CN106167809A - Ocean atrophy bacillus cereus foreign DNA proceed to method - Google Patents

Abstract

The invention provides a method for transferring exogenous DNA of Bacillus marine atrophaeus, comprising the following steps: respectively cultivating a donor strain, an auxiliary strain and a recipient strain, and respectively detecting the culture fluid of the donor strain and the culture fluid of the auxiliary strain and the OD ₆₀₀ of the culture medium of the recipient strain; mix the donor strain, the auxiliary strain and the recipient strain cultivated in the previous step in equal amounts, perform three-parent hybridization, and screen out the strains that successfully hybridize, which are recorded as three-parent hybridization Bacterial strains; Bacillus atrophaeus C89/pRP1028 with plasmid strains were screened out; plasmid pRP1028 was extracted; plasmid pRP1028 was transformed into E. coli, and the plasmid of E. coli was extracted for sequencing verification. The invention has the following beneficial effects: it has reference significance for the transformation of exogenous genes of special culture and difficult-to-transform bacterial strains.

Description

Transfer method of exogenous DNA of Bacillus marine atrophaeus

technical field

The invention relates to a method for transferring exogenous DNA of bacillus marine atrophaeus, belonging to the technical field of microorganisms.

Background technique

Bacillus atrophaeus belongs to the genus Bacillus and forms soluble black colonies on medium containing carbohydrates. The research on the application technology of Bacillus atrophaeus is extensive and in-depth, and a large number of biologically active substances are isolated and purified from Bacillus atrophaeus, among which, the compounds neobacillamide A and bacillamide C are isolated from Bacillus atrophaeus C89 in this laboratory (Yu LL, Li ZY , Peng CS, et al. Neobacillamide A, a novel thiazole-containing alkaloid from the marinebacterium Bacillus vallismortis C89, associated with South China Sea sponge Dysidea avara. Helv Chim Acta. 2009, 92:607-612.). Bacillus atrophaeus can be used as a vaccine carrier and vaccine adjuvant, simulate biological weapons, monitor biological environments, and be used as standard microorganisms for biological control agents and plant promoters, etc. It has been reported in the literature that exogenous genes can be transferred to other strains of the genus Bacillus, such as: Bacillus subtilis, Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus licheniformis , Bacillus stearothermophilus, Bacillus megaterium, Bacillus pumilus, Bacillus sphaericus, Bacillus thuringiensis, Bacillus anthracis. So far, there have been no reports of Bacillus atrophaeus var. globigii, ahistorical biowarfare simulant. PLoS one. 2011, 6:e17836.).

Contents of the invention

Aiming at the defects in the prior art, the object of the present invention is to provide a method for introducing exogenous DNA of Bacillus marine atrophaeus, which is simple and easy to operate.

In order to achieve the above object, the present invention transfers the plasmid pRP1028 with the visible turbo-rfp protein gene into Bacillus marine atrophaeus C89 under the action of the helper plasmid pSS1827 through the method of triparental hybridization, and observes it by antibiotics and naked eyes. After screening, the plasmid was extracted and transformed into Escherichia coli DH5α for further verification of the plasmid.

The present invention is achieved through the following technical solutions:

The invention provides a method for transferring exogenous DNA of Bacillus atrophaeus, which comprises the following steps:

S1: Cultivate the donor strain, the auxiliary strain and the recipient strain respectively, control the cultivation temperature of the donor strain and the auxiliary strain to 37°C, and the cultivation temperature of the recipient strain at 28-37°C, and detect The 600nm absorbance value of the culture medium after the cultivation of the donor strain, the auxiliary strain and the recipient strain;

S2: Mix the donor strain, the auxiliary strain and the recipient strain cultivated in step S1, perform three-parent hybridization, and screen out the strain that successfully hybridizes, and record it as the three-parent hybrid strain;

S3: screening Bacillus atrophaeus C89/pRP1028 carrying a plasmid strain from the three-parent hybrid strain;

S4: Extracting plasmid pRP1028 from the Bacillus atrophaeus C89/pRP1028 carrying the plasmid strain;

S5: Transform the plasmid pRP1028 into Escherichia coli, extract the plasmid of Escherichia coli, and perform sequencing verification.

