DK167883B1 - Stabilisation of plasmid(s) - by insertion of DNA fragment expressing partitioning fragment - Google Patents

Abstract

Plasmid carrying an inserted gene(s) not naturally related to the plasmid as well as an inserted DNA fragment expressing a partitioning fragment is new. Plasmid carrying an inserted DNA fragment shorter than the EcoR1-A fragment of plasmid R1 and contg. a R1 par region is new. Bacterium harbouring a plasmid as defined above is new. Prodn. of a gene prod. of plasmid DNA comprises cultivation of a bacterium as defined above, and the prod. is harvested from the bacterial culture. DNA fragment comprising as its major component a R1 par region is new. Method of screening for the correct insertion of a par region in a plasmid (believed to carry such a region) comprises transformation to a bacterium already harbouring another unrelated plasmid carrying the same par region, as well as mediating a phenotype recognisable on approp. media. The correct insertion is identified as the loss of this phenotype. The stabilised plasmids are useful as cloning or prodn. vectors in recombinant DNA technology for prodn. of genes and their prods., e.g. polypeptides and proteins esp. enzymes and hormones, and nucleic acids.

Description

in DK 167883 B1

The present invention relates to the stabilization of plasmids useful in the field of recombinant DNA technology for the production of genes and products thereof.

The continued preservation of most natural plasmids, even in the absence of selection pressures (factors that ensure only the growth of the organisms containing the plasmids; an example is an antibiotic in the nutrient medium in the case of a plasmid carrying a gene that communicates resistance to the antibiotic), suggests that plasmid conservation functions have been developed to ensure the continued presence of these extra-chromosomal elements with high efficiency. The plasmid conservation functions consist primarily of the replication genes, including their control regions, which regulate the plasmid concentration in the cell. In growing cells, the replication control system monitors the number of plasmid copies and corrects deviations from the mean by increasing or decreasing the probability of plasmid replication. However, no replication control system can prevent cells from producing very few or just one copy of the plasmid, and from such cells the possibility of forming a plasmid-free daughter cell is obvious. This problem is, of course, greatest, in terms of low-capital plasmids. Furthermore, a passive distribution of plasmid molecules by cell division inevitably results in a certain frequency of plasmid-free cells. Since the loss of a plasmid molecule from a cell is irreversible, the consequences of such an unstable situation are that the entire population eventually becomes plasmid-free.

Besides a random distribution of plasmids by cell division, other factors may influence the frequency of loss of plasmids from a culture of cells grown in the absence of selection for preservation of the plasmid. For example, certain plasmids require a specific recombination system to separate two newly replicated molecules (Austin et al. 30, Cell 25, 1981, pp. 729-736). Without separation, multimers (coalescing) will form, and thus even a high copy number of a plasmid may appear as a low copy plasmid in terms of distribution to daughter cells, as the probability of generating plasmid-free cells is increased with increasing numbers of plasmid molecules interconnected in multimers. structures. Another phenomenon commonly observed in recombinant DNA technology is the transformation of a stable cloning vector into an unstable hybrid plasmid as a result of the insertion of a DNA fragment, the presence of which either causes a decrease in plasmid copy or causes a harmful product which adversely affects cell growth.

In all of these cases, plasmid segregation and loss occur at a frequency (high or low) that cannot be easily controlled from the outside.

The stability of natural plasmids and especially low copy plasmids suggests that, in addition to the replication control system, there is another set of conservation functions that actively participate in an ordered distribution of the plasmid molecules by cell division. Such functions have been termed distribution functions, and studies, e.g., Meacock et al., Cell 20, 1980, pp. 529-542, Nordstrom et al., Plasmid 4, 1980, pp. 332-349,

Seelke et al., Plasmid 7, 1982, pp. 163-179, have now shown that these are at least partially encoded by the plasmids themselves (in pairs).

Thus, some plasmid deletion mutants have lost their stable conservation or inheritance despite their normal wild-type replication behavior, suggesting that a plasmid-specific feature ensuring stability has been removed (cf. Nordstrom et al., Op. Cit.).

Since many of the plasmids used as vectors in recombinant DNA technology have had a large amount of DNA removed in comparison to the wild-type stem plasmid, they tend to be unstably inherited. This poses a serious problem, since instability of a plasmid ultimately results in complete loss of the plasmid from the cells and in any case reduces the relative yield of plasmid-encoded gene products. This problem is particularly pronounced in large scale production of gene products where growth of microorganisms under selection pressure, eg an antibiotic, is usually not feasible and is often at least undesirable from an environmental point of view and where the microorganisms are grown in a large number of generations. Vectors that are only present in a few copies per cell, are most likely to be unstable inherited and therefore lost from the cells. Even plasmids, which usually have a relatively high copy number, which ensures a relative stability of the plasmid, 35 have been found to be unstable in some cases when DNA fragments carrying genes not naturally associated with the plasmid , is inserted therein.

The present invention relates to plasmids that replicate in Gram-negative bacteria, which are characterized in that they carry one or more inserted genes that are not naturally associated with the plasmid and which further carry an inserted DNA fragment that exerts a distribution function. which DNA fragment is shorter than 19 kb and which DNA fragment comprises the plasmid stabilizing DNA sequence R 1 pair region A, the plasmid stabilizing DNA sequence R 1 pair region B or 10 both R 1 pair region A and R 1 pair region B The term "inserted" shall mean that the gene or genes or DNA fragment has been introduced into the plasmid at one stage during the construction of the finished plasmid.

In this context, the term "a distribution function" refers to a function which ensures an orderly distribution of the plasmid molecules by cell division and which is encoded by a region of a plasmid which, depending on the type of plasmid expressing the function, may comprise one or more genes. . As mentioned above, such regions are found in naturally occurring or wild-type plasmids, but plasmids expressing a Par + phenotype, viz. on which the distribution genes are present and which also carry one or more inserted genes which are not naturally associated with the plasmid are considered to be novel. In this context, plasmids are defined as naturally occurring extracromosomal elements in microorganisms which can be isolated as such, or derivatives thereof.

The plasmids of the invention can be used as cloning or production vectors in the field of recombinant DNA technology for the purpose of extracting a wide variety of products for technical or medical purposes that are directly or indirectly mediated by the inserted gene (s), in particular polypeptides and proteins or fragments thereof, enzymes and a number of non-proteinous products of reactions with enzymes, low molecular weight products such as hormones, and nucleic acids; products of eukaryotic genes, especially mammalian genes, are of particular interest.

4 UK Ί67883 di In accordance with the present invention, the distribution function is a function that is naturally expressed by a region of the wild-type resistance plasmid R1, and hence is hereinafter referred to as a R1 pair region. According to the present invention, such an area has been found to cause partial or full stability not only in unstable RI mini plasmids carrying one or more genes not naturally associated with the plasmid, but also in frequent segregating plasmids not associated. with the plasmid RI and carrying one or more genes not naturally associated with the plasmid. Another example of a particularly valuable distribution function is the distribution function found on the wild-type plasmid F (Seelke et al., Op. Cit.) Which also imparts a high degree of stability to the recipient plasmid. In the following, reference will primarily be made to the RI Par function, but it is clear that many of the 15 phenomena associated with the RI Par function also apply to other Pair functions.

It has been previously stated that the distribution function of plasmid RI is exerted by the so-called EcoRl-A fragment (cf. Nordstrom et al.,

Plasmid 4, 1980, pp. 332-349). Surprisingly, in the research that has led to the present invention, it has been found that the β coR1-A fragment, which has a length of approx. 19 kb (19,000 base pairs), comprises two and only two distinct regions which confer the recipient plasmid a Par + phenotype. These regions are located at each end of the EcoR1-A fragment and no other DNA sequence in the EcoR1-A-25 fragment is presently believed to have any plasmid stabilizing function. For this purpose, the two regions have been designated pairs A and Pair B, abbreviated to parÅ and parB respectively.

