DD204796A1 - PROCESS FOR THE PREPARATION OF VECTOR PLASMIDES FOR E. COLI - Google Patents

Abstract

The invention relates to a method for obtaining new vector plasmids for E. coli and pursues the goal of providing biochemical tools for genetic engineering and molecular biology research. The task of describing a suitable method is solved in such a way that by means of in vitro recombination of the plasmid pBR322 with DNA of Streptomyces phage SH10 bzw.mit isolated fragments of SH10-DNA, by transformation of E. coli HB101, using Selection and propagation of the recombinant clones and subsequent isolation by density gradient centrifugation new vector plasmids can be obtained. In view of the hereby introduced unic cleft sites for Kpn I and Bgl II, these plasmids represent a new quality among the vectors for E. coli.

Description

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Title of the invention

Process for the preparation of vector plasmids for E. coli

Field of application of the invention

The invention relates to a method for the production of vector plasmids with novel unique restriction site cleavage sites, which are advantageous for the introduction of foreign DNA into E. coli. The invention offers applications in genetic engineering as well as other fields of DNA research and downstream in medicine, plant and animal production, the pharmaceutical industry.

Characteristic of the known technical solutions

It is known that DNA of various origins can be transmitted by in vitro manipulation in microorganisms. The vectors (plasmids, phages) which are absolutely necessary for this transfer, which must possess specific properties such as stable replication, unique restriction-site cleavage sites, easy isolation, selective markers, etc., are prepared by various methods, depending on the host vector vector to be generated. System prepared (I ¹ R 78 17 221, GB 79 19 245 and GB 79 16 377).

An example of a method for obtaining a vector for E. coli is the preparation of plasmid pBR322 (Bolivar et al., 1977, Gene, 2, 95-113). The plasmids pMB9 and pSF2124- are recombined by biochemical manipulations and structurally modified to a plasmid with antibiotics double resistance (ampicillin / tetracycline) and several unikalen cleavage sites for restrictases (BamH I ₁ EcoR I, Sal I, Pst I, Hind III) to obtain , The isolation of the plasmid is carried out according to known.

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Method (Clewell, 1972, J. Bacteriol 110, 667-676). The disadvantage of the said production process is that it leads to a plasmid which has no unique cleavage sites for Bgl II and Kpn I.

Further, laboratory protocols are known which serve to prepare other vectors (Bibb et al., 1980, Nature, 284 _f 526-531, Suarez et al., 1980, Nature, 286, ₁ 527-529).

Object of the invention

The aim of the invention is the preparation of new vector plasmids with unique Bgl II or. Kpn I cleavage sites to avoid the listed deficiency of previous methods to expand the range of vectors useful for genetic engineering by three representatives, in particular to use the plasmids for in vitro manipulation of E. coli and in DNA research to be able to.

Explanation of the essence of the invention

The invention has for its object to describe a biochemical method with which new vector plasmids for E. coli can be obtained.

According to the invention the object is achieved in that the new plasmids p1, p22 and p342 are synthesized by in vitro recombination of the plasmid pBR322 with DNA fragments of Streptomyces phage SH10, which propagated after transfer to E. coli HB101 and recovered by CsCl density gradient centrifugation become. The Streptomyces phage SH10 was isolated in ZIMET and assayed for its properties by microbiological and biochemical methods (ELaus et al., 1979, Mol. Gen. Genet., 172, 319-327, ELaus et al., 1981, Journal of General Microbiology, 1.23 269 "to 279;. Walter et al, 1981, Gene, I ^ 57 to 63) In detail, the process for obtaining the E. coli vectors p1, p22 and P342 characterized in that first the plasmid. pBR322 with BamH I and parallel DNA of the

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Phage SH10 with Bgl II into the 11 fragments Bgl-A, Bgl-B, Bgl-C, Bgl-D, Bgl-E, Bgl-F, Bgl-G, Bgl-H, Bgl-I, Bgl-J, Bgl -K be split. Subsequently, the fragments Bgl-E, Bgl-G, Bgl-K are selected from the SH10 cleavage mixture in a manner known per se. After combining the cleavage products, d. H. of the BamH I-digested pBR322 with the 3 selected SHIO fragments, is incubated with T ^ -egase. The ligation products (recombinants) are then used to transform CaC 1-pretreated cells of E. coli HB1O1.

After culturing the transformed cells on ampicillin

H agar agar is replica-based on the phenotype Amp Tet

τ? ^ checked. Of the recombinant plasmids isolated from Amp Tet clones, those possessing the new vector quality have as insert the fragment SHIO-Bgl-E (ηρη-site) or a combination of either the fragments ShiO-Bgl-E + Bgl-G or from the fragments SHIO-BgI-E + BgI-K (Kpn and Bgl site). The strains are stored in 50% glycerol at -20 ° C. The isolation of the vectors on a preparative scale in a conventional manner after culturing the plasmid strains in liquid cultures in the presence of (chloramphenicol with subsequent cell disruption and density gradient centrifugation in CsCl.

