GB2221908A - Recombinant plasmids and vectors and modified synechocystis cyanobacterium host - Google Patents

Abstract

A first plasmid comprises 6.5 kb; is obtained by treatment of a BamHI - XhoI restriction fragment of plasmid pUF311 with Klenow fragment of DNA polymerase I, with degradation of part of the fragment from the BamHI site and ligation to the SmaI fragment of 2 kb, containing the Sm<r> gene, of the E. coli plasmid pHP45 OMEGA; contains a fragment of 2.3 kb which corresponds to its "cyano-bacterial" part; and is resistant to streptomycin (Sm<r>). A second plasmid of 7.15 kb comprises a fragment containing the "cyanobacterial" part ligated to a 4.4 kb fragment of plasmid pFCLV7 carrying Km<r> and Cm<r> genes. The plasmids are used to transform Synechocystis sp. Active and passive promoter-assessing vectors, and expression vectors incorporating them, are also provided, the host/vector system permitting the expression of polypeptides uniformly labelled with <14>C.

Description

Recotnbinant Plasmids, Vectors comprising said Plaids, Method- for their production and modified strain of Synechocysti Cyanobacterium capable of being host to Sd id Piasm;ds and/or saici Vectors.

The present invention relates to recombinant plasmids, to vectors comprising said plasmids, to their methods of production and to a modified strain of Synechocysts cyanobacterium capable of being host to said plasmids and/or said vectors.

The cyanobacteria are photosynthetic Gramnegative bacteria characterized by a plant-type photosynthesis and, in the case of certain filamentous species, by a capacity to fix atmospheric nitrogen symbiotically or in the free state. A small number of these organisms are naturally transformable by their own DNA or, under certain conditions, by foreign DNA which has first been amplified and extracted from i. coli. The filamentous and nitrogen-fixing species do not seem to be tort-neously transformable, but systems for transferring genetic material exist which are based on the mobilization of plasmids by conjugating plasmids with a broad host range in Gram-negative organisms.

TOVEY K.C. et al. (Biochem. J., 1974, 142, 47-56) showed that certain strains of Synechococcua, in particular the strain 7942, are sufficiently radioresistant to be able to grow in pure loCO2 and hence to permit the production'of biomass uniformly label with 14C, in particular amino acids and sugars. This, coupled with appropriate methods of purification, provides what has become a standard method of producing amino acids and sugars uniformly labeled with 14C.

FRIEDBERG et al. (Mol. Gen. Genet., 1986, 203, 505-510) presented plasmid systems which utilize lambda.

bacteriophage regulation signals to accomplish a regulated expression of genes cloned in a strain derived from Synechococcus R2. These plasmid systems carry the operator-promoter region and the cIte gene of the thermosensitive repressor of the lambda bacteriophage. In said article, to study the function of the lambda regulation signals in R2, FRIEDBERG et al. inserted a DNA fragment carrying the cat gene into a Synechococcus R2 recombinant plasmid. This gene codes for chloramphenicol acetyltransferase and makes it possible, by measurement of the enzymatic activity, to demonstrate that the expression can be regulated by temperature. However, the system proposed by FRIEDEERG et al. does not enable a correct regulation to be obtained, either at low temperature or at high temperature.

TANDEAU de MARSAC et al. (Mol. Gen. Genet., 1987, 209, 396-398) described in said article the expression of the genes for larvicidal polypeptide toxins of Bacillus sphaericus 1593 M in a strain of Synechococcus R2 or PCC7942. Bacillus sphaericus is one of the spore-forming bacteria and synthesizes a powerful entomocidal toxin active against the larvae of different kinds of mosquito.

The strain of Synechococcus R2 has proved to be a preferred cyanobacterium for this expression.

The transformation of the strain of Synechococcus R2 is carried out by Van Den Handel s method.

Thesevarious studies refer to the use of the cyanobacterium Synechococcus R2 as a host cell.

Itsis known to be very difficult to choose an expression vector for an appropriate host. An ideal vector is expected to be stable, to be present in a constant and high number in each cell (so as to increase the number of copies of the gene which are capable of being translated into proteins), to be able to function as a shuttle vector, to contain genes whose products make it possible to exercise effective selection on the cell which contains it, such as resistance to antibiotics, and to possess an expression system which permits the copious production of polypeptides or proteins.

In an article published in 1986 in Mol. Gen.

Genet., 204, 185-191, the inventors showed that another cyanobacterium, namely the cyanobacterium Synechocystis PCC 6803, contains 4 cryptic plasmids (pUS1, pUS2, pUS3 of 2.27 kb and pUS4 of 5.20 kb); pUS2 and pUS3 have a very similar structure and are collectively called pUSA.

From these plasmids they obtained two kanamycin resistance (Kmr) recombinant plasmids (pUF3 and pUF12) by cloning of the HpaI restriction fragments of pUS3 and pUS2 respectively at the HindII site of the ampicillin resistance gene (Apr) of the E. coli plasmid pACYC177.

The plasmids pUF3 and pUF12 carry an HpaI fragment of 1.7 kb originating from the plasmid pUSA and are both capable of replicating in E. coli and of rendering the cyanobacterium Synechocysti 6803 resistant to kanamycin (Kmr).

The adjacent fragments of 1.7 kb and 50 bp cloned from the plasmid pUS2 into the plasmid pUF12 carry all the information necessary for effective replication in Synechocystis 6803.

This does not apply in the case of the HpaI restriction fragment of 1.7 kb cloned from the plasmid pUS3 into the plasmid pUF3, because the plasmid pUF3 is unstable in the cyanobacterium and is replaced with the recominant plasmid pUF311.

This different behavior of the plasmid pUF3 and the plasmid pUF12 may result from the opposite orientation of their HpaI cyanobacterial inserts of 1.7 kb, from the absence in pUF12 of a HindIII site characteristic of pUF3, and from the presence in pUF12, but not in pUF3, of the HpaI fragment of 50 bp.

The inventors found an average number of copies per cyanobacterium of one and twelve molecules for pUF12 and pUF311 respectively. The HpaI fragments of pUS2 of 0.32 kb and 0.25 kb, not cloned into pUF12 but present in pUF311, could be involved in controlling the number of copies of plasmids in Synechocystis 6803.

The recombinant plasmid pUF311 was used to construct the Cmr Kmr shuttle vector pFCLV7 of 9.35 kb, which is equivalent to a plasmid pUF311 (8 kb) with an HaeII fragment of 1.35 kb carrying the Cmr gene of the E.

coil plasmid pACYC184.