Since it is advantageous to operate with as small DNA fragments as possible because small fragments are easier to insert into the plasmids and the resulting plasmids are easier to transform into host cells, the invention relates to plasmids carrying an inserted DNA fragment, which is shorter than the Eco RI fragment from plasmid RI and which contains an RI pair region. This inserted DNA fragment usually comprises as its major component RI pair region A, RI pair region B or both RI 35 pair region A and R 1 pair region B. This means that substantially all of the DNA fragment which is inserted into the host plasmid is constituted by one of the two or both pairs of regions, and the remainder of the DNA on the fragments is preferably present to provide suitable restriction sites which provide the DNA fragments with the desired ends which are immediately compatible with a corresponding or compatible restriction site on the recipient plasmid. Such ends may also be provided by means of linkers. However, it should be noted that the DNA fragment comprising pair region A and the DNA fragment comprising pair region B can be introduced separately and sequentially into the same plasmid, which will then be phenotypically identical to a plasmid. 10 which the phenotype ParA +, ParB + has been established by inserting par region A and par region B on the same DNA fragment. For example, if it is desired to further stabilize a plasmid already carrying a par region such as the par B region, the plasmid can be cleaved with an appropriate restriction enzyme and a DNA fragment having ends compatible with this restriction site and which bearing the second pair area such as the park area can then be inserted. The opposite process, i.e. Similarly, insertion of a parB region onto a plasmid already carrying a parA region can also be performed.

Accordingly, interesting plasmids are plasmids, wherein the inserted DNA fragment, which comprises RI pair region A and RI pair region B, has a length not exceeding ca. 6 kb, especially not approx.

4 kb, in particular not 3 kb. When the inserted DNA fragment comprises RI pair region A, it usually has a length not exceeding approx.

4 kb, especially not approx. 2.5 kb, in particular not approx. 2 kb. When the inserted DNA fragment comprises RI pair region B, it usually has a length which does not exceed approx. 2 kb, especially not approx. 1.5 kb, in particular not approx.

1 kb. The size of the £ coRl-A fragment can be reduced in various ways such as by partially restricting the £ coRl-A fragment with the restriction enzyme PstI, or by cutting each RI pair region from the 30 large fragment and transferring each region sequentially to it. the same DNA fragment which can then be inserted into the plasmid to be stabilized.

The plasmids stabilized in accordance with the principle of the invention may be either plasmids naturally associated with the inserted distribution region, such as R1 mini-plasmids, which are stabilized by an inserted R1 pair region (R1 mini-plasmids have been given DK 167883 B1 6 deleted much of the original RI DNA and therefore does not usually contain in itself an RI pair region) or they may be plasmids that are not naturally associated with the distribution function. It is interesting to note that efficient distribution functions which can be used in accordance with the present invention are capable of ensuring a satisfactory stability of not only plasmids with which they are naturally associated, such as in the case of stabilizing RI mini plasmids with a RI pair region, but also plasmids with which the distribution function is not naturally associated. An example of the latter is insertion of an RI pair region into a non-R1 plasmid such as a pMB1 plasmid or derivative thereof such as a pBR322 plasmid or derivative thereof (these plasmids are usually stable but tend to become unstable once or multiple genes not naturally associated with the plasmid are inserted). Another example 15 is the use of a R 1 pair region for stabilizing certain plasmid types, which at least in one stage during the cultivation of bacteria containing the plasmid, has a low copy number, eg a copy number of 0.5-5 copies per cell such as promiscuous plasmids (plasmids capable of replicating in many different host strains or species; this class includes the so-called shuttle vectors which can replicate in two or more types of microorganisms) with low copy number and derivatives thereof, e.g. RK2. Apart from RI, other plasmids from the incF compatibility group include IncFII which require stabilization, e.g., R100 and R6.

One type of plasmids for which stabilization according to the present invention is important is conditional runaway replication plasmids, i.e. plasmids which, when host microorganisms containing the plasmids, are cultured under certain conditions, have a constant low plasmid copy and which, when the microorganisms containing the plasmids, are cultured under certain other conditions, lose their replication control, so that the plasmid copy number increases exponentially until the host cell ceases to grow. Runaway replication plasmids, for which stabilization according to the invention is particularly important, are runaway replication plasmids with a copy number not exceeding approx. 3-5 copies per cell, and in particular, plasmids whose copy number does not exceed ca. 0.5-1 35 copies per cell (the number 0.5 should denote that the replication rate is less than 1 per cell cycle) when microorganisms containing the plasmids are cultured under such conditions which ensure such a low plasmid copy capital and which when the host microorganisms are cultured under certain other conditions which ensure a substantially increased plasmid copy have a copy capital in the region of at least about 500-1000 copies per cell.

The low plasmid copy capital under certain conditions (typically a low temperature such as a temperature of about 30 ° C) is desirable when the plasmids are used as vectors for insertion of foreign genes encoding products that are partially toxic or deadly to the host cell, since the genes due to the low frequency of replication of the plasmids at, for example, 10 a low temperature are only expressed in small amounts at all, so that the cells remain undamaged in the propagation stage of the culture. However, in plasmids with an extremely low copy number, as mentioned above, under the conditions used in the propagation step, such as culture of cells at a low temperature, the lack of a 15-pair region causes the plasmid to be lost from the microbial population at a frequency of ca. 1% per generation for plasmids with a copy number not exceeding 3-5 copies per cell when grown under conditions which ensure such a low copy capital. The loss rate of plasmids with a copy number not exceeding approx. 0.5-1 20 copies per cell, is approx. 5% / cell / generation. It is clear that the plasmids will be lost from the cells before the cells have reached such density in the nutrient medium that it is economical to induce runaway replication unless they have a distribution function to stabilize them; This is especially true in large-scale production, which requires 25 hundreds of generations of cell growth to come up with a production-size culture.

Such runaway replication can be made conditional upon inserting a regulatable promoter above the natural replication control gene (s) on the plasmid (a detailed description of this phenomenon 30 can be found in the presently filed application entitled "Plasmids with Conditional Uncontrolled Replication Behavior", filed on the same day as this application). Plasmids with runaway replication behavior may be of many different types, but preferred runaway replication plasmids are R1 type plasmids.

In this specification, the term "stability" (and associated terms) denotes a loss rate of the plasmid from the host cell of less than 2 x 10 6 per cell per generation. In fact, plasmids which are equal are possible to obtain as stable as wild-type plasmids, viz.

5 with a loss frequency of less than 3 x 10 6 per cell per generation, which corresponds to the level of mutation frequency of genes, by incorporating a Par function into the plasmid. This loss frequency value (LF) is evident when the plasmids are stabilized with both RI par region A and RI par region B, such as when the entire Eco RI-A fragment has been inserted into the plasmids.

However, as mentioned above, the Par + phenotype of plasmid R 1 has been localized to each separate pair region of the FcoR1-A fragment. It has been found that each of these pair regions has a stabilizing effect on unstable plasmids, which can be defined as plasmids lacking a stabilizing or distributive function. Such phenotypes which are phenotypically Par are usually lost from the host cell at higher or lower frequency; for example, plasmid RI derivatives from which the pair region has been removed are thus lost from the host cell at a frequency of approx. 1.5 x 10 cell per generation. Similarly, plasmid p15 derivatives, which are Par, are lost at a frequency of approx.

1 x 10 6 per cell per generation, and some plasmid pMB1 (pBR322) derivatives (such as described in Example 3.3) can be lost at a frequency of about 6 x 10 6 / cell / generation. plasmids stabilized with either parA or parB alone, an LF value of about 10 per cell per generation corresponding to 100 fold stabilization when a DNA fragment carrying one of these pair regions is inserted into an unstable vector; the numbers for a corresponding p1 5 derivative are about 8 x 10 6 (ParA +), 1 x 10 10 (ParB +), and the numbers for a corresponding pMB1 derivative are 5 x 10 6 (ParB +). Plasmids carrying both the parA and 30 parB regions have an LF value of approx. 10 µm per cell per generation or a 10 µm stabilization. To facilitate clarity, the results for stabilized and unstylized replicons of different origins are shown in Table 1. This shows that each pair region acts independently of the other that the pair regions are about as effective for stabilizing at least RI and p15 plasmids and that their effect is cumulative, this knowledge can be exploited when determining the degree to which an unstable plasmid is to be stabilized.

