DD251158A1 - PROCESS FOR THE PREPARATION OF STREPTOCOCCER VECTOR PLASMIDES WITH POLYLINKER - Google Patents

Abstract

The invention relates to a process for the preparation of novel streptococcal vector plasmids. It aims to provide, in particular, new streptococcal vector plasmids with polylinker for the purposes of genetic engineering. The object assigned to this objective is achieved by firstly adding to the new plasmid pSM6, a deletion mutant of the known streptococcal vector plasmid pSM7, with the E. coli plasmid pUC18, carrier of a polylinker with 8 pSM6-foreign single-stranded cleavage sites chimaeren plasmids pLKM618 and pLKM619 (molecular size 8.3 kb each) fused and then these Zwischenplasmide by separating out the pUC18 fuselage under self-ligation into the new, polylinker-carrying streptococcal vector plasmids pLKM6181 and pLKM6191 (molecular size 5.9 kb) converted. The vector plasmids pLKM6181 and pLKM6191 obtained according to the method each have a modified polylinker with 9 pSM6-foreign singular restriction-cleavage sites.

Description

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Field of application of the invention

The invention relates to a process for the preparation of novel Streptococcus vector plasmids which carry a poly linker and can advantageously be used for the cloning of foreign genes in streptococci.

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

Characteristic of the known technical solutions

The streptococcal vector plasmid pSM7 is known (H. Malke, FEMS Microbiol., Lett., 11, 1881, pp. 27-30). This plasmid is

- by a molecular size of 6,4kb,

- By unique cleavage sites for the restrictases EcoRI, Aval, Avail, Kpnl and Pvull as well

- Characterized by the presence of a resistance marker for erythromycin and lincomycin resistance (Em-Lm ^R ).

In addition, however, the plasmid pSM 7 also has properties that are disadvantageous in its use as a plasmid vector in genetic engineering constructions. In particular, this plasmid has no cleavage sites for such common restriction enzymes as Pstl, BamHI, Sail and Smal.

Object of the invention

The invention aims to provide novel streptococcal vector plasmids with polylinker for the purposes of genetic engineering.

Explanation of the essence of the invention

The invention has for its object to describe a method by means of which, by resorting to a deletion mutant of the streptococcal plasmid pSM7 by successive executed complexes of genetic engineering process steps new streptococcal vector plasmids are to be generated with polylinker.

According to the invention the problem is solved as follows:

Starting from the streptococcus plasmid pSM7 known from the literature, a spontaneous deletion mutant was isolated in a manner known per se, which was referred to as streptococcal vector plasmid pSM6 (see Fig. 1). This strain pSM6 carries the strain Streptococcus sanguis challis 6 (pSM6) deposited in the ZIMET depository for microorganisms, Central Institute for Microbiology and Experimental Therapy AdW, DDR-6900 Jena, Beutenbergstrasse 11 under the registration number ZIMET 11181 as a biologically pure culture.

The plasmid pSM 6 is characterized by a molecular size of 5.6 kb, otherwise it has the properties of plasmid pSM7.

Required amounts of pSM 6 DNA are preferably obtained by isolating, purifying and dissolving in a TrisHCl buffer from a culture of S. sanguis ZIMET 11181 the plasmid in a manner known per se.

From samples of DNA of the plasmid pSM 6 thus provided, the linearized form of the plasmid is obtained by opening with the restriction enzyme EcoRI.

Furthermore, required amounts of the known E. coli plasmid pUC 18 (see Fig. 2), which is characterized

- By a molecular size of 2.7kb

By the presence of a polylinker for the singular cleavage sites of the Hindlll, PstI, Sphl, Sail, Accl, HincII, XbaI, BamHI, Xmal, SmaI, KpnI, SstI and EcoRl restricted genes, including the presence of 8 pSM 6-foreign singulars restrictase-cleavage sites,

and continue

- by the presence of 10 cleavage sites for the restrictase Haelll and

By the presence of a resistance marker for ampicillin resistance,

isolated in a conventional manner from E. coli strain JM101 (pUC18), purified and dissolved in TrisHCl buffer. From samples of DNA of the plasmid pUC18 thus provided, the linear form of the plasmid pUC 18 is obtained by opening with the restriction enzyme EcoRI.

