Cloning and/or sequencing vector

Details Cloning and/or sequencing vector Cloning and/or sequencing vector

1995-07-20

1999-06-08

Elliott, George C.

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

43525233, 4353201, C12N 121, C12N 1570

Patent

active

059104384

DESCRIPTION:

BRIEF SUMMARY
SUBJECT-MATTER OF THE INVENTION

The invention relates to a cloning and/or sequencing vector which enables recombinant clones to be selected directly.
The invention also relates to the procaryotic cell which is transformed by this vector and to the procaryotic host cell for this vector, as well as to the use of this cloning and sequencing vector for selecting and sequencing recombinant clones.


STATE OF THE ART AND TECHNOLOGICAL BACKGROUND UNDERLYING THE INVENTION

Phage (the M13 series) and plasmid (the pUC series) cloning vectors, containing numerous unique cloning sites, were constructed by Messing et al (P.N.A.S. USA, 79, pp. 3642-3646 (1977), by Norrander et al (Gene, 26, pp. 101-106 (1983) and Yanisch-Perron et al (Gene, 33pp. 103 to 119) (1985)).
The multiple cloning sites (MCS-multiple cloning sites) of these vectors are located in the coding sequence of the LacZ gene.
Discrimination between the transformed cells which harbor a recombinant vector and the cells which harbor a non-recombinant vector is achieved using the "blue screen" technique described by Gronenborn and Messing (Methylation of single-stranded DNA in vitro introduces new restriction endonuclease cleavages sites, Nature, 272, pp. 375-377 (1978)).
However, this "blue screen" technique suffers from the disadvantage of using a screening procedure (discrimination) rather than a procedure for selecting the clones.
Discrimination by screening is based on identifying a clone within a population of clones on the basis of a characteristic (color) which differentiates it. Selection has no need of this characteristic, since it is only recombinant clones which are isolated by this method.
The screening procedure is based on the color of the recombinant clones (white color) and of the non-recombinant clones (blue color). This color is based on inactivation of the marker beta-galactosidase, preventing cleavage of X-gal (5-bromo-4-chloro-3-indolyl .beta.-galactoside). The cell colonies harboring a non-recombinant vector produce a functional beta-galacto-sidase and, by hydrolyzing the X-gal substrate, produce a blue coloration. In general, the insertion of a DNA fragment into the .beta.-galactosidase gene prevents cleavage of the X-gal. For this reason, the cells harboring a recombinant vector have a white color.
Moreover, this complex procedure requires the use of the substrate X-gal which is a product which is very expensive, unstable and awkward to use.
On the other hand, various cloning vectors permitting direct selection (positive selection) of recombinant strains have been described in the scientific literature.
Pierce et al (Proc. Natl. Acad. Sci., vol 89. No. 6, 1992, pp. 2056-2060) describe a vector which comprises the lethal gene sac B from Bacillus amylolique-faciens, integrated into a plasmid derived from the bacteriophage P1 and under the control of a specific E. coli promoter.
The promoter of this vector includes a region having several specific cloning sites (cleavage site for a restriction enzyme).
Since the gene sac B encodes levan sucrase, which catalyzes the hydrolysis of sucrose into products which are toxic for E. coli, direct selection of the mutants which incorporate a recombinant plasmid is effected on a culture medium containing sucrose. Since the levan sucrase is toxic, even in the absence of sucrose, it is essential, consequently, to repress its synthesis if one wishes to obtain a large number of plasmid copies in the bacterial cytoplasm.
However, it is difficult, if not impossible, to repress the cytotoxic gene completely, particularly if a large number of copies of the vector are required.
Therefore, the impossibility of repressing the cytotoxic gene leads, in phases of producing the plasmid, to the death of the cell and, as a consequence, to selective pressure towards mutated strains (characterized by an inactive lethal gene).
In this case, in order to ensure that the enzyme encoded by the sac B gene does not kill the host cell, it is necessary to incorporate a CI repressor, which regulates the expression of this gene,

REFERENCES:
Pierce et al. (1992) Proc. Natl. Acad. Sci. USA 89:2056-2060. Mar. 1992.

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