DK 167883 B1 9

If less drastic stabilization is needed, ie. Assuming that the plasmid to be stabilized is not very unstable (with an LF value of less than 10 '/ cell / generation), it may be sufficient to insert a DNA fragment containing one of the pair regions to obtain satisfactory stability, i.e. prevent a gradual loss of the plasmid from a bacterial population to large-scale production over hundreds of generations. On the other hand, if the plasmid is very unstable (with an LF value greater than 10 "), it may be necessary or at least 10 advantageous to insert both pairs of regions to ensure extreme stability of the plasmids.

Table 1

Loss rates / cell / generation of different replicons Loss rate x 10 "/ cell / generation

Type Par phenotype replicon Par Par + ParB + ParA + ParB + R1 150 0.6 1.0 0.04 20 pL5 100 8.0 0.01 0.01 pMB1 (pBR322) 60 ND1 0.5 0.1 ND = no data

These measurable LF values can be utilized when plasmids of one of the above 25 types are to be used as vectors for production of gene products, and thus must contain at least one gene not naturally associated with the plasmid. Thus, in a further aspect, the present invention relates to a method for producing a gene product from plasmid DNA, which method is characterized in that bacteria containing a plasmid according to any one of claims 1-16 are cultured and the gene product of the plasmid is recovered from the bacterial culture. Cultivation as such is conveniently carried out using conventional techniques, including conventional nutritional media, which are known to be optimal for the bacterial species. It is worth noting that, as a result of the stabilization, no specific composition of the nutrient medium is required. The recovery of the gene product is also carried out according to well known methods which are adapted to the nature and characteristics of the particular gene product being produced, the characteristics of the host bacterium, etc. Cultivation continues for at least 100 bacterial generations; on large scale production, the number of cell generations needed to propagate the bacterium can exceed 100 generations. Under these circumstances, the LF value of the plasmid is selected in the presence of a pair of regions therein in such a way that the loss of the plasmid is less than 2 x 10 '10 ‘V / cell. This LF value can usually be obtained by one pair area alone. However, in some cases, it is preferred to obtain LF values for the plasmid of less than 10 "cell / generation, especially less than 5 x 10 6 / cell / generation.

Although in some cases these very low LF values can be achieved by inserting just one pair of regions (especially RI pairs of area B), the presence of both RI pairs of ranges is usually required.

In a further aspect, the invention relates to gram-negative bacteria containing plasmids of the invention. It is a particular advantage of the plasmids of the invention that no particular mutants 20 or strains are required to ensure preservation of the plasmid. Thus, any bacterial species and strain capable of containing such plasmids such as gram-negative bacteria can be used. A specific example of a bacterium in which plasmids of the present invention can replicate and maintain their stability is Escherichia coli.

Finally, the present invention relates to a DNA fragment shorter than 19 kb which is characterized in that it comprises the R 1 pair region A, R 1 pair region B or both R 1 pair region A and R 1 pair region B. This means that substantially the entire DNA fragment inserted into the host plasmid consists of one or both of the pair regions and the remaining DNA is present to provide appropriate restriction sites for insertion into a compatible restriction site on the recipient plasmid. The inserted DNA fragment comprising RI pair region A and RI pair region B should, according to this principle, have a length not exceeding approx. 6 kb, especially not approx.

DK 167883 B1 11 4 kb, in particular not 3 kb. When the DNA fragment comprises RI pair region A, it usually has a length which does not exceed approx. 4 kb, especially not approx. 2.5 kb, in particular not approx. 2 kb. When the DNA fragment comprises RI pair region B, it usually has a length not exceeding 5 2 kb, especially not approx. 1.5 kb, in particular not approx. 1 kb. Surprisingly, it has been found that such small and therefore readily insertable DNA fragments have retained their stabilizing function, since the restriction enzyme mapping parA region has been reduced to a region of about length. 1800 bp (base pair) and the parB range has been reduced from 10 to approx. 900 bp. It is possible that the gene or genes that actually transmit a Par + phenotype are even smaller.

A serious problem in constructing hybrid plasmids with a Par + phenotype has been the lack of rapid and simple methods of screening for this phenotype. Usually, the correct hybrid plasmids 15 can be identified by the Par + phenotype, which results in high stability of the plasmid upon growth of the plasmid without selection pressure. However, this type of screening is prolonged and is in any case only relevant if the stem plasmid is inherited unstable. Alternative screening methods have now been developed as described below.

A) Screening for insertion of ParA + fragments

If two different plasmids (from each incompatibility group), both carrying the pair of A + region, exist in the same cell, they displace each other at a certain frequency (incompatibility), probably because they compete for the cell's distribution mechanisms. This type of pair-25 mediated incompatibility phenotype can be utilized for screening for pairs hybrids. For example, a ParA + plasmid may be transformed into an Alac E. coli strain (e.g. CSH50) containing another plasmid carrying the lac genes and the parA + region. Usually, the incoming plasmid will exert pair + -mediated incompatibility with the ParA + plasmid already present in the cell. Due to the Lac + phenotype of the plasmid already present in the cell, such incompatibility is readily detected if transformants are selected on the basis of a resistance marker found only on the incoming plasmid, whereas the presence or absence of the plasmid that already present in the cell, are observed on indicator substrates such as McConkey lactose plate. Replication plating of colonies from such plates to new corresponding plates even reveals low levels of displacement of the plasmid already present in the cell, in the form of colorless colonies showing the presence of Lac cells. An additional stability test or more comprehensive incompatibility test may be required to test the properties of potential parA + hybrid plasmids. This makes it possible - using the correct (compatible or perhaps just incompatible) combination of incoming and already available plasmids - to quickly screen for insertion of Inc + (Par +) - fragments into any plasmid, whether is unstable or stable.

b) Screening for insertion of parB + fragments

Since it has also been shown that two unrelated plasmids, both bearing the parB + region, are incompatible with each other, the same screening strategy as that described for the construction of parB + hybrids can be used for the construction of parB + hybrids. .

c) Screening for insertion of parA +, B + fragments

In addition to being able to displace both parA + and parB + hybrid plasmids according to the above descriptions, the EcoRL-A fragment with the 20 Tn5 insert enables a direct selection (Km®-) of plasmids carrying the parA +, the parB + fragment. Thus, despite the large size of the fragment, very few hybrids are easily selected. Of course, a smaller DNA fragment comprising both par + regions also incompatibility against both parA + and parB + plasmids.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1-5 and 7-17 show restriction maps of the plasmids described in the examples; 6 shows a detailed map (not drawn to the same scale) of the Psti-D fragment of Ri, and FIG. 18 shows stability curves for different types of replicon bearing different pairs of regions compared to Par replicons.

In FIG. Figures 1-5 and 7-17 show linear restriction maps of the plasmids described in the Examples, wherein the phenotypes and genotypes of the plasmids are shown above the horizontal line denoting the stem plasmid.