The thus prepared linearized forms of plasmids pSM 6 and pUC 18 are closed by ligation via the EcoRI site to the novel chimeric plasmids pLKM618 and pLKM619, respectively, which are then used to transform Escherichia coli HB101.

Intermediate plasmids pLKM618 and pLKM619 have a molecular size of 8.3kb each and are characterized by their ability to function and replicate in both streptococcal and Escherichia coli strains. The plasmids pLKM 618 and pLKM 619 already carry the sequence of the polylinker.

Both plasmids differ from one another with regard to the direction of insertion of the pUC 18 component. In plasmid pLKM 618, the pUC 18 moiety is inserted into the pSM 6 component such that the PstI cleavage site of the polylinker is closer to the avalide cleavage site of pSM 6, whereas in plasmid pLKM 619 this insertion is in such a manner that Pstl site of the polylinker is closer to the Pvull site of pSM6 (see Fig.3).

From the obtained in the above ligation reaction mixture, the chimeric plasmids are separated in such a way that with samples of this mixture cultures of an E. coli recipient strain, preferably cultures of E. coli Hb 101, are transformed and on the basis of the resulting Amp ^R Em ^R Transformants screening for DNA molecules of size 8,3kb is performed. By means of a subsequent restriction analysis, those 8.3 kb DNA portions (which are uniquely assigned to the individual transformant strains) are identified which are assigned to the plasmid pLKM618 or to the plasmid pLKM 619. In this way, among the transformant ions, clearly, those of E. coli type, Hb 101 (pLKM618) can be separated from those of E. coli Hb 101 type (pLKM619). Sufficient amounts of pLKM618 DNA and pLKM619 DNA, as required for subsequent portions of the present method for producing streptococcal vector plasmids with polylinker, are prepared in a manner known per se from E. coli strains Hb101 (pLKM618) and HB101 ( pLKM 619), purified and dissolved in TrisHCl buffer.

The DNA of the plasmids pLKM618 and pLKM619 thus obtained is then subsequently cleaved with the restriction enzyme Haelll. Obtained in this way from each of the plasmids 10 linear DNA fragments. The largest of these fragments are characterized by a molecular size of 5.9kb each. They consist of the complete plasmid pSM6 with the resistance marker for Em-Lm "and a 260 bp portion of the plasmid pUC 18. The 260 bp portion of this fragment carries the polylinker sequence from the plasmid pUC18.

Using the per se known method of agarose gel electrophoresis, the 5.9kb fragment of plasmid pLKM618 is separated from the remaining nine Haelll fragments, isolated, purified and dissolved in TrisHCl buffer. In the same way, the 5.9 kb fragment of plasmid pLKM 619 is recovered.

The thus prepared linear 5.9 kb major fragments of plasmid pLKM618 and plasmid pLKM 619 are closed by blunt end self-ligation to restore the Haelll cleavage site to the new plasmids pLKM6181 and pLKM6191. Subsequently, cells of strain Str. Sanguis challis 6 are transformed with these plasmids. The new streptococcal vector plasmids pLKM6181 and pLKM6191 are characterized (see Fig.4)

- by the presence of a resistance marker for erythromycin and lincomycin resistance (Em-Lm ^R ) as well

- by the presence of a modified polylinker with singular cleavage sites for the now 9 Restrictasen Haelll, Sphl, Pstl, Sail, Xbal, BamHI, Smal, Xmal and Sstl.

Both plasmids differ from each other by the insertion direction of the polylinker. Whereas in pLKM6181 the PstI cleavage site of the polylinker is closer to the Avall cleavage site, the PstI cleavage site of the polylinker is located closer to the Pvull cleavage site on plasmid pLKM 6191.

