SU1453896A1 - Recombinant plasmide dna puk11 coding restrictase and methylase ecor 11, and strain of escerichia coli bacteria as their producer - Google Patents

Abstract

The invention relates to microbiology, in particular, to the technology of recombinant DNA, and makes it possible to obtain restriction enzyme and Eco RII methylase enzymes used in scientific research practice. The aim of the invention is to increase the content of restrictase and Etio RII methipase in bacterial cells, To this end; recombinant pVK I DNA was constructed, containing a DNA fragment with full restriction enzyme genes and Eco RII metipases and the left promoter of phage lambda, ensuring efficient expression of Eco RII genes. pVKlI transformed into Escherichia Eco RII cells of at least 50,000 units. each enzyme per I g of raw cell biomass. 3 sec. f-ly. (L

Description

I.

The invention relates to biotechnology. login, in particular to genetic engineering, and is a pejcoM-binant plasmid DNA, which causes the synthesis of restrictase and methylase Eco RII, the content of this recombinant plasmid DNA and. the strain containing this recombinant plasmid producing restrictase and

methylase eso rii ...

one . ,

Restriction of eDonuclease (restrictase) of Eco RII and DNA metiptransfe-. times (methylase) Eco RII is widely used in research and development (genetic engineering, tactical JNA functional analysis of DNA

study of protein-nucleLein interaction, etc.).

The purpose of the invention is to increase the content of restriction enzyme and EcoLAII methylase in bacterial cells.

1. A recombinant plasmid pVKll was obtained, providing an increased synthesis of restrictase and Eco RI1 methylase. 2. A method has been developed for constructing a recombinant plasmid pVKI1, in which the restriction enzyme and methlase genes of EcoRII are under the left promoter of phage lmbda (P), this ensures the efficient expression of the restriction enzyme and methylase of EcoRII.

3. The obtained Escherichig coli strain VKM CR-288D, which has a higher one.

4ib SL

O9 00 05

    ;, .1453896

the level of synthesis of restrictae and methylase Eco RII compared with the known strains of the producers of enzymes Eco RII.,

, A, Plasmid pVKII, the core of re-. strictase and metipase Eco RII consists of a DIC pLC 2833 (the size of which is 2.8 TPO) containing the left proCellus, straight, rod-shaped (1.2-1, b) x (2.0-6.0) µm, motile, with peritrichialni flagella, gram-negative, nesporonosnye.

Cultural features.

Kpetkn well grow on simple nutrient media ..,

With growth on m with peptoin aga15

20

25

thirty

Motor of phage lmbda (Pb); Pet I - fragment of Fe, nutrient agar Difcr of DNA DNA of plasmid pVK 33 containing the restriction enzyme genes of the Eco RII methylase Xrze | 1р Kotrrohp 5 TPO), and has. unique restriction sites for char. the Eco nucleotide RI, Sal I, Xba I, located at a distance of 0.1 TPO from the P / promoter; selective markers Ar 1shp J host in Escherichia coli.

B. To achieve this, the objectives use a method for constructing a plasmid barcode that encodes a restriction enzyme, and Eco RII methylase, consisting in that plasmid DNA pLG 2833 and DNA pVK Ze are subject to joint hydrolysis with an endonuclease of Pst I restriction. The resulting fragments combine DNA digase, then, a mixture of - | recombinant molecules is transformed by E. coli cells (L) with a recombinant molecule. Transformance was plated on α-cympillin medium and the grown colonies were tested for their ability to restrict and modify lambda phage in vivo. Plasmid DNA designated as pVK M was selected from the selected transformants.

B. - To achieve the goal, Echerichia coli bacterial strain VKM CR-288D was also produced, which produces restrictase and Eeo.EII metipase. The strain was deposited in the All-Union Collection of Microorisms in the Institute of Biomedical Physics, USSR Academy of Sciences under the number V1da CR-288D,

 Stagf - producer of restriction enzyme and yeast E – CO RII was obtained by transforming E. coli B834 / pC1857 cells with pVK 11 pazmid DNA. Selection of strain E, coli B834 (). due to the fact that) the strain does not contain enterichera-tion (impurities with Eco RII enzymes and | co; 5 P plasmids; with the heat-sensitive repressor cI gene of the phage anyway. The restriction enzyme and methylase Eco RII content in the designed strain is 500,000 units. enzyme per 1 g of raw biomass of cells.

The Escherlchia coli strain VKM CR - 288D is characterized by the following features:

1 &) rheologic features.

The scientific research institutes are smooth, round, pressed, shiny, gray, with even edges. With the growth in liquid media - mi-pep ton broth, Difco's nutrient broth forms a smooth intense slime. .I

Physiological and biochemical signs.