Thus, parA represents one of the regions of plasmid R1 that ensures preservation of the plasmid, parB represents the other region of plasmid R1 which ensures preservation of the plasmid; icel represents a gene that mediates immunity to colicin E1; ori or oriV denotes the site of initiation of plasmid replication; bla denotes a gene that encodes ampicillin resistance; IR represents an inverted repetitive structure of 10 Tn5; Km® denotes kanamycin resistance; Cm® represents chloramphenicol resistance; Ap® denotes ampicillin resistance; Te® denotes tetracline clin resistance; repA denotes a gene encoding a protein required for Rl replication; lacZ, lacY and lacA represent the inserted lac operon, of which lacZ encodes β-galactosidase, lacY encodes 15 permease, and lacA encodes transacetylase; repA-lacZ 'represents a fusion between the repA and lacZ genes; copB represents a gene encoding a polypeptide that represses transcription from the repA promoter (on RI plasmids); copA represents a gene encoding an RNA molecule that inhibits translation of RepA RNA; dggy represents a gene coding for a temperature-sensitive λ repressor that controls λΡΡ promoter activity; Pdeo denotes deo promoters. The arrow shows the direction of transcription, and the "triangles" denote insertions of DNA.

The black areas and the white areas denote inserted genes; the dotted line denotes a deletion.

Below the horizontal line, the sites of restriction enzymes are shown, where E represents PcoR1; P represents PstI, Bj ^ represents BamHI except in FIG. 1 and 5, where outside the Tn5 DNA denotes Ball; Hp represents tfpal; Ev represents PcoRV; B2 represents BglII; denotes Sall; H3 represents RindIII; and C represents Clal.

In FIG. 6, the PstI-D fragment has been further mapped; the numbers above the horizontal line show the number of base pairs between the restriction sites. Below the horizontal line, the sites of restriction enzymes are shown where denotes Rsal; P represents PstI; and denotes Hpal.

Fig. 18 shows stability curves for Ri, p15 and pBR322 derivatives, which are constructed and tested in the examples. Each curve represents the average of at least two liquid cultures that have been monitored for over 100 generations. It can be seen from the figure that plasmids containing both parA and parB are inherited extremely stable, as is also the case with plasmids containing only parB, and mini-R1 plasmids stabilized with parA only.

It is apparent from the curves of Par plasmids that the frequency of plasmid-bearing cells is initially reduced exponentially as expected 10 due to a constant loss frequency. In later stages, the frequency of plasmid-bearing cells appears to be reduced more rapidly (shown by the fully drawn lines) due to a slightly faster rate of growth of plasmid-free cells. The dotted line shows the curve which has been corrected for this larger growth rate to show the real loss rate.

In contrast, plasmids which are phenotypically Par are lost at a frequency of 1.5 x 10 6 / cell / generation for RI plasmids, 1 x 10 6 V cell / generation for p15 plasmids, and 6 x 10 cell / generation for certain pBR322 plasmids, an example of which is described in Example 3.3 The loss rates of pI5 Par and pBR322 PI derivatives are in fact surprisingly high considering the copy number of these vectors. 15-20 per cell, therefore, a loss rate of 10 "/ cell / generation can be predicted if the plasmids are assumed to be binomially distributed. However, the observed high loss frequency can be readily explained by the fact that p15 replicons form recA-dependent cohesive molecules, presumably due to the lack of a ΙοχΡ-like separation function (Austin et al.,

Cell 25, 1981, pp. 729-736). Also, pBR322 derivatives are known to form cohesive molecules, and when the copy number decreases due to, for example, 30 large cloned fragments, substantial instability follows.

MATERIALS AND METHODS

The strain of Escherichia coli K-12 used was CSH50 (Δ pro-lac, rpsL; cf. J. Miller, Experiments in Molecular Genetics, Cold Spring DK 167883 B1 15

Harbor, New York, 1972). Several plasmids and bacteriophages were used (Table 2).

The experimental techniques used were standard techniques used in the field of microbial genetics (J. Miller, Experiments in Molecular Genetics, Cold Spring Harbor, New York, 1972) and genetic engineering (Davis, Botstein and Roth, A Manual for Genetic Engineering ; Advanced Bacterial Genetics, Cold Spring Harbor, New York, 1980).

All cells were grown in LB medium (Bertani, J. Bact. 62, 1951, 10 p. 293) with 0.2% glucose and 1 µg / ml thiamine or A + B minimal medium (Clark and Måløe, J. Mol. Biol. 23, 1967, p. 99) enriched with 0.2% glucose and 1% casamino acids. The plates used were LA plates containing LB medium and 1.5% agar.

McConkey lactose indicator plates were prepared as recommended by the manufacturer (Difco), and X-gal plates were prepared by adding 20-40 µg / ml 5-bromo-4-chloroindolyl - [? - D-galactoside to A. + B minimal medium enriched with 0.2% glucose and 1 Mg / ml thiamine.

Physico-chemical methods

Clear lysates were prepared according to that of Clewell and 20 Helinski, Proc. Natl. Acad. Sci. USA 62, 1969, pp. 1159-1166, described method.

Small scale preparation of plasmid DNA was performed according to that of Birnboim et al., Nucl. Acids Res. 7, 1979. pp. 1513-1523, described method.

Large scale preparation and analysis of plasmid DNA was performed using "dye buoyant" density gradient centrifugation according to Stougaard and Molin, Anal. Biochem. 118, 1981, p.181.

Polyacrylamide gel electrophoresis and agarose gel electrophoresis of the DNA preparations were performed essentially as described by Molin and Nordstrom, Methods in Plasmid Biology, University of Odense, 1982.

DK 167883 B1 16

The restriction endonucleases were used in accordance with the instructions given by the manufacturer (Boehringer, Mannheim or Biolabs, New England), at 37 ° C. Double and triple digestion were performed by starting with the enzyme that required the lowest salt-5 concentration, and then adjusting with additional buffer before adding the next enzyme.

Treatment with the exonuclease Bal31 was carried out as follows: 0.1 unit Bal31 was added to 50 μg of linear DNA and samples were taken at 1 ', 2', 4 ', 8', 16 ', 32' and 60 'to 60 mM EDTA, extracted with 10 phenol, ethanol precipitated and resuspended in 20 μΐ TE buffer. Half of the 20 μΐ was digested with the appropriate restriction enzyme subjected to agarose gel electrophoresis to determine the average size of the DNA deletions. To the other half, the appropriate linker was added and the mixture was ligated for 48 hours with an excess of T4 DNA ligase.

Ligation of cleaved plasmid DNA was performed as recommended by the manufacturer with the exception of blunt ligation, where an excess of T4 DNA ligase and ATP was added.

Microbiological Methods 20 Distribution Test I: The construction of Lac + vectors made it possible to determine the Par + phenotype of a plasmid by simply smearing on non-selective McConkey lactose or X-gal plates. Bacteria (Δ lac) containing these plasmids mediate a Lac + phenotype that is readily observed as stained colonies on the indicator plates, whereas plasmid-free cells have a Lac phenotype and act as colorless colonies.

Distribution Test II (used for Lac plasmids): A colony from one selective plate (one plate containing an antibiotic) was plated onto another selective plate. From this plate, one colony was plated on an LA plate to form single colonies. From the LA plate, approx. 10 colonies suspended in 1 ml of 0.9% NaCl to a dilution of 10 "and 10" - *, respectively. The dilutions were plated out on IA plates. From these plates, 50 colonies resistance pattern (200 colonies if slight instability is expected) was tested for the appropriate The loss frequency (LF value) is then calculated on the basis of the formula 5 LF = 1 - (u) ^ / 27) where v stands for the frequency of plasmid-bearing cells and assuming that one colony grows for 27 generations. this method is a major statistical fluctuation.

Distribution Test III: Quantitative Measurements of the Stability of Lac + and 10 Lac Plasmids. An entire colony was taken from a selective plate and resuspended in 1 ml of 0.9% NaCl to a concentration of 10® cells / ml.