The wild type cyanobacteria Synechocystis 6803 are only transformed to a small extent by the shuttle vectors pUF12, pUF311 and pFCLV7; however, when the recipient cyanobacterium already contains a plasmid pUF311 (strain SUF311), the efficacy of the transformation by the plasmid pFCLV7 is increased about 40-fold.

In said article, the inventors developed a cloning system for the cyanobacterium Synechocystis 6803 by constructing a suitable shuttle vector and a highly transformable host strain.

The principal components of this system are: - the Cmr Kmr shuttle vector pFCLV7 of 9.35 kb, which carries three unique restriction sites in the Kmr gene (ClaI, SmaI, XhoI) and one in the Cmr gene (EcoRI), and - the host strain SUF311, which is highly transformable by pFCLV7, said transformation frequency being due to theexistence of a homologous recombination between this vector and the hybrid plasmid pUF311 residing in the strain of cyanobacterium.

Continuing their work in this direction, the inventors consequently set out to provide a novel host strain and an expression vector which are highly compatible and which permit the expression of large numbers of polypeptide molecules. The aim of the inventors was to find a host/vector system which permits the expression of large numbers of polypeptides, and especially hormones, uniformly labeled with 14C This host/expression vector system has a number of advantages, especially as regards the expression and production of uniformly labeled polypeptides, compared with the chemical methods of labeling (in particular the tritium method), which only label a few amino acids.

The present invention thus has the advantage of producing substances which are uniformly labeled and whose metabolism, in particular, can be followed very precisely.

Said expression vector/host cell couple, as defined in the invention, also has the advantage of permitting the industrial production of restriction enzymes, animal toxins or algicides/herbicides which are also of great interest to industry.

The present invention relates to a recombinant plasmid having the following properties: - it comprises 6.5 kb; - it is obtained by the treatment of a BamHI XhoI restriction fragment of the plasmid pUF311 with the Klenow fragment of DNA polymerase I, with degradation of part of the fragment from the BamHI site and ligation to the SmaI fragment of 2 kb, containing the Smr gene, of the E. coli plasmid pHP45R; - it contains a fragment of 2.3 kb which corresponds to its cyanobacterial part; and - it is resistant to streptomycin (Smr).

This recombinant plasmid has been called pFCQ5 by the inventors.

According to the invention, said recombinant plasmid is deposited under no. I-784, dated 19 July 1988, in the National Collection of Microorganism Cultures held by the Institut Pasteur.

The present invention further relates to a recombinant plasmid having the following properties: - it comprises 7.15 kb; - it is obtained by the ligation of a BamHI PstI restriction fragment of 4.4 kb of the plasmid pFCLV7 carrying the kanamycin resistance gene (Km) and chloramphenicol resistance gene (Cmr) and an origin of replication of E. coli, with the BamHI - Psti fragment of 2.75 kb of the recombinant plasmid according to the invention, pFCQ5, which contains the cyanobacterial part of 2.3 kb.

This recombinant plasmid has been called pFC57 by the inventors.

The term "cyanobacterial part" of the plasmid denotes the fragment of 2.3 kb which permits the autonomous replication of the plasmid containing it in a Synechocystls sp. and especially in Synechocystis 6803.

These two recombinant plasmids according to the invention are homologous with the exception of their E. coli part carrying the antibiotic resistance genes.

The present invention further relates to a promoter-assessing vector making it possible to test under appropriate conditions the efficacy of promoter sequences, which vector comprises: (a) a DNA segment containing a functional origin -of replication of the E. coli plasmid pACYC177 and a kanamycin resistance gene (cm), permitting the selection of transformants in the cyanobacterium; (bthe chloramphenicol transacetylase (cat) genz devoid of its promoter; (c) immediately upstream of this gene, a multiple cloning site into which an appropriate promoter sequence can be introduced; and (d) upstream of the multiple cloning site, the transcription-terminating signal of protein 32 of the T4 coliphage, said so-called passive promoter-assessing vector being non-replicative as such in the cyanobacterium, but being capable of producing expression vectors in a cyanobacterium.

This passive promoter-assessing vector has been called pFF11 by the inventors.

The present invention further relates to a promoter-assessing vector making it possible to test under appropriate conditions the efficacy of promoter sequences, which vector comprises: (a) a DNA segment containing an origin of replication functional in E. coli and at least one gene for resistance to an appropriate antibiotic, permitting the selection of transformants in the cyanobacterium; (b) a gene which is devoid of its promoter but whose activity is easily detectable either by a simple enzymatic test, or by the fact that it imparts resistance to an antibiotic, or both; (c) immediately upstream of this gene, a multiple cloning site into which an appropriate promoter sequence can be introduced;; (d) upstream of this multiple cloning site, a transcription-terminating signal which prevents transcription from any other promoter which might be located -upstream of the one introduced at the multiple cloning site, said sequences (a), (b), (c) and (d) forming a promoter-assessing vector which is non-replicative as such in the~cyanobacterium, this vector being referred to hereafter as a passive promoter-assessing vector; and (e) a Synechocystis sp. plasmid fragment of 2.3 kb which corresponds to the cyanobacterial part of said plasmid and ensures its replication in the cyanobacterium, said sequences (a), (b), (c), (d) and (e) forming a promoter-assessing vector which is replicative in the cyanobacterium, making it possible to test the efficacy of promoter sequences in the cyanobacterium and to produce expression vectors, this vector being referred to hereafter as an active promoter-assessing vector.

The term "promoter" is understood as meaning any DNA sequence which is capable of ensuring the effective synthesis of the messenger RNA corresponding to a given gene.

The cloning sites can advantageously be the HindII, SalI, PstI and BamHI sites, but this does not imply a limitation.

The gene for resistance to an appropriate antibiotic can advantageously be the streptomycin resistance gene, the kanamycin resistance gene or the chloramphenicol resistance gene.

According to the invention, the gene devoid of its promoter (b) is selected from the group comprising especially the chloramphenicol transacetylase gene, the 0-galactosidase gene and the streptomycin resistance gene.

In an advantageous embodiment, the active promoter-assessing vector comprises: (1) a passive promoter-assessing vector pFF11, as described above; and (2) a Synechocystis sp plasmid fragment of 2.3 kb which corresponds to the cyanobacterial part of said plasmid.

Thus active promoter-assessing vector has been called pFF16 by the inventors.