Cells grow within A - at an optimum pH of 6.8-7.5. Many carbohydrates, alcohols, organic acids, in particular d-glucose, d-fructose, arabinose, and lactose, trehalose, are used as carbon sources. Does not absorb acetate, adanite, galactose. As a source of nitrogen, mineral salts are used in ammonium and nitrate forms, and in organic form as peptone, amino acids. Nitrates are reduced to nitrites. Gelatin is not diluted. Indole do not form. Urease activity is not detected.

Antibiotic resistance.

They show resistance to ampicillin due to the presence of plasmas, such as pVK P, as well as to kanamycin, due to the presence of the pC 1857 plasmid.

The Y. coli VKM CR-288D strain is immune to the lambda phage. The productivity of the strain is 500,000 units. each enzyme per 1 g of raw biomass.

The invention is illustrated by the following examples.

Example..

The E. coli bacteria cells containing plasmid DNA are grown in Difco broth to a titer of 2x10 cells / ml. The cells are collected by centrifugation and DNA is extracted from them. The obtained DNA preparations of the plasmid pLC 2833 and the plasmid pVK 33 are used to construct the plasmid pVK 11. 2 μg of the DNA of the plasmid pLC 2833 are incubated with the Pst I restriction enzyme (0.5 e). The reaction was carried out at 1 h. 3 µg of plasma DNA pVK 33 was cleaved with the restriction endonuclease Pst I (5 e).

40

45

50

55

Cells, straight, rod-shaped (1.2–1, b) x (2.0–6.0) µm, motile, with peritrichous flagella, gram-negative, non-spore-bearing.

Cultural features.

Kpetkn well grow on simple nutrient media ..,

With growth on m-with-peptoin agar, nourishing agar

five

0

five

0

Re, nutrient agar difkr colo

The scientific research institutes are smooth, round, pressed, shiny, gray, with even edges. When grown in liquid media — m-co-peptone broth, Difco's nutrient broth forms a smooth intense slime. .I

Physiological and biochemical signs.

Cells grow within A - at an optimum pH of 6.8-7.5. Many carbohydrates, alcohols, organic acids, in particular d-glucose, d-fructose, arabinose, and lactose, trehalose, are used as carbon sources. Does not absorb acetate, adanite, galactose. As a source of nitrogen, mineral salts are used in ammonium and nitrate forms, and in organic form as peptone, amino acids. Nitrates are reduced to nitrites. Gelatin is not diluted. . Indole do not form. Urease activity is not detected.

Antibiotic resistance.

They show resistance to ampicillin due to the presence of plasmas, such as pVK P, as well as to kanamycin, due to the presence of the pC 1857 plasmid.

The Y. coli VKM CR-288D strain is immune to the lambda phage. The productivity of the strain is 500,000 units. each enzyme per 1 g. raw biomass.

The invention is illustrated by the following examples.

Example..

Cells of E. coli bacteria containing plasmid DNA are grown in Difco broth to a titer of 2x10 cells / ml. Cells are collected by centrifugation and DNA is extracted from them. The obtained DNA preparations of the plasmid pLC 2833 and the plasmid pVK 33 are used to construct the plasmid pVK 11. 2 μg of the DNA of the plasmid pLC 2833 are incubated with the Pst I restriction enzyme (0.5 e). The reaction was carried out at 1 h. 3 µg of plasma DNA pVK 33 was cleaved with the restriction endonuclease Pst I (5 e).

0

five

0

five

514538966

Samples after incubation Warm up the priyuyu as pVK Pi containing about in its

15

65 With 10 missions. Hydrolysis products are analyzed by electrophoresis in l - HOM agaroe gel.

The connection of plasmid DNA fragments is carried out in a restriction buffer with the addition of ATP and dithiotraitol to the final end of M and 5 mM, respectively, and dac lysis (10 e).

The reaction is carried out for 10-12 hours at IA -. The resulting mixture of plasmids is used to transform E, coli C600 (L) cells. E. coli C600 cells () are added to 10 ml of nutrient broth and grown to a titer of 1x x 10 clTml. The cells are harvested by centrifugation (5000 g, 10 min), suspended in 10 ml of 0.2 M CaC solution and held for 1 hour at 0 ° C. The cells are re-harvested by centrifugation, resuspended in 0.3 ml of 0.08 M CaCl2 () and used for transformation.

