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Streptomyces diastatochromogenes electroporation transformation method

A technology of electric shock transformation and lysozyme, which is applied in the direction of using vectors to introduce foreign genetic material, recombinant DNA technology, etc., can solve the problems of low efficiency of transforming 1628 strains and no reports.

Active Publication Date: 2013-10-02
CHINA JILIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, our previous research found that the efficiency of PEG-mediated protoplast transformation to transform strain 1628 was low, and there is no report on the genetic transformation of strain 1628

Method used

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  • Streptomyces diastatochromogenes electroporation transformation method
  • Streptomyces diastatochromogenes electroporation transformation method
  • Streptomyces diastatochromogenes electroporation transformation method

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Preparation of protoplasts and transformation by electric shock

[0029] Amylase Streptomyces chromogenes spores were transferred to a 250 ml shake flask containing 50 ml of CP medium, cultured at 28°C for two days, and 1.5 ml of the bacterial liquid was transferred to another fresh 250 ml flask containing 50 ml of CP medium In a shake flask, add 0.5% glycine, collect mycelia after 28-32 h, wash twice with 10% sucrose solution, resuspend the bacteria in lysozyme with a final concentration of 1 mg ml -1 5 ml of P buffer, reacted at room temperature for 1 h, and the obtained protoplasts were filtered with absorbent cotton, collected by centrifugation at 2000 rpm for 8 min, washed once with P buffer, and stored at -70°C for future use. 100 μl of protoplasts were mixed with an appropriate amount of plasmid DNA, placed on ice for 10 minutes, and then the mixture was placed in an electric shock cup, 1000 V (25 μF and 200Ω), diluted with P buffer and spread on the R2YE regene...

Embodiment 2

[0032] Electric Shock Transformation of Amylase Streptomyces Chromogenes Treated in Different Ways

[0033] Will S. diastatochromogenes 1628 was pre-cultured in CP medium for 28 hours, and then transferred to fresh CP medium for 1 day. Collect the cells by centrifugation (10,000 rpm, 10 min) at 4°C, and wash with 15 ml of pre-cooled 10% sucrose solution twice. Second-rate. Wash the cells twice with 15 ml pre-cooled 15% glycerol solution, then treat the cells with four different additives, and finally resuspend the competent cells in PGS buffer, so that the concentration of the cells is about 2×10 9 cells ml -1 . 100 μl of competent cells were mixed with an appropriate amount of plasmid DNA, placed on ice for 10 minutes, electric shock parameters were 1200 V (25 μF and 200Ω), and 1 ml of CP medium was quickly added after electric shock, and reacted for 1 hour at 28°C. Spread on R2YE regeneration medium containing antibiotics and culture at 28°C for 3-4 days.

[0034] In...

Embodiment 3

[0045] S. diastatochromogenes Table 3 shows the optimized results of various electroporation transformation conditions for different cell forms. Among them, the pre-treatment of the cells refers to collecting the cells when the complete cells grow to the mid-logarithmic growth stage, and adding 3 μg / ml lysozyme and 20 μg / ml penicillin together to the electroporation transformation buffer to react with the cells for a period of time After a period of time, it will be used as a material for electric shock transformation. Immediately after electroporation transformation, add 1 ml of YEME medium and react at 28°C for 2-3 hours, and then spread R2YE solid medium containing corresponding antibiotics.

[0046] table 3 S. diastatochromogenes Optimization of various electroporation transformation conditions for different cell formats

[0047]

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Abstract

The invention discloses an S.diastatochromogenes electroporation transformation method, which comprises: carrying out a mild wall breaking treatment on cells, and carrying out electroporation transformation, wherein the mild wall breaking treatment adopts an electroporation buffer solution containing 5 mug / ml of lysozyme, or adopts an electroporation buffer solution containing 1% glycine, or adopts an electroporation buffer solution containing 30 mug / ml of penicillinG, or adopts an electroporation buffer solution containing 3 mug / ml of lysozyme and 20 mug / ml of penicillinG, and the electroporation transformation parameters comprise an electric field intensity of 12 kV / cm, a cell concentration of 1*10<9> / ml, and a plasmid DNA concentration of 2 mug; and adopting a YEME culture medium to incubate after electroporation transformation, wherein an incubation time is 2-3 h. According to the present invention, a convenient and reliable method is provided for efficient transformation of S.diastatochromogenes, and a path is paved for S.diastatochromogenes gene engineering.

Description

technical field [0001] The invention belongs to the field of microbial genetic engineering, in particular to a S. diastatochromogenes electric shock transformation method. Background technique [0002] Toyocamycin is one of the important members of the nucleoside antibiotic family. Due to the similarity in nucleotide structure, toyocamycin exhibits a wide range of biological activities, such as anti-tumor, anti-bacterial and anti-fungal activities. Therefore, toyocamycin is considered to be an ideal agricultural antibiotic for the control of agricultural fungal diseases. In our previous research, a strain of Streptomyces with toyocamycin as the main metabolite was screened and identified as amylase-chromogenic Streptomyces Streptomyces diastatochromogenes 1628. However, due to the low output, it is difficult to meet the needs of large-scale industrial production. The usual method to solve this problem is to adopt conventional physical and chemical methods for mutagenesi...

Claims

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Application Information

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IPC IPC(8): C12N15/76
Inventor 马正俞晓平陶立彬申屠旭萍边亚琳郝培应许益鹏
Owner CHINA JILIANG UNIV
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