In a variant, the active promoter-assessing vector making it possible to test under appropriate conditions the efficacy of promoter sequences comprises: (a) a DNA segment containing an origin of replication originating from a ubiquitous plasmid, which is functional both in E. coli and in the cyanobacteria, and at least one gene for resistance to an appropriate antibiotic, permitting the selection of transconjugants in the cyanobacterium; (b) a gene which is devoid of its promoter but whose activity is easily detectable either by a simple enzymatic test, or by the fact that it imparts resistance to an antibiotic, or both; (c) imDediately upstream of this gene, a multiple cloning site into which an appropriate promoter sequence can be introduced; and (d) upstream of this multiple cloning site, a transcription-terminating signal which prevents transcription from any other promoter which might be located upstream of the one introduced at the multiple cloning site, said promoter-assessing vector being replicative in the cyanobacterium, making it possible to test the efficacy of promoter sequences in said cyanobacterium, and being capable of producing expression vectors.

In an advantageous embodiment of this variant, the ubiquitous plasmid is an IncQ plasmid.

The IncQ plasmids have the advantage of avoiding the ccstruction of a plasmid comprising an origin isolated from a cyanobacterial plasmid and of allowing direct replication both in E. coli and in the cyanobacteria.

The ubiquitous IncQ plasmids are described especially in the article by T.J. SCHMIDHAUSER et al.

("Broad host range plasmid cloning vectors for Gramnegative bacteria') published in "Vectors: a survey of molecular cloning vectors and their uses (RODRIGUEZ R.L.

and DENHARDT D.T., editors, 1987, 285-332).

The present invention further relates to a microorganism obtained by genetic transformation, which is obtained by transformation of the cyanobacterium Syne chocystis sp. by the plasmid pFCQ5 according to the invention.

This microorganism has been called strain SFCQ5 by the inventors.

According to the invention, said microorganism is deposited under no. I-783, dated 19 July l9B8, in the National Collection of Microorganism Cultures held by the Institut Pasteur.

According to an advantageous provision, this strain exhibits substantial radioresistance.

The transformation of the microorganism according to the invention is principally due to a homologous recombination event between the resident plasmid and the transforming plasmid.

The present invention further relates to an expression vector which consists of: (a) a promoter-assessing vector according to the invention; and (b) a DNA sequence which includes an appropriate promoter, a so-called SHINE and DALGORNO sequence and a short oligonucleotide sequence which contains at least one nonsense codon in each of the three potential reading frames and prevents the initiation of translation upstream of the start codon provided by the gene itself, which expression vector is said to be unloaded,- i.e.

capable of receiving at least one DNA sequence or one gene coding for a given protein or polypeptide.

In a preferred embodiment of the expression vector, the promoter-assessing vector is a passive promoter-assessing vector.

Insanother embodiment of the expression vector.

the appropriate promoter is a constitutive promoter.

In yet another embodiment of the expression vector, the appropriate promoter is a promoter whose activity is modified as a function of the growth conditions.

According to one advantageous provision of this embodiment, the activity of the promoter is modified as a function of temperature under the effect of a thermosensitive repressor of said promoter, which repressor is included in the above-mentioned DNA sequence (b).

According to an advantageous form of this provision, said promoter is selected from the group com prising the lambda phage promoter PL and the lambda phage promoter PR, the repressor being produced in this case by allele 857 of the lambda coliphage cI gene.

According to another advantageous provision of this embodiment, the activity of the promoter is modified as a function of light.

The recombinant plasmid of 6.5 kb according to the invention is obtained by the treatment of a BamHI XhoI restriction fragment of 6.65 kb of the plasmid pUF311 with the Klenow fragment of DNA polymerase I, with degradation over a length of 2.1 kb from the BamHI site, and ligation to the SmaI fragment of 2 kb, containing the Smr gene, of the E. coli plasmid pHP45Q, followed by the preparation of an Smr clone which possesses the said recombinant plasmid after transformatIon in E. coli.

The recombinant plasmid of 7.15 kb as defined above, called pFC57 by the inventors, is obtained by the ligation of a BamHI - PstI restriction fragment of 4.4 kb of the plasmid pFCLV7 carrying the kanamycin resistance gene (Kmr) and chloramphenicol resistance gene (Cmr) and an origin of replication of E. coli, with the BamHI PstI fragment of 2.75 kb of the recombinant plasmid of 6.5 kb according to the invention, which contains the cyanobacterial part of 2.3 kb.

The passive promoter-assessing vector as defined above is obtained by: (1) treatment of the plasmid pFC57 with at least one appropriate restriction enzyme so as to form a fragment devoid of,its cyanobacterial part; (2) treatment of an E. coli plasmid containing the Smr gene with at least one appropriate restriction enzyme so as to form a plasmid fragment containing all or part of the Smr gene, this fragment subsequently permitting recombination in vivo, in the cyanobacterium, between the passive vector and the region carrying the Smr gene on the plasmid pFCQ5 present in the cyanobacterium;; (3) ligation between the fragments prepared in (1) and (2) so as to prepare a so-called "intermediate" plasmid, which is treated with at least one appropriate restriction enzyme so as to prepare a fragment devoid of the Cmr gene initially present on the plasmid pFC57; (4) treatment of an E. coli plasmid containing the coding sequence of a gene whose expression in the cyanobacterium is easily detectable, such as the chloramphenicol transacetylase gene, the D-galactosidase gene or the streptomycin resistance gene, with at least one appropriate restriction enzyme so as to prepare a fragment containing the coding sequence of said gene, but not its promoter, and having, upstream of this coding sequence, at least one unique cloning site making it possible to introduce the promoter or promoters whose expression it is desired to test in the cyanobacterium; and (5) ligation between the fragments prepared in (3) and (4) so as to prepare a plasmid which is the passive promoter-assessing vector.

Th-ligation of the three constituent fragments of this vector is effected either successively, the order of ligation of the fragments being arbitrary, or simultaneously.

The unique cloning site of step (4) is itself advantageously introduced by an additional ligation or by directed mutagenesis.

The present invention further relates to an intermediate recombinant plasmid for the production of the passive promoter-assessing vector.