With the new ones. Polylinker-carrying plasmids pLKM6181 and pLKM6191 are available for Gram-positive bacteria of the genus Streptococcus two primary, structurally optimized cloning vectors as carrier replicas for subsequent genetic engineering projects. The novel plasmids pLKM6181 and pLKM6191 can be used particularly advantageously for the construction of expression vectors for streptococci, which is made possible by the accumulation of additional singular cleavage sites, including more common than BamHI, PstI, Sail, Haelll and SmaI.

embodiment

1 a) Obtaining the intermediate plasmids pLKM618 and pLKM619

DNA of plasmid pSM 6 is recovered from cells of strain ZIMET11181 in the following manner:

500ml of an overnight culture of this strain cultured in BHI medium with 2M glucose, 0.5M DL-threonine, 2.5% horse serum and oyxg / ml erythromycin are centrifuged at 6000rpm for 15 minutes. These sedimented cells are washed with 5OmMTHsHCl (pH 8.2) and then resuspended in 50 ml of the same buffer. After addition of a lysozyme solution (100 mg lysozyme in 5 ml 5OmM 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, 20 mg Pronase in 2 ml TrisHCl are added, and it is incubated at 37 ⁰ C for 10 minutes.

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 1 M NaOH, whereby the denaturation of the chromosomal DNA is achieved. Subsequently, by ιλΙμμ> ΙΙ / ι 7 ,, ", k" ™ "n, 1M TricUri.l Acnnn oin nU.ohlf" on O G

After the 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 3Μ Na-acetate solution and 2 volumes of 96% alcohol at -20 ⁰ C precipitated, sedimented and dried. The DNS is last added in 5ml 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 10 mM TrisHCl, pH 7.5, and is stored at +4 ° C.

In parallel, DNA of the plasmid pUC 18 is obtained from cells of the strain Escherichia coli JM 101 (pUC18) in the following manner:

500 ml of an overnight culture of E. coli J M101 (pUC18) grown in LB medium with 100 μg of ampicillin are centrifuged for 15 minutes at 6000 rpm, and these sedimented cells are washed with 50 mM TrisHCl and then in 13 ml of 50 mM TrisHCl Buffer with 25% sucrose (pH 8.0), then 20 mg of lysozyme are added followed by a 5 minute incubation of the batch After addition of 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), then 11 ml of a 5 M NaCl solution are added and the mixture is mixed gently and then for 20 minutes chilled on ice.

After 20minütigerZentrifugation at 15000 U / min, the DNA contained in the supernatant is precipitated by addition of 0.6 volumes of isopropanol at -2O ⁰ C overnight. After centrifugation at 15,000 rpm for 20 minutes, the pellet is recovered, dried and resuspended in 4.5 ml of TES buffer.

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

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

The preparation of the new, recombinant intermediate plasmids pLKM618 and pLKM619 is now carried out as follows:

5% each of the DNAs of pSM6 and pUC 18 obtained in the manner described above are linearized in separate batches with 10 units each of the enzyme EcoRI. After the completeness of the cleavage was detected by gel electrophoresis, the EcoRI enzyme is inactivated by heat treatment (7O ⁰ C, 5 minutes). Subsequently, both linearized plasmids are purified by addition of 3 per parts by volume of 96% alcohol like (-7O ⁰ C, 60 minutes), sedimented in an Eppendorf centrifuge, 2 times with 70% alcohol, dried and dissolved in each 8μ.Ι Aqua. solved. Thereafter, the plasmid solutions are combined, mixed with 2μ.Ι ligation buffer (10 times concentrated) and 2 units of T4 DNA ligase (in 2μ, Ι ligase buffer) and left overnight at a temperature of +12 ⁰ C. In addition to the reconstructed starting plasmids (pSM6and pUC18), the ligation solution also contains the new recombinant plasmids pLKM618 and pLKM619. The mixture of these plasmids is introduced by transformation into the plasmid-free strain E. coli Hb 101. The preparation of competent E. coli Hb 101 cells and the transformation are carried out in a manner known per se. Clones carrying the novel recombinant plasmids pLKM618 and pLKM619 are selected on LB agar plates supplemented with 100 μg / ml of ¹ ampicillin and 50 μg / ml of ¹ erythromycin. As a result of this transformation, approximately 100 erythromycin-ampicillin resistant transformant clones were present. Some of these have been subjected to plasmid screening, which proceeds in a manner known per se.

10μ.Ι of these plasmid fast isolates were treated with the enzyme EcoRI and analyzed by gel electrophoresis in a conventional manner in comparison with linearized pSM 6 and pUC18 plasmids. Each 10μ, Ι the same plasmid fast isolates were also treated with the enzymes Pstl, HindIII, BamHI and Sail and also analyzed in a conventional manner.