2 x 0.1 ml of the 10'3 dilution was used to inoculate 2 x 10 ml of LB medium and the inoculation was performed at 30 ° C with shaking. At a cell density of approx. 5 x 10 6 cells / ml, the cultures were diluted 10 ^ 15 and 105 times. 0.1 ml of the 104 dilution (5 x 10 3 cells) was used to inoculate 10 ml of fresh LB medium and 0.4 ml of the 103 dilution was plated on McConkey lactose plates and the plates were seeded at 30 ° C. overnight. The dilution from 5 x 10 6 / ml to 20 5 x 102 / ml corresponds to 20 generations of growth (2), so that the change in the frequency of plasmid-bearing cells from one dilution to the next corresponds to the change that occurs during of 20 generations of growth. More generally, the LF value can be calculated as follows: = (1 - LF) ®1 and v2 = (1 - LF) §2 25 where v ^ and 1/2 are the frequency of plasmid-bearing cells after g ^ and g2, respectively, and LF is the loss rate per day. cell per generation. It follows that j / j / j / 2 = (1 - LF) SrS2

S °

30 LF = 1- (1 ^ / 1/3) (1 / (Sl'S2)) DK 167883 B1 18

Applying this formula avoids errors due to fluctuations in the number of grafting cells at time 0. A more appropriate write-up of this formula is LF = In (i / 1 / i / 2) / (g2-gl) 5 Incompatibility tests

Plasmids were screened for presuming to carry an inserted pair region by utilizing the observation that two otherwise compatible replicons carrying the same pair region are incompatible with each other, causing loss of one of the two plasmids in absence of se-10 lesson pressure. The assay is performed by transforming the plasmid to be tested into a bacterial strain carrying a second plasmid, selecting for both plasmids on double-selective plates. After plating on a double-selective plate (a plate containing two different antibiotics), incompatibility was measured either qualitatively or quantitatively.

For the qualitative incompatibility test, a colony from the double-selective plate was plated onto an LA plate to form single colonies. About 10 colonies from this plate were resuspended in 1 ml of 0.9% NaCl and diluted to 10 "+ and 10" - *, respectively. 0.1 ml of the 20 10 "- and 10" - * dilutions were plated on IA plates. From these plates, 50 colonies (or 200 colonies, if slight incompatibility was expected) were tested on the appropriate selective plates.

If a Lac + plasmid was included in the test, McConkey lactose indicator plates were used instead of replica smears on 25 selective plates. Thus, in the case of Lac + plasmids, the screening was performed by transforming putative Par + plasmids into a strain which already contained a Par + hybrid plasmid mediating a Lac + phenotype. Plasmid incompatibility and thus evidence of a specific Par + phenotype of the incoming plasmid is readily detected by screening for Lac colonies on McConkey plates, showing that the Par + plasmid first found in the cell had been destabilized. An example of this screening procedure is described in Example 4.

DK 167883 B1 19

Quantitative incompatibility measurements were performed by measuring the loss rates of Lac + plasmids after establishing heteroplasmid populations as described above. The LF values were measured as described in the "Distribution Test III" section.

5 Genetic techniques

Transformation of bacteria was performed according to Cohen et al.,

Proc. Natl. Åcad. Sci. USA 62, 1972, pp. 2110-2114, with the modification that when a low transformation rate was expected, treatment of the competent cells with DNA on ice was prolonged for several 10 hours, and the cells were re-cooled on ice for 5 hours. -30 minutes. This treatment significantly increased the frequency of transformation.

Infection with bacteriophage λ was performed by growing 10 ml cultures overnight in LB medium enriched with 0.2% maltose (to induce the 15 malB protein, which is the λ receptor) with shaking. The cells were washed with 0.9% NaCl and resuspended in 2 ml of 0.01 M magnesium sulfate and incubated for 1 hour. Dilutions of the λ suspension (10®, 10 ', 10'2) were prepared and 0.1 ml of the phage dilution was used to infect 0.2 ml of the suspension of starved cells. The 10®, 10-1-20 and 10'2 dilutions of the infectious mixtures were plated on selective plates.

For transposing plasmids with Tn5 (KmR), whose source was the phage λ b221 :: Tn5, whose attachment site had been deleted so as not to lysogenize, a suspension of phage was used to infect cells. which contained the plasmid to which the transposition was desired. The infection was performed as described above and kanamycin resistant cells were selected. Several thousands of kanamycin resistant colonies were collected and 0.1 ml of a 10'2 dilution thereof was used to inoculate 100 ml of LB medium.

The culture was grown overnight and plasmid DNA was prepared from this mixture of cells and used to transform E. coli K12 strain CSH50 into kanamycin resistance.

DK 167883 B1 20

Table 2

Plasmids and phages

Plasmid / phage Source 5 __ pKN184 Nordstrom et al., Plasmid 4, 1980, p. 322.

pSF2124 So et al., Mol. Gen. Genet. 142, 1975, pp. 239- 249.

pJL99 Light & Molin, Mol. Gen. Genet. 184, 1981, pp. 56-106.

pGA46 An & Friesen, J. Bact. 140, 1979, pp. 400-407.

pKN501 Molin et al., J. Bact. 138, 1979, pp. 70-79.

pF1403-II Constructed by inserting the AluI-Wael fragment from the basal replicon of plasmid F into the Sma I-15 site of pMC1403 (Casadaban et al., J. Bact.

143, 1980, p. 971), thereby forming a fusion between the E gene from plasmid F and the lacZ gene from pMC1403.

pHP34 Prentki et al., Gene 17, 1982, pp. 189-196.

20 pMC903 Casadaban et al., J. Bact. 143, 1980, p.971.

pKN1562 Molin et al., J. Bact. 138, 1979, pp. 70-79.

pVH1424 Constructed by P. Valentin-Hansen by inserting a Sau3A fragment carrying the deo promoter from plasmid pVH17 (Valentin-Hansen et al., EMBO J., 25 1982, p. 317) into the BamHI site of plasmid pMC1403 (Casadaban et al., op. cit., p. 971). pSKS104 Constructed by M. Casadaban by inserting a

FvuII fragment containing the lac promoter and translation initiation region of pM13mp7 (Messing 30 et al., Nucleic Acids Res. 9, 1981, p. 309) at the Sma I site of pMC1403 followed by homologous recombination between the lacZ segments. pBR322 Bolivar et al., Gene 2, 1977, p. 95.

pMBl Betlach et al., Fed. Proc. 35, 1976, pp. 2037-35 '2043.

λ b221 :: Tn5 D.E. Berg, "Insertion and excision of the transposable kanamycin resistance determinant Tn5", in DK 167883 B1 21 DNA Insertion Elements, Plasmids and Episomes, (ed. A.I. Bukhari et al.).

EDA4 Dempsey and Willetts, J. Bact. 126, 1976, p. 166.

EXAMPLE 1

Insertion of Tn5 (KmR) into the Eco RI-A fragment of RI

Cells of E. coli K-12 strain CSH50 containing plasmid pKN184, a plasmid consisting of plasmid pSF2124 and the 19 kb EcoRl-A fragment (carrying the parA and parS regions) from plasmid RI (Nordstrom 10 et al. , Plasmid 4, 1980, p. 322), were infected as described in MATERIALS AND METHODS with a suspension of bacteriophage Ab211 :: Tn5.

After selection for kanamycin resistance on LA plates containing 200 µg / ml kanamycin, the colonies were collected and mixed. 0.1 ml of a 10 "dilution thereof was used to seed 100 ml of LB medium.

The culture was grown overnight and plasmid DNA prepared from the culture was used to transform E. coli K-12 strain CSH50, selecting for kanamycin resistance on plates containing 200 µg / ml kanamycin.

Hereby was found a plasmid, pOU1, which mediates resistance to 20 kanamycin and has an insertion of phage DNA corresponding to ca. 5 kb (5000 base pairs). The plasmid had a molecular weight / size of 34 kb, which was measured by producing plasmid DNA and assayed on agarose gels, and the following phenotype: KmR, ApR, ParA +, ParB +.

Plasmid DNA was prepared from pOU1 and the plasmid was mapped by restriction enzymes by purifying the plasmid DNA, cleaving it with one or more restriction enzymes and analyzing the resulting fragments by agarose gel electrophoresis (cf. Fig. 1). Hereby, the λ :: Tn5 fragment inserted into the Eco RI-A fragment of pKN184 was identified as the carrier gene encoding kanamycin resistance, and the location of the fragment was determined as shown in FIG. Plasmid pOU1 was used for further analysis and plasmid constructs.