As a preferred solution, in step S1, the donor strain is cultivated in LB medium containing spectinomycin, the auxiliary strain is cultivated in LB medium containing ampicillin, and the recipient strain is cultivated in LB medium cultivated in.

As a preferred solution, the LB medium is formed by dissolving 10 g of tryptone, 5 g of yeast extract, and 5 g of sodium chloride in 1 L of distilled water.

As a preferred solution, the donor strain is Escherichia coli DH5α carrying pRP1028, the auxiliary strain is Escherichia coli DH5α carrying plasmid pSS1827, and the recipient strain is Bacillus maritimus C89.

As a preferred solution, in step S2, the three-parent hybridization is carried out in beef extract peptone medium.

As a preferred solution, the beef extract-peptone medium is formed by dissolving 10 g of peptone, 3-5 g of beef extract, and 5 g of sodium chloride in 1 L of artificial seawater.

As a preferred solution, the composition and content of the artificial seawater are: 26.518g/L NaCl, 2.447g/L MgCl ₂ , 3.305g/L MgSO ₄ , 1.141g/L CaCl ₂ , 0.725g/L KCl, 0.202g/L NaHCO ₃ and 0.083g/L NaBr, the solvent is distilled water, and the pH of the artificial seawater is 7.0-7.2.

As a preferred solution, the screening of the three-parent hybrid strain is carried out on beef extract peptone solid medium containing polymyxin and spectinomycin.

As a preferred version, the concentration of the polymyxin is 60 Units/mL, and the concentration of the spectinomycin is 100-300 μg/mL.

In this study, the plasmid pRP1028 was transformed into Bacillus marine atrophaeus C89 by the combined transfer method of three-parent hybridization. The technology of transferring exogenous genes into Bacillus atrophaeus is still the first at home and abroad. The present invention realizes the transfer of exogenous genes into Bacillus atrophaeus through a combination of transfer methods, laying a solid foundation for further research on the genetic manipulation of Bacillus atrophaeus. Base.

Compared with the prior art, the present invention has the following beneficial effects:

1. The plasmid pRP1028 was transferred into Bacillus marinum atrophaeus C89 by using the combined transfer method of three-parent hybridization. The technology of transferring foreign genes into Bacillus atrophaeus is still the first at home and abroad;

2. The exogenous gene was transferred into Bacillus atrophaeus through plasmids through combined transfer, which laid the foundation for further research on the genetic manipulation of Bacillus atrophaeus;

3. It has reference significance for the transformation of exogenous genes in special culture and difficult-to-transform strains.

Description of drawings

Other characteristics, objects and advantages of the present invention will become more apparent by reading the detailed description of non-limiting embodiments made with reference to the following drawings:

Fig. 1 is method flowchart of the present invention;

Figure 2 shows B.atrophaeus C89 and B.atrophaeus C89/pRP1028 in LB solid medium;

Figure 3 shows B.atrophaeus C89 and B.atrophaeus C89/pRP1028 in LB liquid medium.

detailed description

The present invention will be described in detail below in conjunction with specific embodiments. The following examples will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

The Bacillus atrophaeus transformed into the plasmid pRP1028 in the present invention has been preserved in the China Center for Type Culture Collection (CCTCC). The address of the preservation unit is: Wuhan University, Wuhan, China, and the postal code is: 430072. The preservation date is: On May 18, 2016, the deposit number is: CCTCC NO: M 2016269 Classification designation: Bacillus atrophaeus C89/pRP1028Bacillus atrophaeusC89/pRP1028.

The source of Escherichia coli in the present invention can refer to Plaut RD and Stibitz S. Improvements to a Markerless Allelic Exchange System for Bacillus anthracis. PLOS ONE, 2015. DOI: 10.1371/journal.pone.0142758.

The beef extract peptone culture medium selected among the present invention is to be made by 10g tryptone, 3-5g beef extract, and 5g sodium chloride is dissolved in 1L artificial sea water. The composition and content of the artificial seawater are: 26.518g/L NaCl, 2.447g/L MgCl ₂ , 3.305g/L MgSO ₄ , 1.141g/L CaCl ₂ , 0.725g/L KCl, 0.202g /L of NaHCO ₃ and 0.083g/L of NaBr, the solvent is distilled water, and the pH of the artificial seawater is 7.0-7.2.

