SU1539205A1 - Recombinant plasmide dna d24 coding synthesis of restrictase and methylase, and strain of escherichia coli bacteria as producer of restrictase and methylase sso ii - Google Patents

Abstract

The invention relates to biotechnology, in particular to genetic engineering, and is a recombinant plasmid DNA that causes the synthesis of restrictase and methylase SSOII and the strain containing this recombinant DNA producing restrictase and methylase SSOII. The purpose of the invention is to increase the level of synthesis of restrictase and methylase SSII. The invention allows to obtain a strain with the production of restriction enzyme and methylase SSOII - 350000 and 400000 units. per 1 g of raw biomass, respectively. 1 tab.

Description

The invention relates to biotechnology, in particular to genetic engineering, and is a recombinant plasmid DNA that causes the synthesis of restrictase and methylase SsoII and a strain containing this recombinant DNA producing restrictase and methylase SsoII.

SsoII restriction enzyme and methylase recognize 5-membered degenerate CCNGG sequence, hydrolyze DNA from. forming 5-membered sticky ends, providing high ligation efficiency, and central cytosine is methylated in the CCNGG recognition sequence, forming the modified base 5-methylcytosine.

The aim of the invention is to increase the synthesis of restrictase and SsoII methylase.

The goal is achieved by plasmid d 24 and E. coli B834 strain containing this plasmid.

Plasmid d 24 restricting enzyme and methylase coding SsoII. consists of the following elements:

the plasmid DNA of the pUC19 vector with a size of 2.69 tpo;

P4 plasmid DNA from S. sormei47-, encoding restrictase and SsoII methylase, which is 4.4 tpo.

Strain producing restrictase and methylase SsoII obtained by transformation of E. coli B834 cells with plasmid d24.

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The content of SsoII restrictase in the designed strain is 350000 units / cell biomass, methylase 400000 units.

Example 1. Plasmid DNA from E. coli XS13 strain containing two plasmids; P4 and P9 are isolated by an alkaline method. The separation of the plasmid P4 DNA from the high molecular weight DNA P9 was carried out by DEAE-cellulose column chromatography.

The resulting preparation of plasmid P4 and the preparation of vector pUC19 are used to construct plasmid d24, which is carried out as follows.

0.5 μg of pUC19 vector DNA is incubated with the restriction enzyme SalGl (5 units) in buffer, A (100 mM NaCl, 50 mM Tris-HCl, pH 7.5; 10 mM MgCl2, 1 mM dithiothreitol). 5 μg of plasmid P4 are incubated with SalGl restriction enzyme (10 units) in buffer A. Reactions are carried out at 37 ° C for 1 hour. After incubation, the samples are heated at 65 ° C for 15 minutes. Analysis of the completeness of hydrolysis was carried out by electrophoresis.

The plasmid and vector DNA were combined in a buffer: 0.05 M Tris-HCl, pH 7.4, 0.01 M 1 mM dithiothreitol, O, 1 mM ATP with the addition of Phage T4 DNA ligase (1000 U / ml) from calculating 0.5 μl of enzyme per 5 μg of DNA for 12 hours at 10 ° C.

. The resulting mixture of combined frag ments is used in the transformation of E. coli PS200 cells. The transformation is carried out as follows. 50 µl of the E. coli PS200 cell suspension is added to 20 µl of liquid nutrient medium LB and grown at 37 ° C to a cell / ml titer. The cells are cooled in ice for 10 minutes and centrifuged at 5000g for 10-15 minutes at 4 ° C, then the supernatant is carefully discarded. the precipitate is suspended in 0.5 ml of 0.1 M CaCl1. The cells were kept in ice for 10-12 hours, after which they were used for transformation.

For 200 µl of suspended E. coli PS200 cells, take less than 20 µl of the connected DNA fragments and incubate for 40 min in ice, then for 90 s at 42 ° C and again for 2 min in ice, add 1.8 ml of LB broth and incubated for 1 h at 37 ° C. Then 2 ml of the suspension of transformed cells are centrifuged for 3 rpm

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Eppendorf reef at room temperature and resuspended in 100 μl LB with 0.01 M MgSO4. A 100 µl suspension of phage b 221 titer 2 is added to the sample and incubated for 20 minutes at 37 ° C. The cells are then plated on ampicillin plates at a concentration of 50 μg / ml.