The passive promoter-assessing vector, called pFF11, is obtained by the following successive steps: (1) the N. coli plasmid pKK232-8 containing the N-terminal region of the cat gene devoid of its promoter is treated with the restriction enzymes BamHI and EcoRI so as to give a BamHI - EcoRI fragment of 0.27 kb of said plasmid; (2) a ligation is effected between the fragment obtained in (1) and the EcoRI - BamHI fragment of 6.3 kb originating from the recombinant plasmid pFC57 of 7.15 kb according to the invention, and carrying the C-terminal region of the cat gene, which is capable of reconstituting, with the above fragment of 0.27 kb, the entire cat gene devoid of its promoter, and the kanamycin resistance gene ( Kmr ), so as to give an intermediate plasmid called pFF4 by the inventors;; (3) said intermediate plasmid is treated with the rrriction enzymes BamHI and FspI so as to give a BamHI - FspI fragment of said intermediate plasmid pFF4; and (4) a ligation is effected between the fragment obtained in (3) and a BamHI - XmnI fragment of 1.7 kb of the B. coli plasmid pHP45Q carrying the streptomycin resistance gene and the transcription-terminating signal of protein 32 of the T4 coliphage.

Thpresent invention further relates to a method of producing the active promoter-assessing vector comprising the plasmid fragment of 2.3 kb of cyanobacterial origin, according to the invention, wherein the plasmid fragment of 2.3 kb, which corresponds to the cyanobacterial fraction of a recombinant plasmid according to the invention, is introduced in vivo, in a strain of Synechocystis according to the invention into a passive promoter-assessing vector, which fragment is introduced by recombination between the homologous regions of the promoter-assessing vector and the recombinant plasmid pFCQ5 present in said strain.

An expression vector according to the invention is obtained by the ligation of an appropriate E. coli plasmid fragment, obtained by suitable treatment with at least one appropriate restriction enzyme, with a promoter-assessing vector according to the invention, linearized by treatment with at least one appropriate restriction enzyme, so as to form an unloaded expression vector ready for receiving at least one foreign DNA fragment which it is desired to express.

The expression vector called pFF17 is obtained by the ligation of the HinPI fragment of 0.87 kb of the E. coll plasmid pMY12-6 with the passive promoterassessing vector called pFF11, at the BamHI site of said assessing vector.

The present invention further relates to a method of loading an unloaded expression vector with at least one foreign DNA sequence, wherein at least one foreign sequence is introduced direct, in vitro, into said expression vector.

In an advantageous way of carrying out this method, the foreign DNA sequence is introduced upstream of the gene devoid of its promoter by cloning into one of the restriction sites downstream of the promoter.

According to a preferred provision of this way of carrying out the method, when the gene devoid of its promoter is the cat gene, cloning is effected into one of its four unique sites, namely BamHI, SalI, HincII and PstI.

The unloaded expression vector pFF17 can directly express the cat gene coding for a chloramphenicol transacetylase activity, for which there is normally no genetic information in the cyanobacterium Synechocystis sp.

The present invention further relates to a socalled loaded expression vector which is formed of an unloaded expression vector according to the invention, into which at least one foreign DNA sequence which it is desired to express has been inserted direct in vitro.

When the expression vector pFF17 is introduced into a Synechocystis sp. cyanobacterium according to the invention, i.e. carrying the plasmid pFCRS, a loaded expression vector, expressing the cat gene and called pFF18 by the inventors, is formed in said cyanobacterium.

The present invention further relates to a procedure for the expression of a foreign gene, which comprises using a loaded expression vector, as defined above, in an appropriate cyanobacterium and especially in a microorganism according to the invention.

In an advantageous embodiment of this procedure, expression is effected in the presence of a medium containing at least 95% of 14C, advantageously in the form of 14C02.

Said expression vector permits in particular the conditional expression of any DNA sequence at a temperature above 37"C. This vector, introduced into a cyanobacterium and especially into the modified cyanobacterium according to the invention, makes it possible to express genes for proteins or polypeptides, and especially for animal ordnes, in order to produce them in the form of polypeptides labeled with 14C.

In addition to the foregoing provisions, the invention also includes other provisions which will become apparent from the following description referring to Examples of how to carry out the method forming the subject of the present invention.

It must be clearly understood, however, that these Examples are given solely in order to illustrate the subject of the invention, without in any way implying a limitation.

EXAMPLE 1= METHOD OP PRODUCING THE PLASMID pFCQ5 (i) About 2 vg of the plasmid pUF311 are digested by the enzymes BamHI and XhoI and deposited on agarose gel. After electrophoresis, the DNA fragment of 6.65 kb is purified by electroelution followed by one extraction with phenol and two extractions with chloroform and then by precipitation in the cold in the presence of 2.5 M ammonium acetate and two volumes of ethanol.

(ii) About 2 vg of the E. coli plasmid pHP45Q (Prentki and Krisch, i984, Gene, 29, 303-313) are digested by the enzyme SmaI and deposited on agarose gel. The fragment of 2.0 kb is purified by electroelution and precipitated as in (i).

(iii) The fragment of 6.65 kb, taken up in a volume of 10 > 1 containing NT buffer (according to Maniatis et al., Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratory, 1982, 545 p.) and the four nucleotides dCTP, dTTP, dATP and dGTP, each at 200 nM, i incubated for 30 minutes at room temperature in the presence of two units of the Klenow fragment of E. coli DNA polymerase I, treated for 5 minutes at 800C and precipitated with ethanol as described in (i).

(iv) About 0.5 Jlg of an approximately equimolar mixture of these two fragments is resuspended in 10 v1 of ligation buffer (according to Maniatis et al., op. cit.) in the presence of five units of the DNA ligase of the T4 coliphage. After one night at 160 C, half this mixture is used to transform E. coli DH5a cells rendered competent by treatment with CaC12. These cells are plated in 100 IZl aliquots on to LB medium containing streptomycin at 20 vg/ml.

(v) After incubation for 24 hours at 370C, streptomycin-resistant colonies are observed. One of them was reisolated and its plasmid DNA was prepared by a rapid method from a 5 ml culture and then by preparation from a 250 ml culture, followed by purification of the plasmid on a CsCl gradient. A physical map of the plasmid was established by single or double digestion with several restriction enzymes, including SmaI, BamHI and XhoI. The corresponding plasmid has the properties described for the plasmid pFCQ5, i.e. it results from a fusion between the SmaI fragment and the BamHI - XhoI fragment present in the ligation mixture (iv), the latter having been partially digested by an exonuclease from the BamHI site over a length of 2.1 kb.

EXAMPLE 2: METHOD OF PRODUCING THE PLASMID pFC57 (i) About 2 Jlg of the plasmid pFCLV7 are digested by the enzymes BamHI and PstI and deposited on agarose gel. After electrophoresis, the fragment of 4.4 kb is purified by electroelution and precipitated with ethanol as described in Example 1 (i).

