Genetic transformation method for paenibacillus polymyxa

Abstract

A genetic transformation method for Paenibacillus polymyxa. The method comprises: inoculating Paenibacillus polymyxa into a liquid culture medium and culturing the Paenibacillus polymyxa to a middle phase of a growth period to prepare a competent cell; and electrotransforming a plasmid into the competent cell, and carrying out resistance screening after resuscitation to obtain a transformant. The method realizes the electrotransformation of plasmid pIC333 containing a Tn10 transposon into Paenibacillus polymyxa and verifies that the plasmid also exhibits transposition effect and temperature-sensitive characteristics in the Paenibacillus polymyxa, thereby facilitating the development of genetic manipulation work related to transposition in the Paenibacillus polymyxa. Therefore, the problem in the prior art of transformation and transposition of exogenous plasmids in Paenibacillus polymyxa is solved.

Description

Genetic transformation method applied to paenibacillus polymyxa TECHNICAL FIELD

[0001] The present application belongs to the field of microbial physiology and biochemistry, and particularly relates to a genetic transformation method applied to Paenibacillus polymyxa. BACKGROUND

[0002] Paenibacillus polymyxa is a gram-positive rod-shaped bacillus, which is the type species of the genus Paenibacillus, and belongs to the order Bacillales, the family Paenibacillaceae and the genus Paenibacillus. The genus Paenibacillus is differentiated from the genus Bacillus, and belongs to the order Bacillales. Therefore, Paenibacillus polymyxa has certain similarities with Bacillus subtilis. Paenibacillus polymyxa is a rhizosphere bacterium that promotes plant growth, and can produce many useful compounds, including cyclic peptide antibiotics, such as polymyxin, which is the “last line of defense” against multiple drug-resistant bacteria.

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

[0004] Darrell H. Mallonee et al. used penicillin treatment of cells to promote plasmid DNA transformation of Bacillus polymyxa ATCC12321, and selected transformants through high-osmotic and non-high-osmotic media, but the transformation frequency was low (10-6), and the transformant protoplasts could not form cell walls outside the cell, and the transformants could not be subcultured. Alexandre Rosado et al. proposed using electroporation to transform B. polymyxa SCE2, and optimized the conditions of electroporation voltage, buffer strength, electroporation buffer, plasmid DNA concentration, plasmid preparation purity and plasmid source to improve the transformation efficiency, and finally achieved a transformation efficiency of 3.2 × 10 5100 cfu / μg DNA. Brito LF et al. established the transformation of E. coli-P. polymyxa shuttle plasmid based on magnesium amino clay in P. polymyxa, but the transformation efficiency was only 100 cfu / μg DNA. Efficient introduction of foreign DNA into cells is the basis and premise of bacterial genetic manipulation. At present, the most commonly used method for introducing foreign DNA into P. polymyxa is electroporation, and its transformation efficiency still needs to be further improved compared with E. coli. Therefore, a perfect and convenient genetic manipulation system needs to be constructed for P. polymyxa. SUMMARY

[0005] In order to solve the problems of low transformation efficiency, poor gene inactivation targeting and weak stability of genetic manipulation system in the prior art genetic transformation method applied to P. polymyxa, the present application adjusts the traditional genetic transformation method for P. polymyxa as the starting strain, so as to provide a transformation method applied to P. polymyxa with high transformation efficiency. On this basis, the present application successfully electroporates the plasmid pIC333 containing Tn10 transposon into P. polymyxa, and verifies that the plasmid also has transposition effect and temperature-sensitive characteristics in P. polymyxa, which is convenient for carrying out genetic manipulation work related to transposition in P. polymyxa. Therefore, the present application effectively solves the problems of transformation and transposition of foreign plasmid in P. polymyxa in the prior art.

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

[0007] The present application first optimizes the electroporation conditions of P. polymyxa. It is found that the transformation efficiency of foreign DNA is higher when P. polymyxa is cultured to the middle of the growth period to prepare electroporation competent cells. The specific optimization process of the electroporation conditions is as follows: picking a fresh P. polymyxa ATCC842 single colony, inoculating in a test tube for overnight culture; inoculating the bacterial solution into LB+ sorbitol culture medium at a 4% inoculation amount, measuring OD 600 nm every 1 h, and drawing a growth curve; selecting P. polymyxa ATCC842 at different growth periods to prepare electroporation competent cells; electroporating under the condition of 1.8 kV voltage, and recovering in a shaking bed for 3 h, 4 h and 5 h respectively; centrifuging the bacterial solution, appropriately diluting and coating, performing antibiotic plate screening, and calculating the electroporation efficiency to determine the optimal electroporation conditions.