Plasmid DNA, designated d 24 and containing the vector pUCl 9, carrying the VG gene, determining the resistance to ampicillin, and plasmid DNA P4, carrying the genome of the restrictase and methylase SsoII, are isolated from the cells of clones grown on ampicillin plates. . The isolated plasmid DNA d 24 is subjected to restriction analysis.

Example 2. Determining the presence of restrictase and methylase SsoII in E. coli strain PS200 containing plasmid d 24.

0.1 g of E. coli PS200 biomass containing plasmid d 24 is suspended in 170 µl of 0.01 mm MJNCli, incubated with shaking for 2 hours at 4 ° C, centrifuged at 10,000 g for 15 min, then 10 µl of the supernatant is added to the sample containing 1 µg of bacteriophage L in 30 µl of buffer A. The sample is incubated for 1 h at 37 ° C, and then analyzed by electrophoresis in 1.2% n-agarose gel. Restriction pattern in the case of strain-E. coli PS200 carrying plasmid d 24 is characterized by the restriction enzyme SsoII cl.

1 μg of plasmid DNA d 24, isolated from E. coli strain PS200, is treated with 10 units. retryptically endonuclease SSoII in buffer A for 2 hours at 37 ° C. Electrophoresis shows that plasmid DNA 24 is not fragmented during such processing, while plasmid pUC19 gives a picture of hydrolysis typical of plasmid pUC19 treated with restriction enzyme SsoII. These data indicate the protection of recognition sites for SsoII restriction enzymes in d 24 by methylation.

The determination of the efficiency of seeding bacteriophage L b 221 on cells of strains synthesizing restrictase and methylase SsoII is carried out as follows.,

The strain E. coli PS200 carrying plasmid d24, determining the synthesis of restrictase and methylase SsoII, reduces the efficiency of seeding bacteriophage

 by 5 orders of magnitude compared with the efficiency of seeding on the untransformed E. coli PS200 strain. After passage on E. coli PS200 strain cells transformed with plasmid d 24, phage b 221 was seeded on E. coli PS200 strain cells carrying plasmid d 24 and E. coli XS13 strain synthesizing SsoII methylase. At the same time, there was no decrease in the seeding efficiency, which indicates that DNA of phage LI 221 was modified in E. coli PS200 cells transformed with plasmid d24.

Example 3. The plasmid d 24 t transform in the strain E. coli B834 according to the method described in example 1, and

SsoII restrictase activity was determined in serial ten-fold dilutions of the supernatant, 1 µg of fa-a DNA, ha A in 30 µl of buffer A (see Example 1) was incubated with 2 µl of each dilution. The activity level of SsoII restrictase is 350000 units per 1 g of biomass.

The activity of methylae is determined in a reaction mixture with a total volume of 100 µl containing 1 µg of acceptor DNA, 0.5 μCi of 3H-S-adenosyl-methionine and 5 µl of each of the tenfold dilutions of the sulernatant. Incubation is carried out for various time periods from J to 24 hours at 37 ° C. Samples are applied to filters.

GF / C, precipitated with 5% trichloroacetus, a strain is obtained - producing restrictase with 20% acid, washed with 70% eta- and methylase SsoIInol, dried and calculated using Escherichiai. coli B834, a non-radioactivity in toluene scintille plasmid d 24 - producing retreator. For the unit of enzymatic rictase and methylase SsoII, the characteristic activity is taken by the amount fermented by the following traits. 25 cops, providing a complete modicultural morphological. The cell line is 1 µg of acceptor DNA, direct, rod-shaped, immobile, 24 h.

gram-negative, lactose-positive. The level of activity of methylase, defined when seeding on plates with 1.3% MPA divided in E. coli strain B834, the lack of growth in the form of individual colonies, which contributes to plasmid d24, corresponds to

400,000 units per 1 g of biomass.

