DD273286A1 - METHOD OF PREPARING POLYLINKER-CARRYING STREPTOCOCCUS-ESCHERICHIA COLI-MULTICOPY-SHUTTLE-PLASMIDE MINIMIZED MOLECULAR SIZE - Google Patents

Abstract

The invention relates to a process for the preparation of novel Streptococcus Escherichia coli multicopy shuttle plasmids of minimized molecular size with polylinker. It aims to provide new universal vector plasmids for the purposes of genetic engineering. The object assigned to this aim is achieved by first opening a polylinker-carrying streptococcal plasmid of the pLKM series in its polylinker and breaking the E. coli phasmid pMc, a polylinker-carrying vector, into two fragments, after which these fragments are in gives a ligation mixture and with this transforms an E. coli recipient strain and then selects among the transformant clones deletion plasmids of the types pLM2 and pLM3, which are characterized by a small molecule size, by the possession of a polylinker and after backtransformation into Streptococcus sanguis recipients by the Characteristics of a multicopy plasmid.

Description

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Field of application of the invention

The invention relates to a method for the production of novel Streptococcus-Escherlchia coli multicopy shuttle plasmids of minimized molecular size, which carry a polylinker and can advantageously be used for the cloning of foreign genes in Streptococcus and Escherlchia coil.

The invention offers applications in genetic engineering and other fields of molecular biology and downstream especially in the pharmaceutical biotechnology industry.

Characteristic of the known state of the art

Known are a number of Streptococcus Escherichia coll-shuttle vectors constructed on the basis of the known streptococcal vector pSM6 and various E. coli vectors, e.g. pSM618 or pSM6322 (published in FERRETTI & CURTISS [Eds.] 1987, Streptococcal Genetics, American Society for Microbiology, Washington, and LAPLACE et al., 1988, J. Basic Microbiology). These plasmids are distinguished

by a molecular size of at least 8.4 kb,

- by unique cleavage sites for a number of restrictases (eg EcoRI, BamHI, Hindill, Sail, Xbal) as well as'

- by the presence of at least two resistance markers (eg for erythromycin-lincomycin resistance and ampicillin resistance).

The disadvantage of their use as plasmid vectors in genetic engineering constructions their relatively large molecular weight and low copy number to book.

Aim of the invention

The invention pursues the goal of providing novel Streptococcui-Eicherlchla coll-multlcopy ehuttle plasmids of minimized molecular size and polylinker for the purposes of genetic engineering.

Explanation of the essence of the invention

The invention has for its object to describe a gentachnisches-mikrobiologlsches process, with the help of successively executed complexes of genetic engineering process steps new Streptococcus E. coll-multicopyshuUle-Plasmlde minimized molecular size can be obtained with polylinker. According to the invention, this object is achieved in its basic feature as follows: Using standard genetic engineering process steps is first

- On the one hand a Streptococcut cloning vector of the pLKM serotype, which in addition to the erythromycin resistance as PhBnotyp marker carries a polylinker with 10 Elngutären restriction sites in itself, by treatment with the Restrngerae Pstl llnearisiert and

on the other hand, the Escherlchla coll plasmid pMc known from the literature (molecular size 3.8 kb, chloramphenicol resistance, see also P.STANSSENS et al., Gene 36, 1985, 211-223), which has a polylinker with 7 slngulo restriction sites, namely for the Restriktasen EcoRI, SmaI, BamHI, Sail, Hindill and Xbal, In itself carries, also by treatment with the Restrktase Pstl into 2 fragments (the part sizes 2,0kb and 1,8kb) decomposed.

Selected Polyllnker-bearing Streptococcus pLKM cloning vectors are now in use

a recent invention of the Central Institute.

The thus obtained 3 PstI-PstI DNA fragments are placed in a Llgationsansaiz, and with this approach

a population of erythromycin-sensitive Escherichia coli reclp strain is transformed. Among the

Erythromycin resistant Escherlcla coli clones recovered as a result of this transformation procedure

then select those which carry recombinant Polyllnker-carrying plasmids of the pLM family of size less than 7.7 kb.

After the plasmid DNA has been isolated from these clones, the plasmid DNA thus obtained is finally inserted Erythromycin-sensitive Streptococcus recipient strain transformed. Among those obtained as a result of this procedure Erythromycin-resistant Streptococcus transformant clonon would still be selected for their pLM plasmids

have the status of a multicopy plasmid, and from the clones of this characterization, the plasmid DNA is isolated in a conventional manner. ,

The basic feature of the method will now be further developed to the effect that in the input step of the method of Streptococcus cloning vector pLKM6181 (Moie 6.III kb, erythromycin resistance) is used. This vector

carries a polylinker, which has slangular cleavage sites among others for the restrictases Haelll, Sphl, PstI, Sail, XbaI, BamHI, Xmal, SmaI and SstI.