[0008] As preferred, the method of inoculating the Paenibacillus polymyxa into the liquid culture medium to culture to the middle of the growth period comprises: inoculating the Paenibacillus polymyxa into the liquid culture medium to culture to OD 600 nm 0.4~0.7. Wherein, the culture method comprises: taking fresh single colony P. polymyxa ATCC842 to inoculate into 5 mL LB test tube, 200 r / min, 37℃ overnight culture. Inoculating into 50 mL LB + sorbitol culture medium at 4% inoculation amount, 200 r / min, 37℃ culture, every one hour interval, measuring OD 600 nm and recording the value, drawing the P. polymyxa ATCC842 growth curve. According to the P. polymyxa ATCC842 growth curve, selecting the bacteria of three periods of the growth period to prepare the competent cells, and storing at -80℃. More specifically, the Paenibacillus polymyxa is inoculated into 50 mL LB + sorbitol culture medium at 4% inoculation amount, 200 r / min, 37℃ culture for 7~8 h, and then OD 600 nm 0.4~0.7 is reached.

[0009] As preferred, the method of electrically transforming the plasmid into the competent cells comprises: dissolving the stored competent cells on ice, adding the plasmid, mixing to form a mixed solution, placing on ice for 25~35 min, adding the mixed solution into the pre-cooled electrode cup, and 1.5~2.0 kV electric shock, more preferably 1.8~2.0 kV electric shock.

[0010] As preferred, the method of resuscitation comprises: adding the preheated resuscitation liquid, resuscitating at 35~40℃, 180~200 rpm for 3~4 h, more preferably 4 h. When the resuscitation time is 4 h, the electric transformation efficiency is the highest, which can reach 1.795×10 5 cfu / μg. Wherein, the resuscitation liquid can be LB liquid culture medium containing D-sorbitol and D-mannitol.

[0011] As preferred, the Paenibacillus polymyxa is Paenibacillus polymyxa ATCC842. It can be understood that, because the Paenibacillus polymyxa is similar to Bacillus subtilis in evolution, and belongs to the same Bacillus genus, and has similar properties, so the method provided by the present application is also applicable to Bacillus subtilis, such as Bacillus subtilis 168.

[0012] As preferred, the plasmid comprises: plasmid pWB980, plasmid pIC333.

[0013] The plasmid pIC333 has a Mini-Tn10 transposon, SPC spectinomycin resistance and Em erythromycin resistance, and the temperature-sensitive replicon is inactivated above 35 DEG C in the gram-positive bacterial host. The present application 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 an antibiotic plate, three subcultures are carried out to detect whether the transposon is transposed in Bacillus subtilis. The experiment proves that the plasmid pIC333 has transposition effect and temperature-sensitive characteristics in Bacillus subtilis, and is an effective mini-Tn10 transposon vector of Bacillus subtilis. However, in the prior art, it is difficult to transform the plasmid pIC333 into Paenibacillus polymyxa by the traditional genetic transformation method. To solve this problem, the inventors' team successfully transformed the pIC333 plasmid into the competent cells of the mid-log phase cells of Paenibacillus polymyxa under the above-optimized Paenibacillus polymyxa electrotransformation system conditions, screened by an antibiotic plate after screening, and carried out multiple subcultures to detect whether the Tn10 transposon is transposed in Paenibacillus polymyxa. Specifically, the method comprises the following steps: inoculating Paenibacillus polymyxa into a liquid culture medium to culture to OD 600 nm To 0.4-0.7 to prepare competent cells; the plasmid pIC333 is electrotransformed into the competent cells, and after 4 h of recovery, resistance screening is carried out again to obtain transformants. The transformants are subcultured for three times, and it is detected that the genome of the transformants contains the transposon of the plasmid pIC333; after being cultured at 35-40 DEG C, the plasmid pIC333 in the transformants is lost. Therefore, it can be determined that the pIC333 plasmid also has transposition effect and temperature-sensitive characteristics in Paenibacillus polymyxa, indicating that the transposon can be applied to the construction of a mutation system of Paenibacillus polymyxa, and lays a certain foundation for the subsequent development of a cell factory in Paenibacillus polymyxa.