The table shows the levels of synthesis of restrictase and SsoII methylase in E. coli strains obtained by transformation of plasmid 35 d 35.

The invention makes it possible to obtain a freak in R-form with jagged edges. Ho-jpomo grows on solid and liquid nutrient media (MPA, BCH, LB-bulon and LB-arap).

Physiological and biochemical. Cells grow in the range of 4-45 C at an optimum pH of 6.8-7.5. The strain decomposes glucose, lactose, mannitol with the formation of acid, does not decompose sucrose, ferments maltose, xylose, sorbitol, dulcite, rhamnose, forms indole and hydrogen sulfide, exhibits resistance to ampicillin (up to 100 µg / ml), due to the presence of plasmid d 24 .

Example 4. The culture of E. coli B834 cells bearing pazmidd 2b is grown in 1 l of LB medium with 50 μg / ml ampicillin at 37 ° C to a titer of 4 10 cells / ml. Cells are pelleted by centrifugation (5000 g, 15 min).

1 g of raw biomass is suspended in 5 ml of buffer containing 50 mM Tris-HCl, pH 7.6; 0.1 M NaCl-, 7 mM 2-mercaptoethanol, 100 μg / ml lysozyme and incubated at 0 ° C for 10 minutes. The cells are destroyed by ultrasound. The extract is obtained by centrifugation at 2 ° C. (48,000) for 1 hour.

The number of synthesis of restrictase and methylase SsoII in the strain containing plasmid d24 is at least 350000 and 400000 units. on 4Q 1 g of biomass, respectively.

Claims (2)

1. Recombinant plasmid DNA 45 d24 encoding the synthesis of restrictase and methylase SsoII mol.m. 4.5 EDD and size 7.1 tpo, containing: plasmid DNA of pUC19 vector, 2.7 tpo; to plasmid DNA P4 with restriction enzyme gene and SsoII methylase of 4.4tp one site of cleavage with endonuclease Bglll, Clal, EcoRV, SacII, PstI, SphI, Findlll, SaGI, Kpnl, Smal, 55 BamHI, Xbalj two sites of recognition of endonucleosis, CfrlOl, Said; four EcoRI cleavage sites; bla gene encoding / g-lactamase; SsoII restrictase activity was determined in serial ten-fold dilutions of the supernatant, 1 µg of fa-a DNA, ha A in 30 µl of buffer A (see Example 1) was incubated with 2 µl of each dilution. The activity level of SsoII restrictase is 350000 units per 1 g of biomass. Methylae activity is determined in a reaction mixture with a total volume of 100 µl containing 1 µg of acceptor DNA, 0.5 McCi of 3H-S-adenosyl-methionine and 5 µl of each of the tenfold dilutions of the sulernatant. Incubation is carried out for various time periods from J to 24 hours at 37 ° C. Samples are applied to filters. The number of synthesis of restrictase and methylase SsoII in the strain containing plasmid d24 is at least 350000 and 400000 units. per 1 g of biomass, respectively. Invention Formula 1. Recombinant plasmid DNA d24, which encodes the synthesis of restrictase and methylase SsoII mol.m. 4.5 EDD and size 7.1 tpo, containing: plasmid DNA of pUC19 vector, 2.7 tpo; P4 plasmid DNA with restriction enzyme gene SsoII 4,4tpo one site of cleavage with endonuclease Bglll, Clal, EcoRV, SacII, PstI, SphI, Findlll, SaGI, Kpnl, Smal, BamHI, Xbalj two sites of recognition of endonucleosis, CfrlOl, Said; four EcoRI cleavage sites; bla gene encoding / g-lactamase; a lac gene fragment linked to a synthetic polylinker located on either side of the cloned DNA of the plasmid PA; t genes restrictase and methylase SsoII, located on the cloned DNA P4 plasmids flanked by SalGl sites, which determine the synthesis of restrictase and methylase SsoII. 2. Bacterial strain Fscherichia coll HYSK-178 - producing restrictase and methylase SsoII.