Genetic transformation method applied to Paenibacillus polymyxa

Abstract

The present invention provides a genetic transformation method applied to Paenibacillus polymyxa, the method comprising: inoculating Paenibacillus polymyxa into a liquid culture medium and culturing to the middle of the growth period to prepare competent cells; electrotransforming a plasmid into competent cells, and then performing resistance screening after recovery to obtain transformants. Using this method, the present invention successfully electrotransformed the plasmid pIC333 containing the Tn10 transposon into Paenibacillus polymyxa, and verified that the plasmid also has a transposition effect and temperature-sensitive properties in Paenibacillus polymyxa, which is convenient for carrying out genetic operations related to transposition in Paenibacillus polymyxa. Therefore, the present invention effectively solves the problem of transformation and transposition of exogenous plasmids in Paenibacillus polymyxa in the prior art.

Description

Technical Field

[0001] The invention belongs to the field of microbial physiology and biochemistry, and in particular relates to a genetic transformation method applied to Paenibacillus polymyxa. Background Art

[0002] Paenibacillus polymyxa is a Gram-positive rod-shaped bacillus. It is the type species of the genus Paenibacillus. It belongs to the order Bacillales, the family Paenibacillaceae, and the genus Paenibacillus. Paenibacillus is differentiated from the genus Bacillus and belongs to the order Bacillales. Therefore, there are certain similarities between the characteristics of Paenibacillus polymyxa and Bacillus subtilis. Paenibacillus polymyxa is a plant growth-promoting rhizobacterium that can produce many useful compounds, including cyclopeptide antibiotics, such as polymyxin, the "last line of defense" against multidrug-resistant bacteria.

[0003] For decades, only a few reports have documented the development of genetic manipulation systems in Paenibacillus polymyxa, and the existing transformation systems of Paenibacillus polymyxa have disadvantages such as low transformation efficiency, poor targeting of gene inactivation, and weak stability of genetic manipulation systems. The restriction modification system in bacteria can resist foreign DNA, resulting in reduced transformation efficiency. Exploring the restriction modification system in Paenibacillus polymyxa can not only overcome this barrier and improve transformation efficiency, but also provide a direction for exploring the diversity of bacterial restriction modification systems. Therefore, the construction of the genetic manipulation system of Paenibacillus polymyxa not only lays the foundation for the cloning and expression of exogenous genes in Paenibacillus polymyxa, but can also be widely used in the study of functional genomics in Paenibacillus polymyxa, and promote the development of more effective and convenient genetic manipulation tools.

[0004] Darrell H. Mallonee et al. used penicillin treatment technology to promote plasmid DNA transformation of Bacillus polymyxa ATCC12321, and screened transformants through hypertonic and non-hypertonic culture media, but the transformation frequency of this method was low (10-6), and the transformant protoplasts could not form cell walls in vitro, and the transformants could not be subcultured. Alexandre Rosado et al. proposed the use of electroporation to transform B. polymyxa SCE2, and improved the transformation efficiency by optimizing the conditions such as electroporation voltage, buffer strength, electroporation buffer, plasmid DNA concentration, plasmid preparation purity and plasmid source, and finally achieved a transformation efficiency of 3.2×10 5cfu / μg DNA. Brito LF et al. established the transformation of Escherichia coli-Bacillus polymyxa shuttle plasmid based on magnesium amino clay in Bacillus polymyxa, but the transformation efficiency was only 100 cfu / μg DNA. The efficient introduction of exogenous DNA into cells is the basis and prerequisite for bacterial genetic manipulation. Currently, the most commonly used method for introducing exogenous DNA into Bacillus polymyxa is electrotransformation, and its transformation efficiency still needs to be further improved compared with Escherichia coli. Therefore, it is necessary to build a complete and easy-to-operate genetic manipulation system for Bacillus polymyxa. Summary of the invention

[0005] In order to solve the shortcomings of low conversion efficiency, poor targeting of gene inactivation and weak stability of genetic operation system in the genetic transformation method applied to Paenibacillus polymyxa in the prior art, the present invention uses Paenibacillus polymyxa as the starting strain, and adjusts the traditional genetic transformation method in a targeted manner, thereby providing a transformation method with high conversion efficiency applied to Paenibacillus polymyxa. On this basis, the present invention successfully uses the method to electrotransform the plasmid pIC333 containing the Tn10 transposon to Paenibacillus polymyxa, and verifies that the plasmid also has transposition effect and temperature-sensitive characteristics in Paenibacillus polymyxa, which is convenient for it to carry out the genetic operation work related to transposition in Paenibacillus polymyxa. Therefore, the present invention effectively solves the problem of transformation and transposition of exogenous plasmids in Paenibacillus polymyxa in the prior art.