As a result, two transformant clones were identified, one carrying the 8.3 kb recombinant plasmid pLKM618 and the other the 8.3 kb recombinant plasmid pLKM 619 (see Fig. 3). Both plasmids possess the polylinker and two resistance markers each (Amp ^R , Em-Lm ^R ).

1b) Construction of polylinker-carrying plasmids pLKM6181 and pLKM6191 Sufficient amounts of purified pLKM 618 DNA and pLKM619 DNA, as required for the subsequent procedures, are prepared from strains E. coli Hb 101 (pLKM618 ) and E. coli Hb 101 (pLKM619).

Each 5 ju.g of this pLKM 618 DNA or pLKM 619 DNA are then cleaved in separate mixtures in a total volume of 50 μ \ with the restriction enzyme Haelll in a conventional manner. The resulting 10 fragments of the plasmid pLKM618bzw. of the plasmid pLKM619 are separated by gel electrophoresis in a conventional manner. The resulting from this cleavage respectively largest fragment of the plasmid pLKM618bzw. The plasmid pLKM 619 is characterized by a molecule size of 5.9 kb each and consists of the complete plasmid pSM6with the resistance marker Em-Lm ^R and a 260 bp portion of the plasmid pUC18 with the polylinker sequence. These 5.9kb DNA fragments are extracted from the agarose gel in a manner known per se. Finally, these DNA fragments are dissolved in 15 μl of 10 mM TrisHCl, pH 7.5.

Their blunt ends are then linked together in separate ligation experiments by self-ligation, whereby the Haelll cleavage site is reconstructed. For this purpose, in each case 15 μΙ DNS solution with 2 / xl ligation buffer (10X concentrated) and 2 units of T4 DNA ligase are added and left overnight at a temperature of +4 ⁰ C.

Competent cells of the plasmid-free Streptococcus sanguis strain Challis 6 are transformed in a manner known per se on the following day using this ligation mixture. Plasmid-carrying transformants from both ligation experiments are selected on BHI agar plates with 5 ^ g erythromycin per ml agar. A certain number of transformant clones are isolated and subjected to plasmid screening in a conventional manner.

To identify the desired new Streptokokken vector plasmids with polylinker each 15μ, Ι the Plasmidschnellisolate with the restriction enzymes, EcoRI, HindIII, PstI, Sail, BamHI, Haelll, Avail and Pvull are cleaved and compared with suitable standard DNA (pSM 6 with EcoRI cleaved; DNA of E. coli bacteriophage λ cleaved with HindIII; pBR322 with Avail cleaved) were analyzed in a manner known per se.

Two transformant clones, each containing a 5.9kb plasmid, showing expected cleavage patterns, but having different insertion directions of the polylinker, are selected for isolation of purified plasmid DNA.

The cleavage of these purified plasmid DNA with the enzymes EcoRI, HindIII, BamHI, PstI, Smal, Sail, Haelll, Avail and Pvull and the subsequent gel electrophoretic size analysis of the resulting fragments allow a clear identification of the new streptococcal vector plasmids pLKM6181 and pLKM 6191. These Analysis confirms that the polylinker fragment of pUC18 is in the expected manner in pLKM6181 and pLKM6191, respectively (see Fig. 4).

The following cleavage sites of the polylinker prove to be unique cleavage sites of the plasmids pLKM 6181 and pLKM6191: SstI, Xmal, Smal, BamHI, XbaI, Sail, PstI, SpHI and Haelll.

Claims (1)

1. A process for the preparation of Streptokokken vector plasmids with polylinker using standard genetic engineering procedures, characterized in that - Plasmid pSM6, a derivative of plasmid pSM7, with the E. coli plasmid pUC18, which carries a 8 pSM 6-foreign, polysaccharide unique polysaccharide cleavage sites, by ligation over the EcoRI cleavage sites to the new chimeric intermediate plasmids pLKM 618 and pLKM.619 (molecular size 8.3 kb each) fused, - From these Zwischenplasmiden by digestion with the Restellase Haelll the pUC18 trunk separates out and - The thus provided 5.9 kb major fragments of the intermediate plasmids in each case by means of self-ligation to the new, polylinker-carrying streptococcal vector plasmids pLKM 6181 and pLKM6191 closes.