(ii) About 4 ssg of the plasmid pFCQS are digested by the same enzymes and deposited on agarose gel. The fragment of 4.4 kb is purified by electroelution and precipitated with ethanol as described in Example 1 (i).

(iii) About 0.5 ssg of an approximately equimolar mixture of the two fragments is taken up in 10 ssl of ligation buffer, treated overnight at 160C with one unit of the DNA ligase of the T4 coliphage and used to transform competent E. coli DH5a cells as described in Example 1 (iv). After these cells have been plated on to LB medium containing kanamycin (50 sZg/ml) and chloramphenicol (30 g/ml), colonies are observed after incubation for 24 hours at 370C. One of these colonies, taken at random, was reisolated and its plasmid DNA was prepared as described in Example 1 (v).A physical map of the plasmid, established by digestion in the presence of various restriction enzymes (including BamHI and PstI), shows that it has the expected structure of the recombinant plasmid pFC57, except that the PstI site has not been reconstituted during cloning.

EXAMPLE 3: METHOD OF PRODUCING THE PASSIVE PRo?4TER- ASSESSING VECTOR pFFll (i) About 2 ssg of the plasmid pKK232-8 (Brosius, 1984, Gene, 27, 151-160) are digested by the enzymes BamHI and EcoRI and deposited on agarose gel. The fragment of 0.27 kb is purified by electroelution and precipitated with ethanol as described in Example 1.

(ii) About 3 ssg of the plasmid pFC57 are digested by the same enzymes and the fragment of 6.3 kb is purified by electroelution and precipitated with ethanol as described in Example 1.

(iii) About 0.5 pg of an approximately equimolar mixture of the two fragments is treated with one unit of the DNA ligase of the T4 coliphage and introduced by transformation intc competent E. coli DH5a cells as described in Example 1. After plating on to LB dishes containing kanamycin (50 ssg/ml), several colonies appear after incubation for 24 hours at 370C.

(iv) One of these colonies, taken at random, con -tains a plasmid whose restriction map (especially after digestion by the enzymes SalI, PstI, BamHI and EcoRI) corresponds to the expected map of a plasmid obtained by recombinatdn in vitro between the two fragments purified above. This plasmid was purified from a 250 ml culture by passage over a CsCl gradient; it was called pFF4.

(v) About 3 ssg of the plasmid pFF4 are digested by the enzymes BamHI and FspI and deposited on agarose gel, and the fragment of 3.7 kb is purified by electroelution and precipitated with ethanol as described in Example 1. The fragment of 1.7 kb, produced by the double digestion of about 4 ug of the plasmid pHP45Q by the enzymes BamHI and XmnI, is similarly prepared.

(vi) About 0.5 1g of an approximately equimolar mixture of the two fragments is treated with 5 units of the DNA ligase of the T4 coliphage and used to transform competent E. coli DH5a cells as described in Example 1.

After plating on to LB containing kanamycin (50 pg/ml) and streptomycin (20 pg/ml), colonies are obtained, most of which are also capable of growing on LB containing streptomycin (20 ssg/ml). The plasmid prepared from one of these colonies, taken at random, has the expected physical map of pFF11.

EXAMPLE 4: METHOD OF PRODUCING THE ACTIVE PRDHoTSR- ASSESSING VECTOR pFFi6 (i) 50 ml of a culture of Synechocystis SFCQS in the exponential growth phase (O.D. of about 1.0 at 580 nm), cultivated beforehand in three successive cultures on modified BG11 medium (Labarre et al., J. Bacteriol., 1987, 169, 4668-4673) containing 5 ssg/ml of streptomycin, at 30 , with orbital stirring (250 rpm) and with illumination at 3500 lux (white light), are harvested by washing in 10 ml of fresh medium and centrifugation at room temperature (10 minutes at 12,000 g).

(ii) These cells are taken up in a volume of fresh medium which is adjusted so that there are about 108 cells per ml. One ml of this suspension is placed in a sterile pyrex tube and 1 zg of the plasmid pFF11 (puri fied as described in Example 3), in 100 ml of a 10 mM Tris-HCl buffer (pH 7.5) which is 0.1 mM in respect of EDTA, is added. The mixture is shaken gently for a few seconds and incubated, without shaking, for 90 minutes under the illumination and temperature conditions described above.

(iii) The cells are plated in 100 ul aliquots on to dishes of the BG11 medium described above and incubated for 24 hours under the illumination and temperature conditions already described, after which 0.4 ml of a stock solution of kanamycin (5 mg/l) is added under the gelose. After incubation for at least one week under the same conditions, isolated colonies appear on the dishes and are purified by reisolation on BG11 medium containing 30 ssg/ml of kanamycin.

(iv) The cells of one of these colonies, taken at random, are inoculated into a 50 ml culture of modified BG11 medium containing kanamycin (50 zg/ml). After three days with orbital stirring under the conditions described above in (i), 1 ml of this culture is transferred to a fresh culture.

(v) From this last 50 ml culture in the exponential growth phase, the cells are harvested by centrifugation and the plasmid DNA is prepared according to the protocol of Chauvat et al., 1986, Mol. Gen. Genet., 204, 185-191. About 0.1 pg of plasmid is obtained and this is taken up in 100 > 1 of the Tris-HCl buffer (pH 7.5) described above. After the transformation of competent E. coli cells as described in Example 1, and plating on to LB containing 50 vg/ml of kanamycin, kanamycinresistant colonies are observed in 24 hours of incubation at 37"C. From one of these colonies, taken at random, the plasmid DNA is prepared as described in Example 1.

Its physical map is established by restriction analysis using various enzymes, including BamHI and EcoRI, and it is seen toorrespond to the expected structure of the plasmid pFF16.

EXAMPLE 5= METHOD OF PRODUCING THE EXPRESSION VECTOR pFF17 (i) About 4 ssg of the plasmid pMY12-6 (Taurimoto et al., 1982, Molec. Gen. Genet., 187, 79-86) are digested by the enzyme HinPI and deposited on electrophoresis gel. The fragment of 0.87 kb is prepared by electroelution, precipitated with ethanol as described in Example 1 and then treated with the Klenow fragment of E. coli DNA polymerase I as specified in Example 1.

(ii) About 1 pg of the plasmid pFF11 is digested by the enzyme BamHI, extracted with phenol and then twice with chloroform, precipitated with ethanol and then treated with the Klenow fragment as described in Example 1.