A Streptococcus sanguls Challis strain transformed with dam vector pLKM is in the depository for Microorganisms of the GDR, Central Institute of Microbiology and Experimental Therapy of the Academy of Sciences, Beutenbergstr. 11, Jena, GDR - 6900 have been deposited under the registration number IMET. At the beginning are required quantities

a) to pLKM 6181 plasmid DNA from a culture of S. sanguls challis 57 (pLKM6181) or

b) anpMc-phasmid-DNA single culture of E. C0IIJMIOI (pMc)

isolated, purified and dissolved in TrisHCl buffer in a manner known per se,

For transformation with the prepared according to the method Ligatlonsansatz, in addition to the Pstl-Pstl DNA fragments above Of 1.8kb and 2.0kb size as a third Pstl-Pstl DNA fragment of 5.9kb size, now becomes the E. coli recipient JM101 used. Subsequent to the transformation reaction, among the obtained erythromycin resistant E. coll JM101- Transformantenklonen a screening for plasmids of minimized molecular size, especially after those with a Molecule size vn less than 7.7 kb performed. These mini-plasmids then re-screen Polylinker-carrying deletion plasmids of type pLM2 (molecular size 4.1 kb) and of type pLM 3 (molecular size 4.6 kb)

performed. Both neoplasmids, both pLM 2 and pLiVO, have a polylinker that is particularly singuarian

Has cleavage sites at least for the Restriktacen Smal, BamHI, Xbal, Sail and Pstl. Clones of E. coli JM101 carrying the desired minimized plasmids of the pLM2 and pLM3 types are cultured,

and from these cultures, the respective Plasmld DNA is isolated in a conventional manner, purified and dissolved in TrisHCl buffer. With samples of this plasmid DNA, for the final step of the procedure, the recipient strain S. sanguls

Challis 6 (re) transformed and selected for erythromycin-resistant challis transformant clones. Out Cultures of these transformant strains are also isolated the plasmid DNA and treated with a collection

Restrictases, in particular with EcoRI, Hindi, Hindi, Avail, Pvull, Small, Sail, Xbal, BamHI, Pstl, Haelll, Hpall and Aval again

a) the molecular size determined as well

b) each detected the presence of a polylinker;

Furthermore, among the pLM shuttle plasmids obtained by this backtransformation, those having a multicopy status are selected.

As a result of these investigations, the following parameters were confirmed in each case for the Streptococcus-Escherlchia coli multicopy shuttle plasmids pLM2 and pLM 3 (see figure below):

Plasmid pLM2: - molecular size 4.1 kb;

The presence of a polylinkera with unique cleavage sites for the above-mentioned restrictases and EcoRI;

- presence of a marker for erythromycin resistance;

4 times increased copy number (26 copies per cell) compared to the starting plasmid pLKM 6181;

PlasmldpLM3: - molecular size 4.6 kb;

- Presence of a polylinker with slgulär cleavage sites for the o. A. Rostriktasen;

- presence of a marker for the fc'rythromycln-Reelsteni;

- fourfold increased copy number Compared to the output plasmid pLKM 6181.

As can be seen in the figure, the shuttle plasmid pLM 2 lacks a DNA segment compared to the plasmid pLM 3, which is characterized by a characteristic arrangement of | a is characterized by an EcoRI and HlncII cleavage site.

With pLM2 and pLM3, the novel polylinker-bearing Streptoeoccus-Escherlchla coll-multlcopy shuttle cloning vectors of molecular size, original, structurally optimized carrier replicas are available for ancillary genetic engineering projects. These two new ehuttle platforms are particularly advantageous for the expression of genes in Gram-positive hosts, due to their small molecular size, the accumulation of slgular cleavage sites, including more common ones such as Pst !, Sail, and Smal; hl is enabled.

Ausführuugsbelsplol

a) Recovery of phasmid DNA pLKM 6181 and plasmid DNA pMc.

DNA of the plasmid pLKM6181 is recovered from cells of the strain S. sanguls challis 6 (pLKM6181) in the following catfish.