22

Wolf Ib / OiSJ b l

The strain E. coli CSH50 / p0Ul is deposited in the Deutsche Sammlung von Microorganism, Grisebachstrasse 8, D-3400 Gottingen, hereinafter abbreviated to DSM, under the deposit number 2712.

The deposit was made in accordance with the Budapest Treaty.

EXAMPLE 2

Construction of a plasmid suitable for cloning pairs of + fragments

Plasmid pJL99 (Light & Molin, Hol. Gen. Genet. 184-, 1981, pp. 56-61) bears a fusion between the repA gene from plasmid RI and the lac operon; The plasmid mediates a Lac + phenotype. The pstI-SalI fragment carrying the repA-lac fusion was excised from pJL99 and inserted into pGA46, which is a pl5 replicon (An & Friesen, J. Bact. 140, 1979, pp. 400-407) to form of the plasmid pJL124. A map of this plasmid is shown in FIG. 2. Plasmid pJL124 also mediates a Lac + phenotype on McConkey lactose indicator plates, but it was found that insertion of DNA fragments with little or no promoter activity into the Psti site of pJL124 interferes with the activity of the repA promoter in such a way that these hybrids no longer appear as Lac + on McConkey lactose indicator plates. However, on the more sensitive X-gal indicator plates, 20 transformed colonies are Lac +, which shows that expression of β-galactosidase is decreased, but not completely suppressed in the hybrids. Therefore, pJL124 can be used to clone Psfcl fragments, since hybrid plasmids are readily detected on McConkey lactose indicator plates such as Lac transforms. Furthermore, since pJL124 is a pl5-25 replicon and thus unstable, which is readily detected as cells from which the plasmid is lost form colorless colonies on lactose indicator plates without selection pressure, it is also possible to screen on X-gal plates for Psfcl fragments capable of to stabilize pJL124.

DK 167883 B1 23

Thus, the program for the isolation of Psti pairs + fragments consists of the following steps: 1) Ligation of the pJL124. gap with PstI and Psti fragments from another plasmid.

2) Transformation to CSH50 with smear on McConkey lactose plates containing chloramphenicol.

3) Testing clones that appear as Lac on McConkey-lac toplates for stable inheritance of the Lac + phenotype on X-gal indicator plates (cf. MATERIALS AND METHODS).

The strain E. coli CSH50 / pJL124 is deposited in the DSM under the deposit number 2760.

The deposit was made in accordance with the Budapest Treaty. EXAMPLE 3

Stabilization of unstable plasmids with the parA and parB regions 15 1. Mini-Ri replicon

Plasmid p0U71 (DSM deposit number 2471), which is a runaway replication derivative of plasmid pKN1562, which includes the basal replicon of plasmid Ri, the λΡΡ promoter and the clgg repressor gene of phag EDX4, the gene encoding β-lactamase from Tn3 the transposon and a unique EcoRl site 20 (a detailed description of the construction of pOU71 can be found in the co-filing application of the present applicant, entitled "Plasmids with Conditional Uncontrolled Replication Behavior" filed on the same day as the present application), was cleaved with EcoRl, and EcoRl -A:: The tn5 fragment from pOUl (cf. Example 1) was inserted followed by ligation and transformation of E. coli strain CSH50, selecting kanamycin resistant cells on LA plates containing 50 pg / ml kanamycin.

The transformants were screened for pOU71's runaway replication phonotype by smearing on LA plates containing 50 µg / ml kanamycin DK 167883 B1 24 and incubation at 42 ° C. Cells containing runaway replication plasmids eventually cease to grow under these conditions.

Hereby plasmid pOU71-184 was identified, which plasmid had a size of 25.5 kb and the following phenotype: ParA +, ParB +, Ap®, Km® ·.

The plasmid was mapped with restriction enzymes as described in Example 1 (cf. Fig. 3). It appears from the restriction map that the PcoRl-A :: Tn5 fragment has been inserted correctly into the EcoRl site of pOU71.

Cells containing the plasmid were grown on LA plates for 100 generations without selection pressure, i.e. without being grown on plates containing an antibiotic; by determination according to the procedure described in MATERIALS AND METHODS (Partition Test II), it was not observed that the cells lose the plasmid, whereas pOU71 was lost from cells grown under similar conditions at a frequency of 1% per day. generation. Thus, it was determined that pOU71-184 is stably inherited with a loss frequency of approx. 10 '^ / cell / generation.

The strain E. coli CSH50 / pOU71-184 is deposited in the DSM under the deposit number 2763.

The deposit was made in accordance with the Budapest Treaty.

2. pl5 replicon 20 Plasmid pJL124 (cf. Example 2), which is a Par p15 replicon, was digested with restriction enzyme Pst I and mixed with plasmid pKN184 (cf. example 1), which had been partially cleaved with PstI, followed by of ligation. The ligation mixture was transformed into E. coli strain CSH50, selected for chloramphenicol resistance on 25 McConkey lactose indicator plates containing 50 Mg / ml chloramphenicol.

The cells containing pJL124 carrying one or more PstI fragments (cf. Example 2) were tested for stable inheritance of the Lac + phenotype on X-gal plates. One of the stably inherited plasmids was found to contain both the parA and parB regions. This plasmid, pOU2, has a size of 24 kb and the following phenotype: Cm®, Lac +, ParA +, ParB +.

DK 167883 B1 25

The plasmid was mapped with restriction enzymes as described in Example 1 (cf. Fig. 4). It is evident from the restriction map that a PstI fragment has been deleted from the PcoR1-A fragment.

The cells containing the plasmid were grown on X-gal plates for 100 generations without selection pressure, and it was determined that pOU2 is stably inherited with an LF value of less than 10 '/ cell / generation as opposed to pJL124, which was lost from the cells at a frequency of 0.5-1¾ / cell / generation.

The strain E. coli CSH50 / PO2 is deposited in the DSM under the deposit number 2713.

The deposit was made in accordance with the Budapest Treaty.

3. pMB1 replicon

Plasmid pF1403-II (cf. Table 2) is an unstable inherited derivative of pBR322 (a pMB1-r epicone), which bears a fusion between a gene from plasmid F and the lac operon, the plasmid mediates an Ap phenotype. The EcoRl-A :: Tn5 fragment from plasmid pOU1 (Example 1) was inserted into the unique? CoR1 site of plasmid pF1403-111 immediately above the gene fusion followed by ligation and transformation to E. coli strain CSH50 selected for Ap® · on plates containing 20 50 µg / ml ampicillin, Km 2 · on plates containing 50 µg / ml kanamycin and

Lac + on McConkey lactose indicator plates.

The resulting plasmid pOU10 was mapped with restriction enzymes as described in Example 1 (cf. Fig. 5) and the insertion of the Eco RI-A :: Tn5 fragment was verified. The plasmid has a size of 25 34 kb and the following phenotype: Km ^, Ap ^, Lac +, ParA +, ParB +.

The plasmid was tested for stable inheritance as described in Example 3.1. Thus, it was shown that pOU110 is stably inherited with an LF value of less than 10 µl / cell / generation, whereas pF1403-l1 went

ISLAND

lost from the cells at a frequency of 6 x 10 generation.

DK 167883 B1 26

The strain E. coli CSH50 / pOU10 is deposited in the DSM under the deposit number Ulk.