[0014] The present application has the following beneficial effects: the present application optimizes the electrotransformation conditions of Paenibacillus polymyxa, provides a genetic transformation method applied to Paenibacillus polymyxa, specifically, electrotransformation competent cells are prepared when Paenibacillus polymyxa is cultured to the mid-log phase, and the method effectively improves the transformation efficiency of exogenous DNA. By using the method, the present application also successfully transforms the temperature-sensitive pIC333 plasmid containing a Mini-Tn10 transposon into Paenibacillus polymyxa, and verifies that the pIC333 plasmid has transposition effect and temperature-sensitive characteristics in Paenibacillus polymyxa, indicating that the transposon can be applied to the construction of a mutation system of Paenibacillus polymyxa, and lays a certain foundation for the subsequent development of a cell factory in Paenibacillus polymyxa. Meanwhile, the method has the advantages of high efficiency and simple operation, and can be used for more in-depth research of other Paenibacillus polymyxa. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is a growth curve of P. polymyxa ATCC 842 in Example 1.

[0016] Figure 2 is the electroporation efficiency of P. polymyxa ATCC 842 in different growth stages in Example 1.

[0017] Figure 3 is the electroporation efficiency of P. polymyxa ATCC 842 in different recovery time in Example 2.

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

[0019] Figure 5 is a verification diagram of the success of transposon transposition in Example 3. DETAILED DESCRIPTION

[0020] The present application will be described in more detail by the following specific embodiments, and other advantages and effects of the present application can be easily understood by those skilled in the art from the contents disclosed in the specification. The present application can also be implemented or applied by other different specific embodiments, and various modifications or changes can be made to the details in the specification based on different views and applications without departing from the spirit of the present application. It should be noted that the following examples and features in the examples can be combined with each other without conflict. The methods used in the examples of the present application are conventional methods, and the reagents used can be obtained from commercial channels, unless otherwise specified.

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

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

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

[0024] Sorbitol LB liquid medium: LB liquid medium containing 0.5 mol / L D-sorbitol.

[0025] 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 liquid: LB liquid medium containing 0.5 mol / L D-sorbitol and 0.38 mol / L D-mannitol.

[0027] Example 1

[0028] The present example provides a genetic transformation method applied to Paenibacillus polymyxa, and the specific method is as follows.

[0029] P. polymyxa ATCC842 was used as the starting strain for culture, and OD 600 nm The growth curve of P. polymyxa ATCC842 was plotted: fresh single colony P. polymyxa ATCC842 was inoculated in 5 mL LB test tube, 200 r / min, 37°C overnight culture; 4% inoculation amount was inoculated in 50 mL LB medium added with sorbitol, 200 r / min, 37°C culture; OD 600 nm was measured every hour, and the value was recorded; the growth curve of P. polymyxa ATCC842 was plotted (shown in Figure 1).

[0030] P. polymyxa ATCC842 was inoculated into liquid medium and cultured to different growth periods to prepare competent cells: according to the growth curve of P. polymyxa ATCC842, the bacteria in the middle of the growth period (OD 600 nm =0.43, OD 600 nm =0.68) and the late growth period (OD 600 nm =0.88) were selected to prepare competent cells, which were stored at -80°C.

[0031] Plasmids were electroporated into competent cells, and after resuscitation, resistance screening was performed to obtain transformants: Prepared *P. polymyxa*ATCC842 electroporated competent cells were dissolved on ice, and 1 μg of plasmid pWB980 was added. The mixture was incubated on ice for 30 min. The mixture was then added to a 1 mm pre-cooled electrode cup, electroporated at 1.8 kV, and 900 μL of pre-warmed resuscitation solution was quickly added. The cells were incubated at 37°C and 200 rpm for 4 h. The bacterial culture was centrifuged at 5000 rpm for 2 min, the supernatant was discarded, and 100 μL was spread onto a plate containing LB resistant Kan (50 μg / mL) and incubated overnight at 37°C. Transformants were obtained, and the transformation efficiency was calculated.

