RU2095413C1 - Method of preparing plasmid from streptomycetes - Google Patents

Abstract

FIELD: genetic engineering of microorganisms, technical microbiology. SUBSTANCE: method involves preparing new plasmids pSCH2 and pSCH3 from Streptomyces chrysomallus VKM Ac-1355 and its protein variant. Plasmids show deletion (size is 1.7 kb) located between restriction sites for restriction endonucleases Kpn I and Eco 147 I. EFFECT: improved method of plasmid preparing. 2 dwg

Description

 The invention relates to biotechnology, and can be used in genetic engineering, molecular biological and microbiological studies.

 Known methods for the isolation of plasmids from various types of streptomycetes / EP, application 0006692 C 12 N 15/00, 1980; EP application 0038156, C 12 N 15/00, 1981 /. The objective of the invention is to isolate new streptomycete plasmids with new properties, determine their size, detect unique restriction sites and construct a restriction map.

 As a closest analogue, the invention contemplates a method for producing plasmids from Streptomyces coelicolor, S. lividans and S. Parvulus / EP, application 0006692, C 12 N 15/00, 1980 /. The invention differs from the closest analogue in that a strain of Streptomyces chpysomallus VKM Ac-1355 and its white variant are used to isolate plasmids.

 The essence of the invention lies in the fact that the cultures of the strains are grown in a nutrient medium, then mycelium cells are lysed with sodium dodecyl sulfate (SDS) and new plasmids pSCH2 and pSCH3 are isolated by centrifugation, respectively.

 In FIG. 1 shows a restriction map of the plasmid pSCH2, and FIG. 2 is a restriction map of the plasmid pSCH3.

 The invention is illustrated by the following example.

 Example.

Cultures of the Streptomyces chrysomallus BKM strain
AC-1355 and its white variant are inoculated individually in 100 ml of sterile medium YEME / 1 / of the following composition:
yeast extract 3 g / l
bacto peptone 5
malt extract 3
glucose 10
also containing 34% sucrose, 0.005 M MgCl ₂ and 0.5% glycine. Cultivate on a rocking chair with vigorous stirring for 48 hours at 28 ^o C.

The mycelium is precipitated by centrifugation at 2,000 rpm / then washed with 0.3 M sucrose. Mycelium is directly used to isolate plasmids or stored at -20 ^o C. The basis for the procedure for isolation of plasmids is the alkaline lysis method / 2 /. Isolation of plasmids is as follows.

1. Mycelium is suspended in isolation medium at pH 7.4 / 10 ml of medium per 1 g of raw mycelium / of the following composition: 0.3 M sucrose, 25 mM Tris-HCl, 25 mM EDTA, lysozyme (1 mg / ml medium)
2. Incubate for 5-15 minutes at 37 ^o C.

3. Add two volumes of an alkaline solution of SDS / 1% SDS, 0.2 M NaOH / and incubated for 10 min at 0 ^o C.

4. Add sodium acetate in an amount of 1.5 of the original volume / 3M, pH, 4.8 /, incubated for 1 h at 0 ^o C, centrifuged at 18000 rpm for 20 minutes

 5. To the supernatant add isopropanol in an amount of 0.6 of the initial volume, stand for 15 min at room temperature, centrifuged at 2500 rpm / min, for 20 min, the precipitate is dried and dissolved in 10 ml of TE-buffer / 10 mm tis-HCl, pH 7.4; 1 mm EDTA, pH 8.0 /.

6. An equal volume of 4 M LiCl is added to the DNA solution, incubated for 1 h at 0 ^° C and centrifuged for 20 minutes at 2000 rpm.

7. 2 volumes of ethanol are added to the supernatant, incubated for 1 h at 0 ^° C, centrifuged for 20 minutes at 2500 rpm, the precipitate is dried and dissolved in 0.5 ml of TE buffer.

8. To the DNA solution add a solution of RNase (preheated) to a final concentration of 100 μg / ml, incubated for 10 min at 37 ^o C.

 9. Add 0.1 volume of sodium acetate and extract the DNA solution with one volume of chloroform / chloroform: isoamyl alcohol, 24: 1 /.

10. Two volumes of ethanol are added to the DNA solution, kept for 1 h at 0 ^° C, centrifuged for 20 minutes at 2500 rpm, the precipitate is washed with 70% ethanol, dried and dissolved in 0.1 ml of double distillate.

 Thus, two plasmids pSCH2 and pSCH3, with sizes of 13.4 and 15.1 kilobases respectively, are isolated from the lysate of cultures of Streptomyces chrysomallus BKM Ac-1355 and its white variant.

Plasmids are characterized by the following features:
1. Have three unique restriction sites for the endonucleases Pvu 11, Kpn 1, Eco 147 1 (Stu 1).

 2. Have three restriction sites for the endonuclease Bam H1.

 3. They are distinguished by a deletion of 1.7 kilobases located between restriction sites for Kpn1 and Eco 147 1 endonucleases.

Information sources:
1. Hopvood DA Bibb MJ Chater KF et al. 1985. Genetic manipulations of Streptomyces. A laboratory manual.

 2. Birnboim H.C. Doly 1979. A rapid al Kaline extraction procedure for screening recombination plasmid DNA. Nucleic Acid. Rec. v. 7, p. 1513.4

Claims (1)

A method for producing plasmids from streptomycetes, which involves culturing them, lysing mycelium and precipitation of plasmid DNA, characterized in that Streptomyces chrysomallus VKM Ac-1355 strain and its white variant are used and plasmid pSCH _{2 is} isolated, respectively, with a mol.m. 13.4 kilobases with the restriction map shown in FIG. 1, and plasmid pSCH ₃ mol.m. 15.1 kilobases with the restriction map shown in FIG. 2.

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