[0006] The technical scheme adopted by the present invention is: a genetic transformation method applied to Paenibacillus polymyxa, the method comprising: inoculating Paenibacillus polymyxa into a liquid culture medium and culturing to the middle of the growth period to prepare competent cells; electrotransforming a plasmid into the competent cells, and performing resistance screening after recovery to obtain transformants.

[0007] The present invention first optimizes the electrotransformation conditions of Paenibacillus polymyxa. Experiments show that when Paenibacillus polymyxa is cultured to the middle of the growth period, the electrotransformation competent cells are prepared, and the exogenous DNA transformation efficiency is higher. The specific optimization process of the electrotransformation conditions is as follows: pick a fresh single colony of P. polymyxa ATCC842 and inoculate it in a test tube for overnight culture; inoculate the bacterial solution in LB+sorbitol medium at a 4% inoculation amount, and measure the OD every 1 hour. 600nm And draw a growth curve; select P.polymyxa ATCC842 at different growth stages to prepare electro-competent cells; electro-transform at a voltage of 1.8kV, and resuscitate in a shaker for 3h, 4h, and 5h respectively; centrifuge the bacterial solution, perform appropriate dilution and coating, screen on antibiotic plates, and calculate the electro-transformation efficiency to determine the optimal electro-transformation conditions.

[0008] Preferably, the method of inoculating Paenibacillus polymyxa into a liquid culture medium and culturing the medium until the middle of the growth period comprises: inoculating Paenibacillus polymyxa into a liquid culture medium and culturing the medium until the OD 600nm The culture method comprises: taking a fresh single colony of P. polymyxa ATCC842 and inoculating it into a 5 mL LB test tube, culturing it at 200 r / min and 37°C overnight. Inoculating it into 50 mL LB+sorbitol medium with a 4% inoculum, culturing it at 200 r / min and 37°C, measuring the OD every one hour. 600nm And record the values ​​and draw the growth curve of P.polymyxaATCC842. According to the growth curve of P.polymyxa ATCC842, bacteria in the early, middle and late growth periods were selected to prepare competent cells and stored at -80°C. More specifically, Bacillus polymyxa was inoculated with 4% inoculum in 50mL LB+sorbitol medium, cultured at 200r / min, 37°C for 7-8h, and the OD 600nm Reach 0.4~0.7.

[0009] Preferably, the method for electroporating the plasmid into competent cells comprises: dissolving the stored competent cells on ice, adding the plasmid, mixing to form a mixed solution, placing on ice for 25 to 35 minutes, adding the mixed solution to a pre-cooled electrode cup, and electroporating at 1.5 to 2.0 kV, more preferably 1.8 to 2.0 kV.

[0010] Preferably, the resuscitation method comprises: adding preheated resuscitation solution, resuscitating at 35-40°C and 180-200 rpm for 3-4 hours, more preferably 4 hours. When the resuscitation time is 4 hours, the electroporation efficiency is the highest, which can reach 1.795×10 5 cfu / μg. Wherein, the resuscitation solution can be LB liquid culture medium containing D-sorbitol and D-mannitol.

[0011] Preferably, the Paenibacillus polymyxa is Paenibacillus polymyxa ATCC 842. It is understandable that since Paenibacillus polymyxa and Bacillus subtilis are evolutionarily close, belong to the genus Bacillus, and have similar properties, the method provided by the present invention is also applicable to Bacillus subtilis, such as Bacillus subtilis 168.

[0012] Preferably, the plasmids include: plasmid pWB980 and plasmid pIC333.