[0032] As shown in Figure 2, the OD of P. polymyxaATCC842 during the mid-growth stage is... 600 nm The highest conversion efficiency, approximately 1.795 × 10⁶, is achieved when the coefficient of performance is 0.68. 5 cfu / μg, and at OD 600 nm Conversion efficiency and D when it is 0.43 600 nm The value is close to 0.68. This indicates that *P. polymyxa*ATCC842 achieves the highest transformation efficiency during the mid-growth phase. Based on the experimental data, the optimal time for competent cell preparation is the mid-growth phase, i.e., OD... 600 nm =0.4~0.7.

[0033] Example 2

[0034] The difference from Example 1 is that the resuscitation time in Example 2 was 3 hours, while the other steps were the same. The results are shown in Figure 3.

[0035] Comparative Example 1

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

[0037] After *P. polymyxa* ATCC842 competent cells were electroporated with 1.8 kV, 1 ml of resuscitation medium was added, and the cells were resuscitated at 37°C for 3, 4, and 5 hours, respectively. The electroconversion efficiency is shown in Figure 3. It can be seen that increasing the resuscitation time from 3 h to 4 h significantly improved the electroconversion efficiency, reaching approximately 1.795 × 10⁻⁶. 5 However, an excessively long recovery time (5 h) actually reduced the electroconversion efficiency to almost zero. Therefore, the optimal recovery time after electric shock is 4 h.

[0038] Control group: Detects whether transposons transpose in Bacillus subtilis.

[0039] First, fresh single colony Bacillus subtilis 168 was inoculated in 5 mL LB test tube, 200 r / min, 37℃ overnight culture; 4% inoculation amount was inoculated in 50 mL LB + sorbitol medium, 200 r / min, 37℃ culture, every hour interval, OD was measured 600 nm and the value was recorded; Bacillus subtilis 168 growth curve was drawn (shown in Figure 4).

[0040] Detection of transposon transposition: the electrotransformation competent cells of Bacillus subtilis 168 in the middle of the growth period were dissolved on ice, 1 μg plasmid pIC333 was added, mixed and then rested on ice for 30 min. The bacterial solution was added to a 1 mm pre-cooled electrode cup, 2.0 kV electric shock, and 900 μL preheated recovery liquid was quickly added, 30℃, 200 rpm, recovery for 3 h; the negative control did not add plasmid, and the positive control added plasmid pWB980. The bacterial solution was centrifuged at 5000 rpm for 2 min, the supernatant was removed, and the remaining 200 μL was taken and 50 μL was spread on the plates containing Spec (100 μg / mL), Erm (25 μg / mL), Spec + Erm, LB, respectively, and cultured at 30℃, 200 rpm, overnight.

[0041] If LB and Spec plates grow transformants and Erm plates do not grow transformants, it indicates that plasmid pIC333 may have been lost, and IS part may have transposed to Bacillus subtilis 168 genome, indicating that the transposon in plasmid pIC333 has transposition ability in Bacillus subtilis 168. If LB and Erm plates grow transformants and Spec plates do not grow transformants, it indicates that plasmid pIC333 has been transformed into Bacillus subtilis 168 but has not transposed.

[0042] The transformants grown on the Erm plate were selected, inoculated in 5 mL LB test tube, and cultured in a 30℃, 200 rpm shaker until the bacterial solution was turbid. Again, inoculate in 5 mL LB test tube, and culture in a 37℃ shaker for 1 hour. Repeat the passage for 3-4 times. Spread on Spec (100 μg / mL), Erm (25 μg / mL), Spec + Erm, LB plates. Culture at 37℃ overnight. If LB and Spec plates grow transformants and Erm plates do not grow transformants, it indicates that the transposon in plasmid pIC333 has transposition ability and temperature-sensitive characteristics in Bacillus subtilis 168.

[0043] According to the resistance plate screening results in Table 1, after the plasmid pIC333 is electroporated into Bacillus subtilis 168, the transposon Tn10 directly transposes under the culture condition of 30℃, and the plasmid pIC333 no longer exists in the bacteria.

[0044]

[0045] Example 3: Detection of whether the transposon transposes in Paenibacillus polymyxa

[0046] The present example provides a genetic transformation method applied to Paenibacillus polymyxa.

[0047] The difference between Example 1 and Example 3 is that, in Example 3, the plasmid pIC333 is transformed into the competent cells of Paenibacillus polymyxa in the middle of the growth period according to the method provided in Example 1, and the cells are recovered for 4 hours after the electric shock; no plasmid is added in the negative control, and the plasmid pWB980 is added in the positive control. After the recovery is completed, the bacterial solution is centrifuged at 5000 rpm for 2 minutes, the supernatant is removed, and the remaining 200 μL is taken to spread on the Spec (100 μg / mL), Erm (25 μg / mL), Spec+Erm, and LB plates, respectively, and cultured at 30℃, 200 rpm, overnight.