The mixture of ligated DNA fragments was incubated with E. coli B83A / pC1857 cells treated with CaCl solution for 1 hour at room temperature (18–20 ° C) for 10 minutes. After ten-fold dilution of the suspension with transforming nutrient broth, 30 cells are grown for 2 h at 28 ° C and plated on agar medium with ampicillin (50 µg / ml). Cells

The composition consists of three fragments that are formed by hydrolysis of gchasmid DNA with the restriction enzyme Pet I.

The isolated pVK 11 DNA is transformed into E. coli B83A / pC1857 “Transformants are selected on medium containing ampicillin (50) -and 10 kanamycin (25 μg / ml). Plasmid

pC1857 carries a thermo-sensitive mutation in the CI repressor gene. Thus, transcription can be increased due to temperature conditions of cultivation.

E. coli B834 / pC1857 / p cells are grown at 28 ° C to a density of 2-4-10 cells / ml, the temperature is dramatically increased and incubated for 4 hours. Cells are harvested by centrifugation, resuspended in a buffer of 0.01 M potassium phosphate (pH 7.0), 1 mK EDTA, 7 mM 2-mercaptoztanal; 0.4 mM Had, sonicate. After the destruction centrifuged (100,000 E. 1 h,). Cell-free extract is used to determine enzyme activity. Methylase activation was determined in a reaction mixture of total volumes of 150 µl containing 40 mM potassium phosphate (pH 7.8), 1 mM EDTA, 7 mM 2-mercaptoethanol, 20 µg DIC K. coli B834, 4 mM S-ada nosyl (methyl- N) methionine and 50 μl

20

25

E. coli B834 / pC1857 / p cells are grown at 28 ° C to a density of 2-4-10 cells / ml, the temperature is sharply increased and incubation is carried out for 4 hours. Cells are harvested by centrifugation, resuspended in a buffer of 0.01 M potassium phosphate (pH 7.0), 1 mK EDTA, 7 mM 2-mercaptoztanal; 0.4 mM Had, sonicate. After the destruction centrifuged (100,000 E. 1 h,). Cell-free extract is used to determine enzyme activity. Methylase reactivity is determined in a reaction mixture of total volumes of 150 µl containing 40 mM potassium phosphate (pH 7.8), 1 mM EDTA, 7 mM 2-mercaptoethanol, 20 µg DIK K. coli B834, 4 mM S-ada nosyl (methyl- N) methionine and 50 μl

grown up on a medium with antibiotics,

checked for restriction and modification of the enzyme at various dilutions.

phage L by the Eco.RII system. The reaction was carried out for 1 h at 37 ° С, and the restriction phase of phage D vir was added, and an equal volume of 0.75 N sodium was obtained

grown on E. coli strain SbOO, op-alkali, incubated for 30 min at 60 C.

alkali.

It is determined by comparing the phage titer when it is sown on E. coli CABOO (A) and E. coli C600 / pVK33 strains and clones containing recombinant plasmids. When phage are plated onto clones containing recombinant plasmids carrying the restriction enzyme genes and Eco RII methylases, they limit the status of the phage compared to the strain CABOO (A) by two orders of magnitude, as on the C600 / pVK33 strain. Feigures grown on these clones containing recombinant DNA are sown on the COOS () and COOP / PPCR strains. Phages grown on clones containing recombinant plasmids, aecymjie genes of Eco RII, give the same titer on both SbOO (D) and C600 / PVK33 strains.

 Of the clones that provide for the restriction and modification of the phage Lvir, plasmid DNA is isolated,

five

0

The composition consists of three fragments that are formed by hydrolysis of gchasmid DNA with the restriction enzyme Pet I.

The isolated pVK 11 DNA is transformed into E. coli B83A / pC1857 “Transformants are selected on medium containing ampicillin (50) -and kanamycin (25 μg / ml). Plasmid

pC1857 carries a thermo-sensitive mutation in the CI repressor gene. In this way, transcription can be increased due to temperature conditions of cultivation.

E. coli B834 / pC1857 / p cells are grown at 28 ° C to a density of 2-4-10 cells / ml, the temperature is sharply increased and incubation is carried out for 4 hours. Cells are harvested by centrifugation, resuspended in a buffer of 0.01 M potassium phosphate (pH 7.0), 1 mK EDTA, 7 mM 2-mercaptoztanal; 0.4 mM Had, sonicate. After the destruction centrifuged (100,000 E. 1 h,). Cell-free extract is used to determine enzyme activity. Methylase reactivity is determined in a reaction mixture of total volumes of 150 µl containing 40 mM potassium phosphate (pH 7.8), 1 mM EDTA, 7 mM 2-mercaptoethanol, 20 µg DIK K. coli B834, 4 mM S-ada nosyl (methyl- N) methionine and 50 μl

0

five

alkali, incubated for 30 min at 60 C.

alkali.