(iii) About 0.5 ssg of an approximately equimolar mixture of the fragment and the linearized plasmid is treated with 5 units of the DNA ligase of the T4 coliphage and introduced by transformation into competent DH5a cells as described in Example 1. This ligation makes it possible to obtain colonies which grow on LB containing chloramphenicol (50 ssg/ml) at 420C and are sensitive to the same antibiotic at 30"C. At least one of these colonies was found to correspond to cells carrying the plasmid pFF17, which corresponds to ligation of the HinPI fragment of 0.87 kb with pFF11, with the reconstitution of a single BamHI site on the two expected sites, the reconstituted site being the one immediately downstream of the lambda coliphage promoter PR carried by the HinPI fragment, as demonstrated by restriction analysis of the corresponding plasmid.

EXAMPLE 6: METHOD OF LOADING THE EXPRESSION VECTOR pFF17 WITH A FOREIGN DNA (i) About 3 yg of the plasmid pFF17 are treated with the enzyme BamHI, extracted with phenol and then with chloroform (two extractions) and precipitated with ethanol as described in Example 1. The DNA residue is redissolved in 48 l of CIP buffer (according to Maniatis, op. cit., p. 133) and treated for one hour at 37 in the presence of 0.02 unit of commercial calf intestine phpsphatase (CIP). After a further series of extractions with phenol and then with chloroform, the DNA is reprecipitated with ethanol.

(ii) About 2 ssg of the plasmid pMC1871 (Shapira et al., 1983, Gene, 25, 71) are digested by the enzyme BamHI and deposited on agarose gel. The fragment of 3.2 kb containing the sequence of the lacZ gene (coding for E. coli -galactosidase, devoid of the first eight amino acids and containing three stop codons immediately at the end of the coding sequence) is purified by electroelution, extractions with phenol and then with chloroform and precipitation with ethanol, as described in Example 1.

(iii) 0.5 pg of an approximately equimolar mixture is used to transform competent cells of the strain DH5a. After plating on to LB medium containing kanamycin (50 pg/ml) and streptomycin (20 ssg/ml), the colonies obtained after incubation for 24 hours at 370 are reisolated. From clones analyzed at random, it is shown (by digestion with various restriction enzymes, including BamHI, which should produce a fragment of 3.2 kb and a fragment of 6.3 kb) that at least one of them contains a plasmid of 9.5 kb having the expected restriction map of a plasmid resulting from integration of the lacZ gene into the plasmid pFF17.Analysis by ClaI digestion makes it possible to identify the orientation in which the lacZ fragment has been integrated, the orientation which permits expression under the control of the thermoregulatable promoter PR being that which produces a ClaI fragment ol-4.3 kb and one of 5.2 kb.

(iv) At least 100 1Ig of plasmid are prepared from a 250 ml culture of the corresponding clone on LB medium containing kanamycin (50 pg/ml), with purification of the plasmid on a CsCl gradient. This DNA is used to transform a 50 ml culture of Synechocystis SFCQ5 under the experimental conditions described in Example 4. After the transformed cells have been plated on to BG11 medium supplemented with kanamycin, colonies are observed after incubation for 24 hours as described in Example 4, and colonies are also observed after incubation for at least one week.

After reisolation, one of these colonies is inoculated on to the same 50 ml liquid culture medium and its plasmid DNA is prepared according to the protocol of Chauvat et al. (1986, Mol. Gen. Genet., 204, 185-191) and then used to transform competent E. coli DH5a cells.

After transformation on LB supplemented with kanamycin, colonies are.obtained after incubation for 24 hours at 37".

A plasmid of 12.5 kb is shown to be present on some of the corresponding clones (by means of the restriction map using various enzymes, but at least BamHI and ClaI), said plasmid resulting from recombination in vivo between the transforming plasmid (carrying the lacZ gene integrated into pFF17) and the plasmid pFCQ5 present in the strain of Synechocystis.

EXAMPLE 7: EXPRESSION OF CHwRJL'SPHENICOL TRANSACETYLASE IN THE EXPRESSION VECTOR pEF17 (i) About 1 Jlg of the plasmid pFF17 is used to transform a 50 ml culture of Synechocystis pFCQ5 according to the protocol described in Example 4.

After incubation for at least one week at 300 under 3000 lux of white light, kanamycin-resistant colonies are observed. A number of these colonies are reiso'ated the same medium and then transferred to dishes containing chloramphenicol at 10 ssg/ml and incubated at 30 and 39 under the conditions described above.

The majority of the colonies initially selected for their kanamycin resistance acquired chloramphenicol resistance at 39 , while at the same time remaining sensitive to this antibiotic at 300.

These properties indicate that the plasmid pFF11 (which carries the Kmr gene as well as the cat gene, the latter being under the control of the lambda coliphage promoter PR and its thermosensitive repressor) has acquired an origin of replication (corresponding to the fragment of 2.3 kb of cyanobacterial origin which is present in pFCQ5) functional in Synechocystis sp. by homologous recombination with the plasmid pFCQS initially present in the strain SFCQ5. Thus the cat gene can express (in a thermosensitive manner) in the cyanobacterium. This recombinant plasmid is called pFF18 by the inventors.However, the transformed strains have retained the plasmid pFCQ5, since a fraction of the molecules of pFCRS has not participated in recombination in vivo and has therefore remained intact.

(ii) To enrich the clones of cyanobacteria resulting from the transformation with plasmid pFF18, and to facilitate the spontaneous loss of the existing plasmid pFCQS, one of the clones resistant to kanamycin (and to chloramphenicol at 39 ) is streak-plated again on to medium containing kanamycin (300 pgSml) but devoid of streptomycin, and incubated for 5 days at 30". This operation is repeated five times. A 50 ml culture of BG11 medium containing kanamycin is inoculated using the last subculture. The Synechocystis plasmid DNA is extracted from this last culture by the method described in Example 4.The plasmid DNA is used to transform competent F coli cells (see Example 1), which are plated on to LB medium containing streptomycin (20 vg/ml) and incubated for 24 hours at 37". The selection on streptomycin corresponds to E. coli colonies which have acquired either the plasmid pFCQS or the recombinant plasmid pFF18. As the latter is also kanamycin-resistant, the percentage of molecules of plasmid pFF18 (relative to all the molecules of pFF18 and pFCQS in the cyanobacterium from which the DNA has been extracted) is determined by measuring the fraction of the streptomycin-resistant E. coli colonies which are also resistant to kanamycin when they are transferred on to LB medium containing the latter antibiotic.This percentage is typically of the order of 75%. Finally, it is shown that the E. coli colonies which are resistant to kanamycin and streptomycin (and which therefore supposedly harbor the plasmid pFFl8) are resistant to chloramphenicol when they are incubated at 390 or 420, but not at 300.

