ACTIVE PROMOTER VECTOR GAUGE IN CYANOBACTERIA

Abstract

An active promoter gauge vector in cyanobacteria, capable of providing expression vectors. This active promoter gauge vector comprises: - a gene lacking its promoter, but whose activity is readily detectable, either by a simple enzymatic test, or by the fact that it confers resistance to an antibiotic, or both; - immediately upstream of this gene, a multiple cloning site, into which a suitable promoter sequence can be introduced; - upstream of this multiple cloning site, a transcription terminator signal, which prevents transcription from any other promoter located upstream of the one introduced at the cloning site, and further comprises a DNA segment containing an origin of replication from a ubiquitous plasmid, functional in both E. coli and cyanobacteria, and at least one suitable antibiotic resistance gene, allowing the selection of transconjugators in cyanobacteria.

Description

This Addition relates to a new calf vector gauge of active promoter in cyanobacteria rie, capable of providing expression vectors. One of the actlf promoter gauge vectors conforming to the main patent is characterized by the fact that it comprises: (a) a DNA segment containing an origin of functional replication in E. col and a re- gene resistance to an appropriate antibiotic, allowing se- (b) a gene lacking its promoter, but whose activity is detectable either by a simple enzymatic assay, or by the fact that it confers resistance to an anti-inflammatory drug, or both; (c) immediately upstream of this gene, a multiple cloning site into which a suitable promoter sequence can be introduced; (d) upstream of this multiple cloning site, a transcription terminator signal that prevents transcription from any other promoter located upstream of the cell introduced into the multiple cloning site; and (e) a Synechocyst plasma fragment sp. of 2.3 kb, which corresponds to the cyanobacterial part- nothing of said plasma, said promoter gauge vector being replicative in the cyanobacterium, allowing testing of promoter sequences in said cyanobacterium and being able to provide expression vectors. According to the main patent, the active promoter gauge vector as defined therein constitutes part of a gene expression vector for proteins or polypeptides in a cyanobacterium to produce them as 14C-labeled polypeptides. Active promoter gauge vectors conforming to the main patent necessarily include a 2.3 kb fragment of a Synechosystzs sp. plasmid. which corresponds to the cyanobacterial part of said plas- mide. However, it would be interesting to have vectors that multiply in both E. coli and cyanobacteria while not containing cyanobacterial fragments; such vectors would be in particular, easier to build. The present invention aims to provide an active promoter gauge vector that replicates in both E. coli and Synechocystils sp. and provides expression vectors conforming to the main patent. The present invention relates to an active promoter gauge vector for testing in appropriate conditions for the effectiveness of promo- sequences tors in cyanobacteria and being able to provide expression vectors in a cyanobacterium, compre- nant: - a gene lacking its promoter, but whose activity is easily detectable, either by a simple enzymatic test, or by the fact that it confers resistance to an antibiotic, or both; immediately upstream of this gene, a multiple cloning site, into which an appropriate promoter sequence can be introduced; - upstream of this multiple cloning site, a transcription terminator signal, which prevents transcription from any other promoter that would be located upstream of the one introduced at the cloning site, ca- ractérlsé in that it further comprises a segment of DNA containing a replication origin from a plas- a ubiquitous medium, functional in both E. coli and cyanobacteria, and at least one gene for resistance to a suitable antibiotic, allowing the selection of transconjugators in cyanobacteria. According to an advantageous embodiment of said vector, the ubiquitous plasmid is an Inc Q plasmid. The ublqulitaires Inc Q plasmldes are notably- described in the article by TJ SCHMIDHAUSER et al., ("BroadHost Range Plasmid Cloning vectors for Gram-negative bacteria") published in "Vectors: a survey of molecular cloning vectors and their uses" (RODRIGUEZ RL and DENHARDT DT Eds, 1987, 285-332). Plasmas Inc Q have the advantage of avoiding the construction of a plasma diagram including an iso- origin derived from a cyanobacterial plasma and to directly allow replication in both E. coli and in cyanobacteria. In addition to the foregoing provisions, the invention also includes other provisions, which will emerge from the description that follows. The invention will be better