Biochemical properties of p1, p22 and p342

The plasmids p1, p22 and p342 have a molecular weight of 3.97 Md, 4.25 Md and 3 _f 8 Md, respectively. In the right orientation (clockwise) of the EcoR I cleavage site are the distances of the BgI II or Kpn I- Splitting locations as follows:

pi: Bgl II 515 bpi Kpn I 16i5bp

'p22' :: Bgl II '1O4-5bp, Kpn Γ 1465bp

p342 r Kpn T W5bp ·

In addition, the plasmids have unique cleavage sites for Pst X, EcoR I, Hind III.

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The plasmid-carrying strains E. coli HB101 (pi), E. coli HB101 (p22) and E. coli HB1O1 (p342) are in the St aminsmation of the Central Institute of Microbiology and Experimental Therapy (ZIMET) under the numbers ZIMET 10749, ZIMET 10750 , ZIMET 10 ^ 511 · has been deposited.

embodiments

1. First, 60 / ng of SH10 DNA in a volume of 100 μl (100mM Tris-HCl, pH 7.4, 10mM ₂ -mercaptoethanol, 10mM MgCl ₂ ) - containing 10 units of Bgl II and parallel 1, ug pBR 322 in a volume of 25, ul TM with 1 unit BamH I for 2 hours at 37 ⁰ C incubated. The reaction is stopped by heating to 65 ⁰ C for 10 minutes. Thereafter, the SHIO-split mixture is separated in an i, 2% horizontal agarose gel for 16 hours at a voltage of 2V / cm. After staining with ethidium bromide (1 μg / ml H 2 O), the gel is irradiated with UV light (254 nm) and the bands corresponding to the fragments Bgl-E ₁ Bgl-G and Bgl-K are excised , The DNA is isolated from these samples by freezing (Thuring et al., 1975 * Anal. Biochem., 66, 313-220), extracted twice each time with xsoamyl alcohol or ether, and extracted for 6 hours against TE buffer (10 mM Tris-HCl, pH 7 »5, 1 mM EDTA, 10 mM NaCl). The ligation of the fragments with pBR322 (BamH J cleaved) is carried out in batches of 50 / al ligase buffer (60 mM Tris-HCl, pH 8.0, 100, uM ATP, 10 mM dithiothreitol)

a) 0.3 PBR322 b) 0.3 / Ug pBR322 c) 0.3 / Ug pBR322 O ₁ 2 / Ug Bgl-E 0.2 / Ug Bgl-E 0.2 / Ug Bgl-E

0.1 / Ug BgI-G 0.05 / Ug BgI-K

in the presence of 1 unit T ^ ligase for 2 hours at 12 ⁰ C. After heating the samples to 65 ⁰ C for 5 minutes, 10 / ul 5x concentrated TM buffer and 1 unit of BamHI is added and 30 minutes at 37 ⁰ C. incubated. Following heating to 65 ⁰ G for 5 minutes 3 hours against TE buffer is dialyzed and transformed with these samples 0aCl ₂ -treated cells of E. coli HB101. The transformation

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Mix on Nutrient broth agar (NB agar, Composition 8 g NB and 15 g agar / 1,) with 30 / ug ampicillin / ml and spiked 22 hours at 37 ⁰ C incubated. Replicas of 500 Amp ^R colonies each are applied to NB agar with ampicillin (30 μg / ml) or tetracycline (20 μg / ml) and incubated again at 37 ^° C. for 22 hours. Ampere colonies are cultured in 5 ml of liquid cultures (10 g bacto-tryptone, 5 g yeast extract and 5 g NaCl per liter) with 30, ug / ml ampicillin for 20 hours at 37 ⁰ C and the plasmids in microscale ( Birnboim et al. 1979, Nucleic Acids Res., 2, 1513-1523). By separating the plasmids in 0.9% agarose gels, those plasmids having a molecular weight greater than that of the vector plasmid pBR322 (2.78 Md) are selected and tested for the presence of Bgl II or Kpn I cleavage sites. All recombinant plasmids with one Kpn H or one Kpn U and Bgl II cleavage site located in the positions listed under "Biochemical Properties" correspond to the vectors p1, p22 and p342.

2. The procedure is as in Example 1, but using the entire SH10 cleavage mixture for ligation and selected for plasmids having a molecular weight of up to 4.25 Md, which are then tested for Bgl II or Kpn I cleavage sites.

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Claims (2)

_ β m. 238975 1 Erfinäungsanspruch. 1. A process for the preparation of Vektorplasraiden for E, coli _s characterized in that is recombined by genetic engineering-biochemical standard operations plasmid pBR322 with fragments BgI-E, BgI-G and BgI-K of Streptomyces phage SH1O, with these recombinants CaCl2-pretreated Cells are transformed from E. coli HB1O1, by selection of these transformed and on ampicillin-containing agar cultured E. coli cells and by characterization with restrictive endonucleases, the plasmids p1, p22 and p342 are generated, which have unique sites for BgI II and Kpn I cleavage sites can be obtained on a preparative scale by purification in the CsOl density gradient « 2. The method according to item 1, characterized in that the SH10-Eragmente BgI-E, BgI-G and BgI-K are obtained by treatment of this phage with the restrictase Bgl II and subsequent separation of the cleavage mixture in the horizontal agarose gel ·