(iii) Using such an E. coli colony resistant to three antibiotics (conditionally as far as chloramphenicol is concerned), LB medium containing kanamycin is inoculated and the corresponding plasmid DNA is prepared as described in Example 1. This plasmid is analyzed by digestion with the appropriate restriction enzymes (but at least -BamHI and XhoI) in order to show that it does indeed have the expected structure of pFF18 (digestion by BamHI gives a band of 2.9 kb and a band of 6.5 kb).

(iv) Using the kanamycin-resistant Synechocystis clone subcultured in (ii), 100 ml of BG11 medium containing kanamycin are inoculated under 3000 lux of white light at 30 , as described above, until the optical density of the culture at 580 nm is about 1.0. Half the culture is then mixed with 50 ml of fresh medium preheated to 48 , and transferred at 39 for 20 hours, the other half being diluted twofold in fresh medium and cultivated's above at 30".

(v) Two ml of each culture are harvested by centrifugation in a microcentrifuge at 15,000 rpm (two minutes) and taken up in the same volume of fresh medium.

This operation is repeated, the cell residue this time being taken up in two ml of 50 mM Tris-HCl buffer (pH 8.0). The cells are frozen in a mixture of solid carbon dioxide and ethanol and crushed in an Eaton press under a pressure of 100 kg/cm2. The extract is gradually thawed in an ice bath and centrifuged for two minutes at 10,000 rpm at 4" and the supernatant is kept for determining proteins (by Bradford s method, 1976, Anal. Biochem., 72, 248-254) and chloramphenicol transacetylase (according to Shaw, 1983, Meth. in Enz., 43, 737-755).The 30 culture contains no detectable chloramphenicol transacetylase activity (background: < 20 units), whereas the 39 culture corresponds to a chloramphenicol transacetylase activity of 4200 units (one unit = one nmol of substrate degraded per minute and per mg of protein).

EXAMPLE 8: VECTOR CONTAINING A UBIQUITOUS PLASMID The IncQ plasmid used is the vector pKT210 described by BAGDASARIAN et al. (GENE, 1981, 1S, 237-247); this plasmid carries a streptomycin resistance gene (Smr) and a chloramphenicol resistance gene (Cmr).

Said vector is transferred in its entirety to the cyanobacterium, where it replicates autonomously and where the two antibiotic resistance genes are expressed so as to irntart a resistance phenotype to the cyanobacterial host, as well as a high level (1900 units of specific activity) of chloramphenicol transacetylase.

This transfer is effected by conjugation using, as the donor strain, a strain of E. coli which harbors, in addition to said plasmid, a mobilizing plasmid advantageously belonging to the class of the IncP plasmids and containing at least the genes necessary for plasmid transfer b-intercellular conjugation. The mobilizing plasmid is especially the plasmid pRK2013 constructed by FIGURSKI & HELINSKI (Proc. Natl. Acad. Sci. USA, 1979, 76, 1648-1652), the characteristic feature of which is to possess the transfer functions of the IncP plasmids combined with an origin of replication of the colK type, which is specific for replication in f. coli and which therefore prevents replication of the mobilizing plasmid in the cyanobacterium. The conjugation between donor cells (E. coli) and recipient cells (cyanobacteria) is effected by direct contact in a medium which permits a substantially equal growth rate of f. coli and cyanobacterium (for example the BG11 medium generally used for the cyanobacteria, with the addition of glucose at 50 mg/l to provide a source of carbon which can be metabolized by E. coli).

This coculture can be carried out either in a moderately agitated liquid medium for one to two days, or in a solid medium on a Petri dish, or by the more elaborate techniques of filter transfer which are commonly used for intercellular conjugation experiments in the laboratory. The cyanobacterial cells which have acquired the vector pKT210 are selected by growth on BG11 medium which is devoid of glucose but to which chloramphenicol has been added (10 mg/l), and are reisolated on the same medium after incubation for one week at 300C with illumination under the customary conditions for growing the cyanobacteria.The plasmid pKT210 is then extracted by the method described in Example 4 and used to transform competent E coli cells, which are plated on to LB medium containing streptomycin (20 mg/l) and incubated for 24 h at 37"C. The plasmids present in said transformants also impart chloramphenicol resistance and their restriction map is identical to that described by BAGDASARIAN et al.

(1981) for pKT210.

Said vector pKT210, associated with a thermoregulated promoter such as defined above, is an expression vector for protein in the cyanobacterium.

As is apparent from the foregoing description, the invention is in no way limited to those methods of execution, embodiments and methods of application which have now been described more explicitly; on the contrary, it encompasses all the variants thereof which may occur to those skilled in the art, without deviating from the framework or the scope of the present invention.

Claims (27)

WHAT TS crPATMRD TS

1. A recombinant plasmid having the following properties: - it comprises 6.5 kb; - it is obtained by the treatment of a BamHI XhoI restriction fragment of the plasmid pUF311 with the Klenow fragment of DNA polymerase I, with degradation of part of the fragment from the BamHI site and ligation to the SmaI fragment of 2 kb, containing the Smr gene, of the E. coli plasmid pHP45a; - it contains a fragment of 2.3 kb which corresponds to its cyanobacterial part; and - it is resistant to streptomycin (Smr).

2. A plasmid according to claim 1 which has been called pFCQ5 and which is deposited under no. I-784, dated 19 July 1988, in the National Collection of Icrornism Cultures held by the Institut Pasteur.

3. A recombinant plasmid having the following properties: - it comprises 7.15 kb; - it is obtained by the ligation of a BamHI PstI restriction fragment of 4.4 kb of the plasmid pFCLV7 carrying the kanamycin resistance gene (Kmr) ) and chloram- phenicol resistance gene (Cmr) and an origin of replica ~ tion of E. coli, with the BamHI - PstI fragment of 2.75 kb of the recombinant plasmid according to claim 1, which contains the cyanobacterial part of 2.3 kb.

4. A promoter-assessing vector making it possible to test under appropriate conditions the efficacy of promoter sequences, which vector comprises: (a) a DNA segment containing a functional origin of replication of the f. coli plasmid pACYC177 and a kanamycin resistance gene (Kmr), permitting the selection of transformants in the cyanobacterium; (b) the chloramphenicol transacetylase gene devoid of its promoter; (c) immediately upstream of this gene, a multiple cloning site into which an appropriate promoter sequence can be introduced; and (d) upstream of the multiple cloning site, the transcription-terminating signal of protein 32 of the T4 coliphage, said so-called passive promoter-assessing vector being non-replicative as such in the cyanobacterium, but being capable of producing expression vectors in a cyanobacterium.