understood with the help of the The following additional description refers to an example of the implementation of the process that is the subject of the pre- sense of invention. It must be clearly understood, however, that this example is given solely as an illustration of the object of the invention, and does not constitute in any way a representation of it. a limitation in a way. Example: Vector containing a ubiquitous plasmid. The Inc Q plasmid used is the pKT210 vector described in BAGDASARIAN et al. (GENE, 1981, 1U, 237-247). This plasmid carries a streptomycin resistance gene (Sm') and a resistance gene. chloramphenicol (CmR). The said vector is transferred into its integral found in cyanobacteria, where it replicates autonomously and where both antibiotic resistance genes are expressed in such a way as to confer a resistance phenotype to the cyanobacterial host, as well as a high level (1900 specific activity units) of chloramphenicol transacetylase. This transfer is achieved by conjugation in using as a donor strain a strain of E. coli he- containing, in addition to said plasmid, a mobilizing plasmid advantageously belonging to the class of IncP plasmids and containing at least the genes necessary for plasmid transfer by intercellular conjugation. A convenient example of a mobilizing plasmid is the pRK2013 plasmid constructed by FIGURSKI & HELINSKI (Proc. Natl. Acad. Sci. USA, 76, 1648-1652, 1979), whose characteristic is to possess the transfer functions of IncP plasmids combined with a colK-type origin of replication, which is specific to replication in E. coli and which therefore prevents the replication of the mobilizing plasmid in cyano- bacteria. Conjugation between donor cells (E. coli) and recipient cells (cyanobacteria) is achieved by direct contact in a medium that allows for a roughly equal growth rate of E. coli and the cyanobacteria (for example, the BG11 medium generally used for cyanobacteria, with the addition of 50 mg / l of glucose to provide a source of C metabolizable by E. coli). This co-culture can be performed in either liquid medium with moderate agitation for one to two days, solid medium on a Petri dish, or using the more elaborate filter transfer techniques commonly employed in laboratory intercellular conjugation experiments. Cyanobacteria cells bearing the pKT210 vector are selected by growth on glucose-free BG11 medium supplemented with chloramphenicol (10 mg / L) and re-isolated on the same medium after one week of incubation at 3°C ​​under illumination, under standard cyanobacterial growth conditions. The plasmid – pKT210 – is then extracted according to the procedure described in Example 4 and used to transform competent E. coli cells, which are plated and incubated for 24 h at 37°C on LB medium containing streptomycin (20 mg / L).The plasmids present in said transformants also confer resistance to chloramphenicol and have a restriction map identical to that described. by BAGDASARIAN et al. (1981) for pKT210. In accordance with the main patent, the said vec- The pKT210 teur associated with a thermoregulated promoter as defined in the Main Patent is a vector protein expression in cyanobacteria. As is clear from the foregoing, the invention is by no means limited to those modes of implementation, realization and application which... need to be described more explicitly, she en- On the contrary, it encompasses all the possible variations that can be seen. to keep in mind the technical expert's perspective on the matter, without straying from nor of the framework, nor of the scope, of the present invention. DEMAND 1') Active promoter gauge vector allows- so much to test under appropriate conditions the effectiveness of promoter sequences in cyanobacteria and being capable of providing expression vectors in a cyanobacterium, according to claim 5 of the main patent, comprising: a gene lacking its promoter, but whose activity is readily detectable, either by a simple enzymatic test, or by the fact that it confers resistance to an antibiotic, or both; - immediately upstream of this gene, a multiple cloning site, into which a suitable promoter sequence can be introduced; - upstream of this multiple cloning site, a transcription terminator signal, which prevents transcription from any other promoter that would be located upstream of the one introduced at the cloning site, ca- characterized in that it further comprises a segment of DNA containing a replication origin from a plas- a ubiquitous, functional antibody in both E. coli and cyanobacteria, and at least one gene for resistance to a suitable antibiotic, allowing the selection of transconjugators in cyanobacteria. 2) Vector according to claim 1, character- sterized in that the ubiquitous plasmid is an Inc Q plasmid.