The selected LB medium was prepared by dissolving 10 g of tryptone, 5 g of yeast extract and 5 g of sodium chloride in 1 L of distilled water.

The method for transferring exogenous DNA of Bacillus marine atrophaeus of the present invention, the technological process as shown in Figure 1, comprises the following steps:

1. Strain culture:

Transform the vector pRP1028 into E.coli DH5α by conventional methods, and culture in 5 mL of LB medium plus 100 μg/mL Spectinomycin (Spectinomycin, Spc) at 37°C with shaking at 200 r/min; inoculate the auxiliary strain pSS1827/DH5α in 5 mL LB medium plus 100 μg/mL ampicillin (Ampicillin, Amp) in 37 ° C, 200 r/min shaking culture; inoculate the recipient strain B. atrophaeus C89 in 5 mL LB medium, 28 ° C ~ 37 ° C, 200 r /min shaking culture. The above three strains were respectively transferred to 5 mL LB medium containing corresponding antibiotics at 28°C-37°C with 0.5-1% inoculation amount, and cultured with shaking at 200r/min until the _OD600 was about 0.5-1.0.

2. Combine transfer:

Take 1 mL of each of the three cultured bacterial solutions in a sterile EP tube, centrifuge at 5000 r/min for 2 minutes, discard the supernatant, and resuspend and wash each bacterial cell three times with fresh 1 mL LB medium to ensure that the residual antibiotics are washed away. , and finally resuspended with 500 μL of LB medium. Take a certain amount of each bacterial solution in the EP tube, mix by pipetting, and spot on the center of the LB non-resistant solid plate. Then the plate was transferred to a constant temperature incubator at 28°C for static culture for 24 hours (face up). Use the tip of a pipette to scrape all the plaque on a plate and transfer it to a sterile centrifuge tube containing beef extract peptone medium. After mixing by pipetting, the mixture was diluted and spread on a beef extract peptone solid plate containing 60 Units/ml polymyxin (Pmx) and 100-300 μg/mL spectinomycin (Spc), and cultured at a constant temperature of 28 °C Keep it in the box for 2-3 days. After red single colonies grow on the plate, use a small pipette to pick the bacteria in 5 mL of LB (Spc300+Pmx60) liquid medium, and culture overnight at 28°C until it turns red (as shown in Figure 2 and Figure 3).

3. Extraction of plasmids:

Plasmid pRP1028 was extracted using Kangwei Century Bioplasmid DNA Extraction Kit (CWBIO PurePlasmid Mini Kit). Take 1 mL of Bacillus atrophaeus containing plasmid pRP1028 and add it to an EP tube, centrifuge at 8000 r/min for 3 min, and discard the supernatant. Add 250 μL of Buffer P1, vortex and mix; add 250 μL of Buffer P2, and gently invert 4 to 6 times; add 350 μL of Buffer N3, and gently invert 4 to 6 times; centrifuge at 13000 r/min for 1 min. Take the supernatant and put it into the adsorption column, centrifuge at 13000r/min for 1min and discard the waste liquid. Add 750 μL of Buffer PW to the adsorption column, centrifuge at 13000 r/min for 1 min and discard the waste liquid; centrifuge again at 13000 r/min for 2 min, air-dry at room temperature, put the adsorption column into a new EP tube, add 30 μL of 65 °C pre- Hot ddH ₂ O, let stand at room temperature for 5 minutes, centrifuge at 13,000 r/min for 1 minute, discard the adsorption column, and store at -20°C. 1% (w/v) agarose gel electrophoresis detection.

4. Plasmid verification:

Plasmid transformation into Escherichia coli verification: Mix 50 μL of DH5a competent cells (trans 5a) with 10 μL of pRP1028 plasmid, heat shock at 42°C for 45 s after 30 min in ice bath, add 500 μL of LB medium to resuspend after 2 min in ice bath, and resuspend in Incubate at 37°C and 200r/min on a shaker for 1 hour, take 100 μL and spread it on an LB ampicillin-resistant plate, and cultivate overnight at 37°C. After a single red colony grows on the plate, use a small tip to pick the bacteria into 5 mL of Spc-containing Incubate the resistant LB overnight at 37°C.