The deposit was made in accordance with the Budapest Treaty. EXAMPLE 4 Cloning of Psti fragments from the Eco RI-A fragment which stabilizes pJL124

The RcoR1-A fragment was cleaved with a number of restriction enzymes and the resulting physical map is shown in FIG. 1. It is apparent from this figure that the fragment consists of many Psti fragments. In order to reduce the size of the fragment mediating the Par + phenotype, attempts were made to subclone pair regions, possibly carried on one or more of the Psti fragments, into the screening vector pJL124 (cf. Example 2). Analysis of multiple clones with the correct phenotype - Lac on McConkey lactose plates and stable inheritance in the absence of selection pressure -15 showed that the Psti-D fragment (cf. Fig. I) mediated this phenotype.

No other single Psti fragment was able to stabilize pJL124. The region responsible for stabilizing pJL124, which was located within the 1.8 kb Ps 10 fragment, was designated parB.

To further analyze the Psti-D fragment, the fragment was cloned into the unique Pst1 site of the bla gene of pBR322, thereby forming pOU93 (cf. Fig. 7; the plasmid is deposited in DSM under deposition number 2724). Restriction mapping of this plasmid showed that the PstI-D fragment contains three Rsa I sites (cf. Fig. 6). Rsal forms 25 blunt ends, and therefore the 900 bp Rsal fragment was inserted into the SmaI site of plasmid pHP34 (Prentki et al., Gene 17, 1982, pp. 189-196) as follows. pOU93 was cleaved with Rsal and mixed with pHP34 which had been cleaved with SmaI, followed by ligation. The ligation mixture was transformed into E. coli strain CSH50, which already contained plasmid pOU94 (a p15 derivative phenotypically Lac + and ParB +; cf. Fig. 8; the plasmid is deposited in DSM under depot number 2725) , for ampicillin resistance was selected on plates containing 50 µg / ml ampicillin.

Due to the incompatibility expressed by parB + regions carried on different plasmids, pOU94 is lost when another 5 plasmid, which is phenotypically ParB +, is introduced into the E. coli cells, making it possible to select for correct inserts and transforms by ironing the cells onto McConkey plates and screening for colorless colonies (Lac). One such incompatible plasmid pHP34 derivative, which was found to contain the 900 bp Rsal fragment, was designated pOU13. The plasmid has a size of 5.3 kb and the following phenotype: TcR, ApR, ParB +.

The plasmid was mapped with restriction enzymes as described in Example 1 (cf. Fig. 9). The mapping revealed that the Rsal fragment had been converted to a 900 bp EcoRl fragment, with the SmaI site 15 of pHP34 flanked by two EcoRl sites.

Tribes £. coli CSH50 / pOU13 is deposited in DSM under deposit number 2716.

The deposit was made in accordance with the Budapest Treaty.

The 900 bp £ coRl fragment from pOU13 was inserted into EcoRl site 20 on pOUlOl, which is an ApR, CmR runaway mini-Rl derivative (a detailed description of the construction of pOUlOl can be found in the present application's simultaneously filed titled " Conditional uncontrolled replication behavior plasmids, filed on the same day as the present application), with a unique? CoR1 site in the cat gene (the gene encoding CmR) to generate plasmid pOU14 having a size of 8 , 2 kb and the following phenotype: Cm ^, ApR, ParB +.

The plasmid was kc. prepared with restriction enzymes as described in Example 1 (cf. Fig. 10).

The plasmid was tested for stable inheritance as described in Example 30 3.1. Thus, it was demonstrated that at 30 ° C, pQU14 is stably inherited with DK 167883 B1 28 an LF value of less than 1 x ICT 2 / cell / generation, whereas pOU101 is lost from the cells at a frequency of 1% per day. generation.

The strain E. coli CSH50 / pOU14 is deposited in the DSM under the deposit number 2717.

5 The deposit was made in accordance with the Budapest Treaty. EXAMPLE 5

Stabilization of mini-R1 plasmids with the parB fragment

Plasmid pOU90, which is a runaway replication derivative of pKN1562, which includes the basal replicon of plasmid RI, the AP 1 promoter and the clg ^ -10 repressor of phag EDA4, the gene encoding the β-lactamase from the Tn3 transposon and the fcoR1 A fragment from pKN184 (a detailed description of the construction of p0U90 can be found in the co-filed application of the present applicant, entitled "Plasmids with Conditional Uncontrolled Replication Behavior" filed on the same day as the present application) into which the derivative FcoR1-A fragment from pKN184 has been inserted , was cleaved with SalI and ligated to generate plasmid pOU61. The plasmid was transformed into E. coli strain CSH50, selecting for Lac phenotype on McConkey plates.

20 pOU61 was mapped with restriction enzymes as described in Example 1 (cf. Fig. 11). From the restriction map, it appears that pOU61 carries the 3.6 kb right end of the EcoR1-A fragment containing the parB region. The plasmid has a size of 10 kb and the following phenotype:

ParB +, ApR, Lac. The plasmid was tested for stable inheritance as described in Example 3.1. Thus, it was shown that pOU61 is inherited stably with an LF value of less than 1 x 10 6 / cell / generation.

The strain E. coli CSH50 / pOU61 is deposited in the DSM under the deposit number 2723.

The deposit was made in accordance with the Budapest Treaty.

Example 16

Stabilization of plasmid pF1403-II with the parB fragment

Plasmid pOU1 (cf. Example 1) was cleaved with SalI and mixed with plasmid pF1403-1, which had also been cleaved with SalI followed by 5 ligation and transformation to E. coli strain CSH50, selected for ampicillin resistant colonies on McConkey lactoseindika Torp-lets. Some of these Lac + clones were screened for stable inheritance of the Lac + phenotype on McConkey plates as described in MATERIALS AND METHODS.

The stably inherited plasmids were mapped with restriction enzymes as described in Example 1 (cf. Fig. 12). It is evident from the restriction map that they carry the SalI fragment from pOU1 on which the parB region is located. The plasmid consisting of pF1403-II and the SalI fragment from pOU1 was designated pOU12. It had a size of 16 kb and 15 following phenotype: ParB +, Lac +, Ap ^. pOU12 was inherited stably with an LF value of less than 5 x 10 6 / cell / generation.

The strain E. coli CSH50 / pOU12 is deposited in the DSM under the deposit number 2715.

The deposit was made in accordance with the Budapest Treaty.

EXAMPLE 7

Stabilization of p15 plasmids by the parA fragment

Plasmid pMC903 (Casadaban et al., J. Bact. 143, 1980, p. 971) was cleaved with EcoRL, and the FcoR1 fragment from plasmid pOU43 (cf. Figs.

13; DSM deposit number 2720) carrying the para region was inserted 25 followed by ligation and transformation to E. coli strain CSH50.

The resulting plasmid was designated pOU45 and had a size of 13.4 kb and the following phenotype: ParA +, Lac, Ap® ·, Km ^ ·.

30 UK Tb / bbJ b l

The plasmid was mapped with restriction enzymes as described in Example 1 (cf. Fig. 14). The restriction map shows that the parA region has been inserted into the lac operon, which is thereby inactivated.

When the plasmid was tested for stability as described in MATERIALS AND 5 METHODS, it was found to be inherited stably with a LF value of 8 x 10 6 / cell / generation, whereas pMC903 has a LF value of 1% / cell / generation.

The strain E. coli CSH50 / pOU45 is deposited in the DSM under the deposit number 2721.

10 The deposit was made in accordance with the Budapest Treaty. EXAMPLE 8

Stabilization of mini-R1 plasmids with the parA region £ coR1 fragment of pOU43 was inserted into a mini-R1 plasmid, pOU82 (DSM accession number 2482), comprising the basal replicon of plasmid RI, the λΡ ^ promoter and the cl repressor gene. from the phage EDA4, the gene encoding β-lactamase, from the Tn3 transposon, the deo promoter and the amino-terminal end of the lacZ gene from pVH1424, and the rest of the lac operon from pSKS104 (a detailed description of the construct of pOU82 is found in the present applicant's application at the same time filed application 20, entitled "Plasmids with Conditional Uncontrolled Replication Behavior", filed on the same day as this application).

