance 200 Ω. Perform electroporation once, and the electroporation time is 4.5-4.8 ms. The electroporation buffer is a mixture of sorbitol, mannitol, glycerol, and water. The concentrations of sorbitol, mannitol, and glycerol in the electroporation buffer are 0.5 mol / L, 0.5 mol / L, and 10 g / L, respectively. Step (2), taking a recovery medium and adding it to an electric shock cup, resuspending the competent cells of Bacillus pasteurianus, and shaking and culturing at 30°C and 180-220 rpm for 2-3 hours to obtain a bacterial solution, wherein the recovery medium is a mixture of 10 g / L (NH4)2SO4, 20 g / L yeast extract powder, 15.748 g / L Tris, 0.8 mol / L sorbitol and water; Step (3), applying the bacterial solution to a strain selection medium for culture, wherein the colonies grown in the strain selection medium are transformants, wherein the method for obtaining the strain selection medium is: uniformly mixing (NH4)2SO4, yeast extract powder, Tris, agar and water to make (NH4)2SO4 10 g / L, yeast extract powder 20 g / L, Tris 15.748 g / L, agar 15 g / L, sterilizing, and then adding kanamycin solution to make the final concentration of kanamycin 100 μg / mL; In the step (1), the method for obtaining the competent cells of Bacillus pasteurianus comprises the following steps: S1, inoculate Bacillus pasteurianus ATCC 11859 into YE liquid medium and culture at 30 °C and 180-220 rpm until OD 600 The value was set to 2.0-2.2 to activate Bacillus pasteurianus ATCC 11859 and obtain the activated bacterial solution. The activated bacterial solution was inoculated into SYE liquid medium at a 5% inoculum amount and cultured at 30°C and 180-220 rpm until the OD 600 The value is 1.4-1.8, and the culture solution is obtained, wherein the SYE liquid culture medium includes 10 g / L (NH4)2SO4, 20 g / L yeast extract powder, 15.748 g / L Tris, 0.3 mol / L sorbitol and water; the YE liquid culture medium includes 10 g / L (NH4)2SO4, 20 g / L yeast extract powder, 15.748 g / L Tris and water; S2, adding lysozyme solution to the culture medium so that the final concentration of lysozyme is 10 μg / mL, shaking and culturing at 30°C and 180-220 rpm for 30 min to enzymatically hydrolyze part of the cell wall of Bacillus pasteurianus ATCC 11859, collecting the bacterial solution after enzymatic hydrolysis of part of the cell wall in a sterile centrifuge tube after ice bath, ice bathing for 10-15 min to stop bacterial growth, centrifuging at 4°C after ice bathing, discarding the supernatant, and collecting the bacterial bodies; S3, wash the bacteria 4-5 times repeatedly, add 80-100 μL of electroporation buffer to resuspend, and obtain Bacillus pasteurianus competent cells in electroporation buffer.

2. The method for constructing the genetic transformation system of Bacillus pasteurianus according to claim 1, characterized in that: In S3, washing the bacteria each time includes: adding an electroporation buffer that has been pre-bathed on ice to the bacteria, resuspending by pipetting, centrifuging the resuspended bacteria at 4°C, and collecting the bacteria.

3. The method for constructing the genetic transformation system of Bacillus pasteurianus according to claim 1, characterized in that: The method for obtaining a kanamycin solution comprises: dispersing kanamycin in water to make the concentration of kanamycin be 100 mg / mL, filtering and sterilizing to obtain a kanamycin solution.

4. The method for constructing the genetic transformation system of Bacillus pasteurianus according to claim 1, characterized in that: The method for obtaining the lysozyme solution comprises: dispersing the lysozyme in water so that the concentration of the lysozyme is 10 mg / mL, filtering and sterilizing, and obtaining the lysozyme solution.

5. The method for constructing the genetic transformation system of Bacillus pasteurianus according to claim 1, characterized in that: In the method for obtaining the strain selection medium in step (3), the pH of the strain selection medium is a natural value.

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