[0013] Plasmid pIC333 carries Mini-Tn10 transposon, Spc spectinomycin resistance and Em erythromycin resistance, and the temperature-sensitive replicon is inactivated above 35°C in a Gram-positive bacterial host. The present invention prepares B. subtilis 168 electrotransformation competent cells, extracts the pIC333 plasmid containing the Tn10 transposon and transforms it into Bacillus subtilis, and after screening by antibiotic plates, performs three subcultures to detect whether the transposon transposes in Bacillus subtilis. Experiments have confirmed that plasmid pIC333 has a transposition effect and temperature-sensitive characteristics in Bacillus subtilis, and is an effective mini-Tn10 transposon vector for Bacillus subtilis. However, in the prior art, it is difficult to transform plasmid pIC333 into Paenibacillus polymyxa by traditional genetic transformation methods. To solve this problem, the inventor team successfully transformed the pIC333 plasmid into competent cells of the mid-growth period of Paenibacillus polymyxa under the conditions of the optimized Paenibacillus polymyxa electrotransformation system, and carried out multiple subcultures after screening with antibiotic plates to detect whether the Tn10 transposon transposed in Paenibacillus polymyxa. Specifically, the method comprises: inoculating Paenibacillus polymyxa into a liquid culture medium and culturing until OD 600nm The pIC333 was 0.4-0.7, and competent cells were prepared; plasmid pIC333 was electrotransformed into competent cells, and resistance screening was performed 4 hours after recovery to obtain transformants. The transformants were subcultured 3 times, and it was found that the genome of the transformants contained the transposon of plasmid pIC333; the plasmid pIC333 in the transformants was lost after the transformants were cultured at 35-40°C. Therefore, it can be determined that the pIC333 plasmid also has transposition effect and temperature-sensitive properties in Paenibacillus polymyxa, indicating that this transposon may be used to construct a mutation system in Paenibacillus polymyxa, laying a certain foundation for the subsequent development of cell factories in Paenibacillus polymyxa.

[0014] Beneficial effects of the present invention: The present invention first optimizes the electrotransformation conditions of Paenibacillus polymyxa, and provides a genetic transformation method applied to Paenibacillus polymyxa, specifically, preparing electrotransformation competent cells when Paenibacillus polymyxa is cultured to the middle of the growth period, and the method effectively improves the conversion efficiency of exogenous DNA. Using this method, the present invention also successfully transforms the thermosensitive pIC333 plasmid containing Mini-Tn10 transposon into Paenibacillus polymyxa, and verifies that the pIC333 plasmid has a transposition effect and thermosensitive properties in Paenibacillus polymyxa, indicating that this transposon can be applied to the construction of the mutation system of Paenibacillus polymyxa, and lays a certain foundation for the subsequent development of cell factories in Paenibacillus polymyxa. At the same time, the method has the advantages of high efficiency and simple operation, and can be used for more in-depth research on other Paenibacillus polymyxa. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the growth curve of P.polymyxaATCC842 in Example 1.

[0016] Figure 2 It is the electroporation efficiency of competent cells of P. polymyxa ATCC842 at different growth stages in Example 1.

[0017] Figure 3 The electroporation efficiency of P. polymyxa ATCC842 at different recovery times in Example 2.

[0018] Figure 4 Growth curve of Bacillus subtilis 168 in LB-sorbitol medium in the control group.

[0019] Figure 5 This is a diagram for verifying the successful transposition of the transposon in Example 3. DETAILED DESCRIPTION

[0020] The following is an explanation of the embodiments of the present invention by specific specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied by other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, in the absence of conflict, the features in the following embodiments and embodiments can be combined with each other. In the embodiments of the present invention, the methods used are all conventional methods unless otherwise specified, and the reagents used can be obtained from commercial sources.

[0021] In the following examples, unless otherwise specified, the final concentration of erythromycin in the culture medium is 25 mg / L, the final concentration of spectinomycin in the culture medium is 100 mg / L, and the final concentration of spectinomycin in the culture medium is 50 mg / L.

[0022] LB liquid culture medium: sodium chloride 10 g / L, peptone 10 g / L, yeast powder 5 g / L, pH = 7.0, sterilization at 121°C for 20 min.

[0023] LB solid medium (g / L): LB liquid medium plus 1.5-2g agar.

[0024] LB liquid medium containing sorbitol: LB liquid medium containing 0.5 mol / LD-sorbitol.

[0025] Competent medium preparation: Solution A: 0.5 mol / L D-sorbitol, 0.5 mol / L D-mannitol, 10% glycerol, dissolved in deionized water; Solution B: 0.5 mol / L D-trehalose, 0.5 mol / L D-sorbitol, 0.5 mol / L D-mannitol, 10% glycerol, dissolved in deionized water;

[0026] Recovery solution: LB liquid culture medium containing 0.5 mol / LD-sorbitol and 0.38 mol / LD-mannitol.

[0027] Embodiment 1:

[0028] This embodiment provides a genetic transformation method applied to Paenibacillus polymyxa. The specific method is as follows.

[0029] P. polymyxa ATCC842 was used as the starting strain for cultivation and OD was measured. 600nm The growth curve of P. polymyxa ATCC842 was drawn: a fresh single colony of P. polymyxa ATCC842 was inoculated into a 5 mL LB test tube, cultured at 200 r / min, 37°C overnight; a 4% inoculum was inoculated into 50 mL LB medium supplemented with sorbitol, cultured at 200 r / min, 37°C; OD was measured every hour 600nm And record the values; draw the growth curve of P.polymyxaATCC842 ( Figure 1 shown).