5. A promoter-assessing vector making it possible to test under appropriate conditions the efficacy of promoter sequences, which vector comprises: (a) a DNA segment containing an origin of replication functional in E. coli and at least one gene for resistance to an appropriate antibiotic, permitting the selection of transformants in the cyanobacterium; (b) a gene which is devoid of its promoter but whose activity is easily detectable either by a simple enzymatic test, or by the fact that it imparts resistance to an antibiotic, or both; (c) immediately upstream of this gene, a multiple cloning site into which an apprcriate promoter sequence can be introduced;; (d) upstream of this multiple cloning site, a transcription-terminating signal which prevents transcription from any other promoter which might be located upstream of the one introduced at the multiple cloning site, said sequences (a), (b), (c) and (d) forming a socalled passive promoter-assessing vector which is nonreplicative as such in the cyanobacterium; and (e) a Synechocystis sp. plasmid fragment of 2.3 kb which corresponds to the cyanobacterial part of said plasmid and ensures its replication in the cyanobacterium, said sequences (a), (b), (c), (d) and (e) forming a promoter-assessing vector which is replicative in the cyanobacterium, making it possible to test the efficacy of promoter sequences in the cyanobacterium and to produce expression vectors, this vector being referred to hereafter as an active promoter-assessing vector.

6. A promoter-assessing vector according to claim 5, wherein the gene devoid of its promoter (b) is selected from the group comprising the chloramphenicol transacetylase gene, the 8-galactosidase gene and the streptomycin resistance gene.

7. A promoter-assessing vector according to claim 5 which comprises: (1) a passive promoter-assessing vector according to claim 4; and (2) a Synechocystis sp. plasmid fragment of 2.3 kb which corresponds to the cyanobacterial part of said plasmid.

8. An active promoter-assessing vector making it possible to test under appropriate conditions the efficacy of promoter sequences, which vector comprises: (a) a DNA segment containing an origin of replication originating from a ubiquitous plasmid, which is function both in E. coli and in the cyanobacteria, and at least one gene for resistance to an appropriate antibiotic, permitting the selection of transconjugants in the cyanobacterium; (b) a gene which is devoid of its promoter but whose activity is easily detectable either by a simple enzymatic test, or by the fact that it imparts resistance to an antibiotic, or both; (c) immediately upstream of this gene, a multiple cloning site into which an appropriate promoter sequence can be introduced; and (d) upstream of this multiple cloning site, a transcription-terminating signal which prevents trans cription from any other promoter which might be located upstream of the one introduced at the multiple cloning site, said promoter-assessing vector being replicative in the cyanobacterium, making it possible to test the efficacy of promoter sequences in said cyanobacterium, and being capable of producing expression vectors.

9. A promoter-assessing vector according to claim 8, wherein the ubiquitous plasmid is an IncQ plasmid.

10. A microorganism obtained by genetic transformation, which is obtained by transformation of the cyanobacterium Synechocystis sp. by the recombinant plasmid of 6.5 kb according to claim 1.

11. A microorganism according to claim 10 which has been called SFCQS and is deposited under no. I-783, dated 19 July 1988, in the Nationa' Collection of Microorganism Cultures held by the Institut Pasteur.

12. A microorganism according to claim 10 which exhibits substantial radioresistance.

13. An expression vector which consists of: (a) a promoter-assessing vector according to any one of claims 4 to 9; and (b) a DNA sequence which includes an appropriate promoter, a scalled SHINE and DALGORNO sequence and a short oligonucleotide sequence which contains at least one nonsense codon in each of the three potential reading frames and prevents the initiation of translation upstream of the start codon provided by the gene itself, which expression vector is said to be unloaded, i.e.

capable of receiving at least one DNA sequence or one gene coding for a given protein or polypeptide.

14. An expression vector according to claim 13 which is a passive promoter-assessing vector.

15. An expression vector according to claim 13, wherein the appropriate promoter is a constitutive promoter.

16. An expression vector according to claim 13, wherein the appropriate promoter is a promoter whose activity is modified as a function of the growth conditions.

17. An expression vector according to claim 16, wherein the activity of the promoter is modified as a function of temperature under the effect of a thermosensitive repressor of said promoter, which repressor is included in the above-mentioned DNA sequence (b).

18. An expression vector according to claim 17, wherein said promoter is selected from the group comprising the lambda phage promoter PL and the lambda phage promoter PR, the repressor being produced in this case by allele 857 of the lambda coliphage cI gene.

19. An expression vector according to claim 16, wherein the activity of the promoter is modified as a function of light.

20. A method of producing the active promoter-assessing vector according to claim 5, wherein the plasmid fragment of 2.3 kb, which corresponds to the cyanobacterial fraction of a recombinant plasmid according to any one of claims 1 to 3, is introduced in vitro, in a strain of Synechocystis according to any one of claims 10 to 12, into a passive promoter-assessing vector, which fragment is introduced by recombination between the homologous regions of the promoter-assessing vector and the recombinant plasmid pFCQ5 present in said strain.

21. A method of loading an unloaded expression vector according to any one of claims 13 to 19 with at least one foreign DNA sequence, wherein at least one foreign sequence is introduced direct, in vitro, into said expression vector.

22. A method according to claim 17, wherein the foreign DNA sequence is introduced by cloning into one of the restriction sites downstream of the promoter.

23. A method according to claim 21 or claim 22, wherein, when the gene devoid of its promoter is the cat gene, the foreign DNA sequence is introduced upstream of said gene of said vector by cloning into one of its four unique sites, namely BamHI, SalI, HincII and PstI, downstream of the promoter.

24. A loaded expression vector which is formed of an unloaded expression vector according to any one of claims 13 to 18, into which at least one foreign DNA sequence which it is desired to express has been inserted direct in vitro.

25. A procedure for the expression of a foreign gene, which comprises using a loaded expression vector according to claim 24 in an appropriate cyanobacterium and especially in a microorganism according to any one of claims 10 to 12.

26. A procedure according to claim 25, wherein expression is effected in the presence of a medium containing at least 95% of 14C, advantageously in the form of 14C02.

27. A labeled protein which is obtained by carrying out the expression procedure according to claim 25 or claim 26 and which is uniformly labeled.