Cloning vector

Details Cloning vector Cloning vector

2000-01-03

2002-01-08

Jones, W. Gary (Department: 1655)

Chemistry: molecular biology and microbiology

Vector, per se

C435S004000, C435S006120, C435S069100, C435S091100, C435S071200, C435S252100, C424S093210, C424S094610, C536S023200, C536S023700, C536S024100

Reexamination Certificate

active

06337208

ABSTRACT:

The present invention relates to cloning vectors, cloning methods and cloning kits. More particularly, the present invention relates to a cloning vector comprising a nucleic acid sequence encoding a polypeptide which when expressed is lethal for a bacterial host cell and at least one cloning site wherein the insertion of a foreign nucleic acid insert in the cloning site causes a disruption of the expression of the lethal polypeptide.
For many molecular biology applications DNA fragments are inserted into cloning vectors, transformed into bacterial cells and plated onto selection medium for individual colony isolation. Frequently, however, problems occur because host cells containing the desired recombinant vector containing the foreign DNA are only obtained with low frequency, particularly when blunt-ended DNA fragments are inserted into the vectors.
U.S. Pat. No. 5,843,656 discloses a recombinant clone selection system for expression in a host cell comprising a repressor gene coding for a repressor, a promoter for promoting the expression of the repressor gene, a restriction endonuclease cleavage insertion site located within the repressor gene or its associated promoter such that when a foreign nucleic acid is inserted at the insertion site, expression of the repressor gene is insertionally inactivated, a surface-expressed moiety gene and an operator functionally linked to the expression of the surface-expressed moiety gene such that when the repressor is bound to the operator expression of the surface-expressed moiety gene is repressed. Transformed host cells containing the foreign nucleic acids may be isolated via the expression of the surface-expressed moiety gene. The method, however, is elaborate and requires compared to standard cloning methods a considerably larger amount of time.
U.S. Pat. No. 6,891,687 claims a vector for cloning a DNA sequence into adenylate-cyclase-positive host cells, the vector containing a selection gene comprising a modified CAP gene which codes for a modified CAP protein with reduced DNA binding specifity compared to a wild-type CAP protein, the expression of which selection gene is lethal in a portion of the host cells, wherein a restriction site for the DNA sequence is located within the selection gene and upon insertion of the DNA sequence at the restriction site, the expression of the selection gene in the host cells is prevented, wherein the modified CAP gene is modified such that the glutamic acid at position 181 of the wild-type CAP protein is substituted by glutamine. A disadvantage of this system is that it is restricted to adenylate-cyclase-positive host cells.
The problem underlying the present invention was thus to provide a new cloning system avoiding the disadvantages of the prior art. This problem is solved by providing a cloning vector comprising (a) a nucleic acid sequence encoding a polypeptide which is lethal for a bacterial host cell wherein the nucleic acid sequence is operatively linked to a regulatable expression control sequence, wherein said polypetide is selected from the
Anabaena flos
-
aquae
GvpA protein or a polypeptide which is homologous thereto, and (b) at least one cloning site wherein insertion of a foreign nucleic acid insert in the cloning site causes a disruption of the expression of the lethal polypeptide.
The cloning vector of the present invention is a tool for high-efficient cloning of DNA fragments and high-level expression of proteins in bacterial hosts, e.g.
E. coli.
The positive selection that makes the cloning so easy is based on the nucleic acid sequence encoding for a small protein which is located on the vector. Over expression of this polypeptide is toxic for
E. coli
and leads to cell death of bacteria carrying the nucleic acid sequence. When a DNA fragment is inserted into the cloning site of the vector, which may be located within the protein coding sequence or between the expression control sequence and the protein coding sequence, the expression of the lethal gene is disrupted. Thus, only transformants with insert-containing vectors will grow, whereas transformants containing the vector without insert cannot grow. The vector is particularly useful for the construction of nucleic acid, e.g. cDNA or genomic libraries, e.g. for sequencing projects and the cloning of PCR products.
Preferably, the vector of the present invention is a bacterial vector, i.e. it contains nucleic acid sequences which allow a propagation in prokaryotic host cells, particularly
E. coli.
Thus, a vector comprises at least one origin of replication which may be a bacterial origin of replication such as colE1 or p15A or a bacteriophage origin such as the f1 origin. In a particularly preferred embodiment, the vector comprises at least two origins of replication, wherein at least one origin is a bacterial origin and at least one orgin is a bacteriophage origin. Furthermore, it is preferred that the vector comprises a selection marker gene, which allows selection on transformants containing the vector. The selection marker gene may be an antibiotic resistance gene such as the ampicillin, kanamycin or tetracyclin resistance gene, In a particularly preferred embodiment the antibiotic resistance gene is the ampicillin resistance gene, i.e. the beta-lactamase gene.
An essential feature of the present invention is the presence of a nucleic acid sequence in the vector encoding a polypeptide which is conditionally lethal for the bacterial host cell. This nucleic acid sequence is operatively linked to a suitable expression control sequence, which is regulatable in a manner which allows propagation of the vector under conditions, wherein the expression control sequence is substantially inactive. Preferably, the expression control sequence is regulatable by a repressor. A specific example is a phage lambda promoter, e.g. the P
L
or P
R
promoter including operator sequences which allow effective binding of lambda repressor and thus substantially inactivating the expression control sequence under appropriate conditions, e.g. in a host cell, which produces the lambda repressor in sufficient amount. Furthermore, the expression control sequence may comprise a translation enhancer, e.g. the T7 translation enhancer to allow effective translation of any heterologous nucleic acid sequence which is inserted into the cloning site.
The vector may also contain at least one nucleotide sequence which is complementary to a sequencing and/or amplification primer. These complementary nucleotide sequences are preferably located adjacent to the cloning site and thus allow efficient sequencing and/or amplification of any foreign nucleic acid fragment which is inserted in the cloning site. Moreover, the vector may be a shuttle vector, which allows propagation in different host cells, e.g. it may additionally comprise nucleic acid sequences which allow a propagation in eukaryotic host cells.
The lethal polypeptide is selected from the
Anabaena flos
-
aquae
GvpA protein (Genbank accession No. M32060) or a polypeptide homologous thereto, which may be a protein from
Fremyella diplosiphon
(Genbank accession No. P07060), a protein from Calothrix (Genbank accession No. AAB23332), a protein from Pseudoanabaena spec. (Genbank accesssion No. P22453), a protein from
Planktothrix rubescens
(Genbank accession No. CAB59543 and CAB59546), a protein from
Aphanizomenon flos
-
aquae
(Genbank accession No. SVFZ), a protein from Microcystis spec. (Genbank accession No. P08412), a protein from
Oscillatoria agardhii
(Genbank accession No. P80996), a protein from
Thiocapsa pendens
(Genbank accession No. P80998), a protein from
Amoebobacter pendens
(Genbank accession No. AAB23337), a protein from
Bacillus megaterium
(Genbank accession No. AAC38419, AAC38416 and AF053765), a protein from Spirulina spec. (Genbank accession No. P80997), a protein from
Haloferax mediterranei
(Genbank accession No. P23761 and Q02235), a protein from
Halobacterium halobium
(Genbank accession No. P08959, S07323, P08959 and P24374), a protein from
Dactylococcopsis salina

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