[0030] P.polymyxaATCC842 was inoculated into liquid culture medium and cultured to different growth stages to prepare competent cells: according to the growth curve of P.polymyxaATCC842, the middle growth stage (OD 600nm =0.43, OD 600nm =0.68) and late growth phase (OD 600nm =0.88) were used to prepare competent cells and stored at -80°C.

[0031] The plasmid was electrotransformed into competent cells, and resistance screening was performed after recovery to obtain transformants: the prepared P. polymyxa ATCC842 electrotransformed competent cells were dissolved on ice, 1 μg of plasmid pWB980 was added, mixed to form a mixed solution, and placed on ice for 30 min; the mixed solution was added to a 1 mm pre-cooled electrode cup, electroshocked at 1.8 kV, and 900 μL of preheated recovery solution was quickly added, and the culture was recovered at 37°C, 200 rpm for 4 h; the bacterial solution was centrifuged at 5000 rpm for 2 min, the supernatant was removed, 100 μL was taken to coat an LB resistance plate containing Kan (50 μg / mL), and cultured at 37°C overnight; transformants were obtained and the transformation efficiency was calculated.

[0032] Depend on Figure 2 It can be seen that P. polymyxa ATCC842 in the middle of the growth period OD 600nm When the conversion efficiency is 0.68, it is the highest, about 1.795×10 5 cfu / μg, and at OD 600nm When the conversion efficiency is 0.43, D 600nm It is close to 0.68. It can be seen that the transformation efficiency of P. polymyxa ATCC842 is the highest in the middle of the growth period. Comprehensive experimental data show that the best time for preparing competent cells is in the middle of the growth period, that is, OD 600nm =0.4~0.7.

[0033] Embodiment 2:

[0034] The difference from Example 1 is that the recovery time in Example 2 is 3 hours, and the other steps are the same. Figure 3 .

[0035] Comparative Example 1:

[0036] The difference from Example 1 is that the recovery time in Comparative Example 1 is 5 hours, and the other steps are the same. Figure 3 .

[0037] After the P. polymyxa ATCC842 competent cells were electroporated at 1.8 kV, 1 ml of resuscitation solution was added and the cells were revived at 37°C for 3, 4, and 5 hours. The electroporation efficiency was as follows: Figure 3 As shown in the figure, it can be seen that after the recovery time was increased from 3h to 4h, the electroporation efficiency was significantly improved and reached about 1.795×10 5 cfu / μg, but too long a recovery time (5h) would reduce the electroporation efficiency to almost 0. Therefore, the optimal recovery time after electroporation is 4h.

[0038] Control group: Detect whether transposon transposition occurs in Bacillus subtilis

[0039] First, take a fresh single colony of Bacillus subtilis 168 and inoculate it into a 5 mL LB test tube, culture it at 200 r / min, 37°C overnight; inoculate it into 50 mL LB + sorbitol medium at a 4% inoculum, culture it at 200 r / min, 37°C, and measure the OD every hour. 600nm And record the values; draw the growth curve of Bacillus subtilis 168 ( Figure 4 shown).

[0040] Detect whether transposon transposition occurs: Take electroporated competent cells of Bacillus subtilis 168 in the middle of the growth period and dissolve them on ice, add 1μg plasmid pIC333, mix well and let stand on ice for 30 minutes. Add the bacterial solution to a 1mm pre-cooled electrode cup, shock at 2.0kV, and quickly add 900μL preheated recovery solution, 30℃, 200rpm, and recover for 3h; no plasmid is added for negative control, and plasmid pWB980 is added for positive control. Centrifuge the bacterial solution at 5000rpm for 2min, remove the supernatant, and the remaining 200μL is taken. 50μL is coated on Spec (100μg / mL), Erm (25μg / mL), Spec+Erm, and LB plates respectively, and culture at 30℃, 200rpm, overnight.

[0041] If transformants grow on LB and Spec plates, but not on Erm plates, it means that plasmid pIC333 may have been lost, and the IS part may have been transposed to the genome of Bacillus subtilis 168, indicating that the plasmid pIC333 transposon has the ability to transpose in Bacillus subtilis 168. If transformants grow on LB and Erm plates, but not on Spec plates, it means that plasmid pIC333 has been transformed into Bacillus subtilis 168 but no transposition has occurred.