Plasmid pOU82 mediates the Ap® and Lac + phenotypes and has a unique EcoRl site that is useful for cloning βcoRl fragments. This plasmid is lost at a frequency of 1% per day. generation in the absence of 25 selection pressures.

A plasmid into which the 2.4 kb long CoRl fragment had been inserted in one orientation was designated p0U47. The plasmid had a size of 14 kb and the following phenotype: ParA +, Lac +, Ap® \ DK 167883 B1 31 pOU47 was mapped with restriction enzymes as described in Example 1 (cf. Fig. 15).

The strain E. coli CSH50 / pOU47 is deposited in the DSM under the deposit number 2722.

5 The deposit was made in accordance with the Budapest Treaty.

The EcoRl fragment carrying the parA region was further reduced by 400 bp by the exonuclease Bal31; The deletion procedure was performed as described in MATERIALS AND METHODS using the unique BamHI site on p0U47. The engineered plasmid was designated 10 pUU472. The plasmid had a size of 13 kb and the following phenotype:

ParA +, Lac +, Ap® ·.

pOU472 was mapped with restriction enzymes as described in Example 1 (cf. Fig. 16). It appears from the restriction map that pUU472 carries the 2.0 kb EcoRl fragment generated by the BaI31 deletion.

When the plasmid was tested for stability as described in MATERIALS AND METHODS, it was found to be inherited stably, meaning that the entire parA region is contained in the 2.0 kb EcoRl fragment.

Strain E, coli CSH50 / pOU472 is deposited in DSM under deposit number 2726.

20 The deposit was made in accordance with the Budapest Treaty. EXAMPLE 9

Construction of a mini-R1 plasmid carrying the parA region

Plasmid pOU90 (cf. Example 5) was digested with BamHI and partially with Sau3A to delete a portion of the Eco RI-A fragment, preserving the 6 kb located to the far left, followed by ligation. The resulting plasmid pOU91 was transformed into E. coli strain GSH50. pOTJ91 has a size of 18.75 kb and the following phenotype: Pair +, DK 167883 B1 32

ApR, Lac +. The plasmid was mapped with restriction enzymes as described in Example 1 (cf. Fig. 17).

Plasmid stability was determined by culturing cells containing pOU91 (and as control cells containing pOU82) on McConkey 5 plates without selection pressure at 30 ° C for 25 generations; the cells transformed with pOU91 formed red colonies (Lac +), whereas cells transformed with pOU82 formed both red and white colonies, showing that during the selection-free period, pOU82 was lost from some of the cells.

The strain E. coli CSH / pOU91 is deposited in the DSM under the deposit number 2483.

The deposit was made in accordance with the Budapest Treaty. EXAMPLE 10

Construction of a parA +, parB + mini-RI plasmid 15 Plasmid pOU82 (cf. Example 8) is cleaved with EcoR1, and the EcoR1 fragment from pOU43 (cf. Example 7 and Fig. 13) is inserted into the parA region. From this EcoRl fragment, the 500 bp located at the far left, including one EcoRl site, is deleted by the exonuclease BaI31.

In the unique Eco RI site of this plasmid is inserted the Eco RI fragment from pOU13 20 (cf. Example 4 and Fig. 9). The resulting plasmid which mediates a ParA +, ParB + phenotype can then be transformed into E. coli strain CSH50, which already carries plasmid pOU94, and screened for essentially as described in Example 4 except that the pOU82 derivative mediates a weak Lac + phenotype, meaning that colonies of cells containing this plasmid will only be red in the center and colorless at the edge when grown on McConkey lactose indicator plates.

Claims (27)

1. Plasmid replicating in gram-negative bacteria, characterized in that it carries one or more inserted genes that are not naturally associated with the plasmid and which are stabilized by an inserted DNA fragment shorter than 19 kb, which DNA fragment comprises the plasmid stabilizing DNA sequence 20 RI pair region A, the plasmid stabilizing DNA sequence RI pair region B or both RI pair region A and RI pair region B. Plasmid according to claim 1, characterized in that the inserted DNA fragment comprises R 1 par region A and R 1 par region B and has a length not exceeding 25 6 kb, especially not 4 kb, in particular not 3 kb. A plasmid according to claim 1, characterized in that the inserted DNA fragment comprises RI pairs region A and has a length not exceeding 4 kb, especially not 2.5 kb, in particular not 2 kb. DK 167883 B1 34 A plasmid according to claim 1, characterized in that the inserted DNA fragment comprises RI pairs region B and has a length not exceeding 2 kb, in particular not exceeding 1.5 kb, in particular not 1 kb. A plasmid according to any one of claims 1-4, characterized in that it further comprises a gene encoding antibiotic resistance. A plasmid according to claim 1, characterized in that it is a pI5 plasmid or a derivative 10 thereof or a high copy plasmid with a broad host spectrum, or a derivative thereof. A plasmid according to claim 1, characterized in that it is a pMB1 plasmid or a derivative thereof, such as a pBR322 plasmid or a derivative thereof. A plasmid according to claim 1, characterized in that it has a low copy number at least in one stage during the cultivation of bacteria containing the plasmid. A plasmid according to claims 8, 20, characterized in that it has a copy number of approx. 0.5-5 copies per cell. A plasmid according to claim 8 or 9, characterized in that it is selected from plasmids of the incFII incompatibility group, including RI and derivatives thereof; 25. and derivatives thereof; and low-copy plasmids with a broad host spectrum and derivatives thereof. A plasmid according to claim 8 or 9, characterized in that it is a conditional runaway replication plasmid. DK 167883 B1 35 A plasmid according to claim 11, characterized in that when host microorganisms containing the plasmid are grown under conditions which ensure a low, constant plasmid copy, it has a copy number which does not exceed approx. 3-5 5 copies per cell, and, when the host microorganisms are cultured under other conditions which ensure a substantially increased plasmid copy, have a copy number in an area of at least about 500-1000 copies per cell. A plasmid according to claim 12, 10, characterized in that it has a plasmid capital of not more than ca. 3-5 copies per cell at one temperature, and a plasmid copolymer, which is in the region of at least ca. 500-1000 copies per cell at a higher temperature. Plasmid according to claim 12 or 13, 15, characterized in that the plasmid copy number, when it does not exceed approx. 3-5 copies per cell, is in the range of approx. 0.5-1 copy per cell. A plasmid according to claim 1, 12, 13 or 14, characterized in that an adjustable promoter is inserted above 20 for the native replication control gene R. of the plasmid. A plasmid according to any one of claims 12-15, characterized in that it is an RI-type plasmid. Gram-negative bacterium, characterized in that it contains a plasmid according to any one of the preceding claims. A bacterium according to claim 17, characterized in that it is Escherichia coli. A method of producing a gene product of plasmid DNA, characterized in that bacteria containing a plasmid 30 according to any one of claims 1-16 are cultured and the gene product of the plasmid is recovered from the bacterial culture. DK 167883 B1 36 Method according to claim 19, characterized in that the cultivation is carried out for at least 100 bacterial generations. Method according to claims 20, 5, characterized in that the loss of the plasmid is less than 2 x 10 6 / cell / generation. A method according to claim 21, characterized in that the loss of the plasmid is less than 10 "/ cell / generation. Method according to claim 22, characterized in that the loss of the plasmid is less than 5 x 10 6 / cell / generation. DNA fragment shorter than 19 kb, characterized in that it comprises RI pair region A, R15 pair region B or both RI pair region A and RI pair region B. The DNA fragment of claim 24 comprising the RI pair regions A and B, characterized in that it has a length not exceeding 6 kb, in particular not 4 kb, in particular not 3 kb. The DNA fragment of claim 24 comprising RI pair region A, 20 characterized in that it has a length not exceeding 4 kb, especially not 2.5 kb, in particular not 2 kb. The DNA fragment of claim 24 comprising the RI pair region B, characterized in that it has a length not exceeding 2 kb, in particular not 1.5 kb, in particular not 1 kb.