Then 2 ml of water and 2 ml of 10% trichloroacetic acid are added to the samples. The precipitated DNA is applied to glass fiber filters, pressed with 0.01 M HC1 and alcohol. After drying the filters consider the radioactivity. For one; Reduced activity of Eco methylase RII. - take the amount of enzyme, which in these conditions includes 1 pcM CHj - groups in DNA for 1 h at. The Eco RII restrictase activity is determined in a mixture of 20 mM Tris-HCl (pH 7.5), 10 mM MgCli, 50 mM NaCl, 1 µg of plasmid DNA pBR 322 (from E. coli B834 / pBR322) and various dilutions of the enzyme NIL. The reaction is 15 minutes. at 37 ° C. The products of DNA hydrolysis are analyzed on a 1% agar gel. For a unit of activity, take the amount of the enzyme restrictase Eco RII, which is necessary for the full

, 7 U53896. , eight

clinging. I μg of pBR 322 DNA for 15 minutes of replication of plasmid pBR 3221 ce, - .. A «« .1- .. - l

at. . Invention, no9Bcuuet get stash - producing pectpuRTadbi and me-, Tipaz Eco. RII with synthea level in these e {cops is not less than 500000 units. each per 1 g of raw biomass cell), that 10 times I will drink levels of synthesis of these enzymes in comparison with the best of fo. known strains t

Lectic Markers; itran; host in - Escherichia eelI.

2, The method for constructing plasmid DNA pVK 11 that encodes a restriction enzyme and Eco RII methylase, consisting in the DNA of the plasmid pLC 2833 and the DNA of the plasmid pVK 33, is subjected to hydrolysis with the restriction endonuclease Pst 1, the resulting Fragments are mixed and co- -. are combined with DNA ligase, then a mixture of recombinant cells is transformed

Ф о р му а N and 3 о. b th e

1, the H-recombinant plasmid DNA (BC II, encoding restriction enzyme and Eco RII methylase, 7.8 tons in size, b.p., containing the DNA of the pLC 2833 vector, 2.8 kb in size, with the left promoter

lambda (P) Pst G phages - DNA fragment of plasmid pVK 33 with restriction gene and Eco RII metlases 5.0 t, p. {unique restriction sites for Eco RI endnucleases, XBa I, Sal 1, section 6b : married on RDS

.standards O, .4 t, p.o from promoter Hz;

og1 plasmid replication pBR 3221 ce 1- .. - l

Lectic Markers; itran; host in - Escherichia eelI.

2, A method for constructing plasmid DNA pVK 11 that encodes a restriction enzyme and Eco RII methylase, consisting in the DNA of the plasmid pLC 2833 and the DNA of the plasmid pVK 33 are subjected to hydrolysis with the restriction endonuclease Pst 1, the resulting Fragments are mixed and combined DNA-ligase, then a mixture of recombinant cells is transformed

1 year E. coli COOL (L), transformants plated On Wednesday with amyitzshina and the grown clones are tested for their ability to restrict and modify lambda phage from Clones, which have restricted and modified lambda phage with specificity of the Eco RII system, plasmid DNA pVK 11.,

3i The strain of the bacteria Escherichia so11 VKM CR-288D - producing restrictase and methylase Eco Rli,

Claims (3)

1, Recombinant plasmid DNA i> VK 11, coding for ECO RII restriction enzyme and methylase 7.8 kb, 'containing pLC 2833 vector DNA, 2.8 kb, o, with left promoter phage lambda (P $ jj Pst I is a DNA fragment of plasmid pVK 33 with the restriction gene Tase and methylase Eco RII 5.0 kbp {unique restriction sites for endonuclease Eco RT, Xba I, Sal I, located at a distance of 0 , 4 kb from the Pl} ⁸ ori promoter of the replication plasmid pBR 322J cElective Markers: Ap ² j limn the host is Escherichia eoll. 2. The method of constructing plasmid DNA pVK 11 encoding re'striktase and methylase Eco RII, which consists in the fact that the DNA of plasmid pLC 2833 and / 10 DNA of plasmid pVK 33 is digested with restriction endonuclease Pst I,. the resulting Fragments are mixed and with ·. unite with DNA ligase, then 15 E. coli cells of SbOO (A *) are transformed with a mixture of recombinant MBLEcules, transformants are seeded onto ampicillin medium and grown clones are tested for the ability to restrict and modify phage lambda ir clones that restrict and modify lambda phage with specificity Eco RII systems secrete plasmid DNA pVK 11. ' ¹ ' 3. The bacterial strain Escherichia coli VKM CR-288D producer of restriction enzyme. And methylase Eco RII,