Plasmid sequencing verification: Sequencing (Suzhou Jinweizhi Biotechnology Co., Ltd.) with primers 5'CTTACCCGTCTTACTGTCGGGAA3'(SEQ ID NO:1) and 5'GTGCGAATAAGGGACAGTGAAGAAGG3'(SEQ ID NO:2) of pRP1028, the sequencing results are as follows: S E Q ID NO: 3. The blast homology analysis was carried out on http://blast.ncbi.nlm.nih.gov/Blast.cgi, which was consistent with the sequence of pRP1028.

In the present invention, the combined transfer method of three-parent hybridization is used to transfer the plasmid pRP1028 into Bacillus atrophaeus C89; the technology of transferring exogenous genes into Bacillus atrophaeus is still the first at home and abroad; The gene was transferred into Bacillus atrophaeus through the plasmid, which laid the foundation for further research on the genetic manipulation of Bacillus atrophaeus; it has reference significance for the transformation of exogenous genes in special culture and difficult-to-transform strains.

Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art may make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims (9)

1. an ocean atrophy bacillus cereus foreign DNA proceed to method, it is characterised in that comprise the steps:

S1: cultivate F+strain, supplementary strain and F-strain respectively, controls described F+strain and supplementary strain Cultivation temperature is 37 DEG C, and the cultivation temperature of described F-strain is 28～37 DEG C, and detect respectively F+strain, supplementary strain and The 600nm absorption value of culture fluid after F-strain cultivation；

S2: F+strain, supplementary strain and the mixing of F-strain equivalent after cultivating in step S1, carries out triparental mating, And filter out the successful bacterial strain of hybridization, it is designated as triparental mating bacterial strain；

S3: filter out the atrophy bacillus cereus C89/pRP1028 with plasmid-bearing strains from described triparental mating bacterial strain；

S4: extract plasmid pRP1028 from the described atrophy bacillus cereus C89/pRP1028 with plasmid-bearing strains；

S5: described plasmid pRP1028 is transformed in escherichia coli, and extracts colibacillary plasmid, carry out sequence verification.

2. ocean atrophy bacillus cereus foreign DNA as claimed in claim 1 proceed to method, it is characterised in that in step S1, F+strain is to carry out cultivating in the LB culture medium containing spectinomycin, and supplementary strain is at the LB containing ampicillin Carrying out in culture medium cultivating, F-strain is cultivated in LB culture medium.

3. ocean atrophy bacillus cereus foreign DNA as claimed in claim 1 proceed to method, it is characterised in that described LB trains Foster base is dissolved in 1L distilled water is formed by 10g tryptone, 5g yeast extract, 5g sodium chloride.

4. ocean atrophy bacillus cereus foreign DNA as claimed in claim 1 proceed to method, it is characterised in that described donor Bacterial strain is the bacillus coli DH 5 alpha with pRP1028, and described supplementary strain is the bacillus coli DH 5 alpha with plasmid pSS1827, Described F-strain is ocean atrophy bacillus cereus C89.

5. ocean atrophy bacillus cereus foreign DNA as claimed in claim 1 proceed to method, it is characterised in that in step S2, Triparental mating is carried out in beef-protein medium.

6. ocean atrophy bacillus cereus foreign DNA as claimed in claim 5 proceed to method, it is characterised in that described beef Cream protein culture medium is by 10g peptone, 3～5g Carnis Bovis seu Bubali cream, and 5g sodium chloride is dissolved in 1L artificial seawater formation.

7. ocean atrophy bacillus cereus foreign DNA as claimed in claim 6 proceed to method, it is characterised in that described manually The component of sea water and content is: the MgCl of NaCl, 2.447g/L of 26.518g/L₂, the MgSO of 3.305g/L₄, 1.141g/L CaCl₂, the NaHCO of KCl, 0.202g/L of 0.725g/L₃And the NaBr of 0.083g/L, solvent is distilled water, this artificial seawater PH be 7.0～7.2.

8. ocean atrophy bacillus cereus foreign DNA as claimed in claim 1 proceed to method, it is characterised in that described three parents The screening of this hybrid strain is to carry out on the beef extract-peptone solid medium containing polymyxin and spectinomycin.

9. ocean atrophy bacillus cereus foreign DNA as claimed in claim 8 proceed to method, it is characterised in that described glue more The concentration of rhzomorph is 60Units/mL, and the concentration of described spectinomycin is 100～300 μ g/mL.