[0048] If the transformants grow on the LB and Spec plates, and no transformants grow on the Erm plate, it indicates that the plasmid pIC333 may have been lost, and the IS part may have transposed to the genome of P. polymyxa ATCC842, indicating that the transposon in the plasmid pIC333 has the transposition ability in P. polymyxa ATCC842. If the transformants grow on the LB and Erm plates, and no transformants grow on the Spec plate, it indicates that the plasmid pIC333 has transformed P. polymyxa ATCC842 but has not transposed.

[0049] The transformants growing on the Erm plate are selected, inoculated into 5 mL of LB test tube, and cultured in a 30℃, 200 rpm shaker until the bacterial solution is turbid. Again, the bacterial solution is inoculated into 5 mL of LB test tube and cultured in a 37℃ shaker for 1 hour. The subculture is repeated for 3-4 times. The bacterial solution is spread on the Spec (100 μg / mL), Erm (25 μg / mL), Spec+Erm, and LB plates, and cultured at 37℃ overnight. If the transformants grow on the LB and Spec plates, and no transformants grow on the Erm plate, it indicates that the transposon in the plasmid pIC333 has the transposition ability and temperature-sensitive characteristics in P. polymyxa ATCC842.

[0050] The transformants were inoculated in Spec (100 μg / mL), Erm (25 μg / mL), Spec+Erm, LB tubes and incubated at 37°C. According to the culture results of the transformants inoculated in different antibiotic tubes (Figure 5), it can be seen that the transposon has been transposed in P. polymyxa ATCC842. It shows that Tn10 has transposition function in P. polymyxa. Because P. polymyxa and B. subtilis are similar in evolution and belong to the same genus of Bacillus, they have similar properties. Therefore, the transposon in plasmid pIC333 can exert transposition effect in B. subtilis 168 and P. polymyxa ATCC842, and has temperature-sensitive characteristics.

[0051] The above-described embodiments are merely preferred embodiments of the present application and are not intended to limit the scope of the present application. Various modifications and improvements to the present application made by those skilled in the art, without departing from the design spirit of the present application, should fall within the scope of the present application.

Claims

1. A method for genetic transformation of Paenibacillus polymyxa, characterized by, The method comprises inoculating Paenibacillus polymyxa into a liquid culture medium to culture to the middle of the growth period to prepare competent cells; and electrically transforming a plasmid into the competent cells, and then performing resistance screening after recovery to obtain transformants.

2. The method of claim 1, wherein, The method for inoculating Paenibacillus polymyxa into a liquid culture medium to culture to the middle of the growth period comprises inoculating Paenibacillus polymyxa into a liquid culture medium to culture to OD600 nm of 0.4-0.

7.

3. The method of claim 1, wherein, The method for electrically transforming a plasmid into the competent cells comprises dissolving the stored competent cells on ice, adding the plasmid, mixing to form a mixed solution, placing on ice for 25-35 min, and adding the mixed solution into a pre-cooled electrode cup and performing 1.5-2.0 kV electric shock.

4. The method of claim 1, wherein, The method for recovery comprises adding pre-heated recovery liquid, and recovering at 35-40 DEG C and 180-200 rpm for 3-4 h.

5. The method of claim 4, wherein, The recovery time is 4 h.

6. The method of claim 1, wherein, The Paenibacillus polymyxa is Paenibacillus polymyxa ATCC 842.

7. The method of claim 1, wherein, The plasmid comprises plasmid pWB980 and plasmid pIC333.

8. The method of claim 7, wherein, The method comprises inoculating Paenibacillus polymyxa into a liquid culture medium to culture to OD600 nm of 0.4-0.7 to prepare competent cells; and electrically transforming plasmid pIC333 into the competent cells, and then performing resistance screening after 4 h of recovery to obtain transformants.

9. The method of claim 8, wherein, The genome of the transformants contains the transposon of plasmid pIC333.

10. The method of claim 8, wherein, The plasmid pIC333 in the transformants is lost after the transformants are cultured at 35-40 DEG C.

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