[0042] Select the transformants grown on the Erm plate, inoculate them into 5mL LB test tubes, and culture them in a shaker at 30℃ and 200rpm until the bacterial solution becomes turbid. Inoculate them again into 5mL LB test tubes and culture them in a shaker at 37℃ for 1 hour. Repeat the subculture 3-4 times. Spread them onto Spec (100μg / mL), Erm (25μg / mL), Spec+Erm, and LB plates. Culture them at 37℃ overnight. If transformants grow on LB and Spec plates, there will be no transformants on the Erm plate. This indicates that the transposon in plasmid pIC333 has transposition ability and temperature-sensitive properties in Bacillus subtilis 168.

[0043] According to the resistance plate screening results in Table 1, after the plasmid pIC333 was electroporated into Bacillus subtilis 168, the transposon Tn10 directly transposed under the culture condition of 30°C, and the plasmid pIC333 no longer existed in the bacteria.

[0044] Table 1. Electroporation of plasmid pIC333 into Bacillus subtilis 168

[0045]

[0046] Note: + indicates the presence of transformants; - indicates the absence of transformants

[0047] Example 3: Detecting whether transposition of transposon occurs in Paenibacillus polymyxa

[0048] This embodiment provides a genetic transformation method applied to Paenibacillus polymyxa.

[0049] The difference from Example 1 is that in Example 3, plasmid pIC333 is transformed into competent cells in the middle of the growth period of Paenibacillus polymyxa according to the method provided in Example 1, and the cells are revived for 4 hours after electroporation; no plasmid is added to the negative control, and plasmid pWB980 is added to the positive control. After the recovery is completed, the bacterial solution is centrifuged at 5000rpm for 2min, the supernatant is removed, and 200μL is left. 50μL is taken and coated on Spec (100μg / mL), Erm (25μg / mL), Spec+Erm, and LB plates respectively, and cultured at 30°C, 200rpm, and overnight.

[0050] If transformants grow on LB and Spec plates, but not on Erm plates, it means that plasmid pIC333 may have been lost, and the IS part may have been transposed to the genome of P.polymyxa ATCC842, indicating that the plasmid pIC333 transposon has the ability to transpose in P.polymyxaATCC842. If transformants grow on LB and Erm plates, but not on Spec plates, it means that plasmid pIC333 has transformed P.polymyxaATCC842 but no transposition has occurred.

[0051] Select the transformants grown on the Erm plate, inoculate them into 5mL LB test tubes, and culture them in a shaker at 30℃ and 200rpm until the bacterial solution becomes turbid. Inoculate them again into 5mL LB test tubes and culture them in a shaker at 37℃ for 1 hour. Repeat the subculture 3-4 times. Spread them on Spec (100μg / mL), Erm (25μg / mL), Spec+Erm, and LB plates. Culture them at 37℃ overnight. If transformants grow on LB and Spec plates, there will be no transformants on the Erm plate. This indicates that the transposon in plasmid pIC333 has transposition ability and temperature-sensitive properties in P.polymyxaATCC842.

[0052] The transformants were inoculated into Spec (100 μg / mL), Erm (25 μg / mL), Spec+Erm, and LB test tubes and cultured at 37°C. Figure 5) It can be seen that: it is further verified that the transposon has transposed in P. polymyxa ATCC842. It shows that Tn10 has transposition function in Bacillus polymyxa. Because Bacillus polymyxa and Bacillus subtilis are close in evolution, they belong to the genus Bacillus and have similar properties. Therefore, the transposon in plasmid pIC333 can play a transposition effect in B. subtilis 168 and P. polymyxa ATCC842, and has temperature-sensitive properties.

[0053] The embodiments described above are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope of the present invention.

Claims

1. A genetic transformation method for Paenibacillus polymyxa, characterized in that: The method comprises: inoculating Paenibacillus polymyxa into a liquid culture medium and culturing until the OD600 nm is 0.4-0.7 to prepare competent cells; electrotransforming the plasmid pIC333 into the competent cells, and performing resistance screening 4 hours after recovery to obtain transformants; The method for electrotransforming the plasmid into competent cells comprises: dissolving the stored competent cells on ice, adding the plasmid, mixing to form a mixed solution, placing on ice for 25 to 35 minutes, adding the mixed solution to a pre-cooled electrode cup, and electroporating at 1.8 kV; The polymyxa bacillus is Paenibacillus polymyxa ATCC842.

2. The method according to claim 1, characterized in that The resuscitation method includes: adding preheated resuscitation fluid, and resuscitating at 35-40° C. and 180-200 rpm for 3-4 hours.

3. The method according to claim 1, characterized in that The genome of the transformant contains the transposon of plasmid pIC333.

4. The method according to claim 1, characterized in that After the transformant was cultured at 35-40° C., the plasmid pIC333 in the transformant was lost.

Images