600ml of an overnight culture of this strain, grown in BHI-Meäium containing 2M Glucoso, 0, BM DL-threonine, 2.6% horse serum and 5pg / ml erythromycin, are centrifuged at 600OUAnIn for 15 minutes. The nucleated cells are washed with 50 mM TrisHCl (pH 8.2) and then resuspended in 60 ml of the same buffer. After addition of a lysozyme solution (100 mg lysozyme in 5 ml öOmM TrisHCl, pH 8.2) is incubated at 37 ⁰ C for 90 minutes. The cells are then centrifuged off and resuspended in 16 ml of 50 mM TrisHCl. Subsequently, 20mg pronase are added in 2 ml of Tris-HCl, and incubated for 20 minutes at 37 ⁰ C.

Cell lysis is carried out by adding 2 ml of a 10% SDS solution. Thereafter, the pH of the solution is adjusted to 12.1 by the addition of 1M NaOH, whereby the denaturation of the chromosomal DNA is achieved. Subsequently, a rapid pH addition of a 2 M TrisHCl solution to pH 8.5. After addition of 0.6 g of crystalline NaCl finally followed by deproteinization each extraction with a volume of NaCl-saturated phenol and chloroform-isoamyl alcohol mixture (24: 1). Subsequently, the DNA solution with 0.1 volume parts of a 3M Na acetate solution and 2 volumes of 96% alcohol at -2O ⁰ C precipitated, sedimented and dried. The DNA is last taken up in 5 ml of TES buffer.

For the separation and purification of the plasmid DNA from contaminating chromosomal DNA, an ethidium bromide-cesium chloride density gradient centrifugation is carried out in a manner known per se. Finally, the purified circular plasmid DNA is present in a solution in 1OmM Tris-HCl (pH 7.5) and stored at +4 ⁰ C. Furthermore, the DNA of the phasmid pMc is obtained from cells of the strain E. coli JM101 (pMc) in the following manner: 500 ml of an overnight culture of E. coli JM101 (pMc) were obtained in the following manner: 500 ml of an overnight culture of E. coli JM101 (pMc) that wurdu grown in LB medium with 25 \ ISHR \ chloramphenicol at 37 ⁰ C, centrifuged for 15 minutes at 6000U / min. The sedimented cells are washed with 50 mM TrisHCl and then resuspended in 13 ml of a 50 mM TrisHCl buffer with 25% sucrose. Thereafter, 20 mg of lysozyme are added, followed by a 5-minute incubation of the mixture. After adding 3 ml of a 0.25 M EDTA solution, the cells are cooled on ice for 5 minutes. The lysis is carried out by the addition of 21 ml of a lysis medium (50 mM TrisHCl, 62 mM EDTA, 1% SDS, pH 8.0). Thereafter, 11 ml of a 5 M NaCl solution are added and the batch is mixed gently and then cooled on ice for 20 minutes.

After 20 minutes centrifugation at 15000 U / min, the DNA contained in the supernatant is precipitated by addition of 0.6 parts by volume of isopropanol at -20 ⁰ C overnight. After centrifugation at 15,000 rpm for 20 minutes, the sediment is recovered, dried and resuspended in 4.5 ml of TES buffer.

Proteins remaining in the solution are separated by centrifugation at 12,000 rpm for 15 minutes. For the separation and purification of the phasmid DNA of contaminating chromosomal DNA, an ethidium bromide-cesium chloride density gradient centrifugation is carried out in a manner known per se.

Finally, the purified circular phasmid DNA is present in a solution of 1OmM Tris-HCl (pH 7.5) and stored at +4 ⁰ C.

b) Construction of the novel multicopy shuttle plasmids pLM 3 and pLM 2.

The construction of the novel shuttle plasmids pLM3 and pLM2 is now carried out as follows: 2 μg of the above-described isolated plasmid DNA from pLKM6181 are linearized with 10 units of the enzyme PstI. After the completeness of the cleavage was detected by gel electrophoresis, the enzyme PstI is inactivated by heat treatment (70 ⁰ C, 10 minutes). The linearized plasmid DNA is then precipitated by addition of 1 part by volume of 3M Na-acetate and 3 volumes of 96% alcohol (-7O ⁰ C, 30 minutes), sedimented in the Eppendorf centrifuge (12,000 rpm, 10 minutes), twice washed with 70% alcohol, dried and distilled in 10 μΙ of distilled water. solved.

In parallel, 5 pg of the phasmid DNA of pMc isolated as described above is cleaved with 20 units of the enzyme PstI. After the completeness of the cleavage in turn by the gel electrophoresis detection of the resulting 2 fragments (2.0 kb + 1.8 kb) was observed, the enzyme PstI is inactivated by heat treatment (7O ⁰ C, 10 minutes). The plasmid DNA is then precipitated in the manner already described for pLKM 6181, washed, dried and distilled in ΊΟμΙ of water. resuspended. Thereafter, the solutions are combined, with 1.5μΙ ligation buffer (10 times concentrated) and 4 units of T4 DNA ligase (in 2.5 μΙ ligase buffer) and left overnight at a temperature of 12 ⁰ C. The plasmid-free recipient-ion strain E. coll JM101 is transformed with this ligation mixture. The

Production of competent E. coli JM101 cells and the transformation itself are carried out in well-known catfish. On LB agar plates supplemented with 10 μg / ml erythromycin, about 100 transformant clones were selected. These transformant clones were, in turn, subjected to plasmid scrambling. Of these plasmid fast sols, 6 μl each were electrophoresed, and among them were selected those which are characterized by the smallest molecular size. Jo 10 .mu.Ι of this Schnelllsolate small molecule size were treated with the enzymes EcoRI, Smal, BamHI, Xbal, Sail and PstI and analyzed by gel electrophoresis in a conventional manner

 In the result was

a) a transformant clone carrying the 4.6 kb plasmid pLM 3, which has a polylinker with the cleavage sites Smal, BamHI, XBaI, Sail and PstI and dio determinant for erythromycln resistance, and

b) another transformant clone carrying the 4.1 kb plasmid pLM 2, which has a polylinker with the cleavage sites EcoRI, Smal, BamHI, Xbal, Sail and PstI and the determinant for the Rythromycln-Reslstenz,

identified.

The proof of the replication ability of both plasmids in Streptococcus was furnished by plosmide-free one Reziplenten strain S. sanglus challis β with 30μΙ each of a pLM 3- or pLM 2-Schnellisolats In a bekrnntor way

transformed and then Erythromycln-resistant clones were isolated.

Some of these S. sanguls transformant clones were subjected to piasmide screening in a manner known per se. The

Plasmid fast isolates obtained were treated with the enzymes EcoRI, SmaI, BamHI, XbaI, Sail, PstI and then analyzed by gel electrophoresis in a manner known per se. The identity of these plasmid rapid isolates with the

Plasmlden pLM3 or pLM2 be detected. In a manner known per se, the copy number of the plasmids pLM3 and pLM 2 in Streptococcus was determined. It was

found that both plasmids have a fourfold higher copy number than the starting plasmid pLKM 6181 when replicated in

Have strains of the genus Streptococcus.

Claims (6)

1. A process for the preparation of Polyllnker-carrying Streptococoue-Eecherlchla coll-multlcopy-ehuttlo-Plosmide minimized molecular size using standard genetic engineering procedures, characterized in that first on the one hand linearizes a polylinker-bearing Streptococcus cloning vector of the pLKM series by treatment with the Restrikiase PstI, on the other hand, the Polyllnker-term Escherichia coli plasmid pMc is broken down into two DNA fragments by treatment with the Pstl restrictase, - dia obtained in this way 3 DNA fragments in a Ugalionsansatz and transformed with this approach an erythromycin-sensitive Escherichla colt recipient strain, among the erythromycin-resistant Escherlchla coll transformant clones obtained, those carrying recombinant pLM family polylinker-carrying plasmids of less than 7.7 kb in size and isolating the plasmid DNA from these clones, - Subsequently transformed with the thus provided plasmid DNA an erythromycin-sensitive Streptococcus recipient strain and - Among the obtained erythromycin-resistant Streptococcus transformant clones selected those whose pLM plasmids carry the status of a multicopy plasmid, and isolated from these clones the plasmid DNA. 2. The method according to claim 1, characterized in that one uses in the input step the polylinker-bearing Streptococcus cloning vector pLKM6181. 3. The method according to claim 1 and 2, characterized in that one uses the recipient Escherichla coil JM101 for transformation with the ligation batch. 4. Method according to claims 1 and 3, characterized in that among the erythromycin-resistant Escherichia coli transformant clones, preferably among the erythromycin-resistant E. coli JM101 transformant clones, those which carry pLM 2-type deletion plasmids (polylinker-bearing; 1 kb) or of type pLM 3 (polylinker-bearing, molecular size 4.6 kb). 5. The method according to claim 1 and 4, characterized in that the rearrangement of the pLM2 and the pLM3 plasmid DNA, the recipient strain Streptococcus sanguls challis 6 uses. 6. The method according to claim 1 and 5, characterized in that among the obtained erythromycin-resistant Streptococcus sanguis Challis 6 transformants selects those whose pLM2 or pLM3 plasmid each carry the status of a multicopy plasmid.