U.S. patent application number 11/842131 was filed with the patent office on 2008-12-04 for cloning and/or sequencing vector.
Invention is credited to Philippe Bernard, Philippe Gabant.
Application Number | 20080299661 11/842131 |
Document ID | / |
Family ID | 3886385 |
Filed Date | 2008-12-04 |
United States Patent
Application |
20080299661 |
Kind Code |
A1 |
Bernard; Philippe ; et
al. |
December 4, 2008 |
CLONING AND/OR SEQUENCING VECTOR
Abstract
A cloning and/or sequencing vector enables recombinant clones to
be selected directly. The vector encodes a fusion protein which
includes a protein poison.
Inventors: |
Bernard; Philippe;
(Bruxelles, BE) ; Gabant; Philippe; (Bruxelles,
BE) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET, FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
3886385 |
Appl. No.: |
11/842131 |
Filed: |
August 20, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09634039 |
Aug 8, 2000 |
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11842131 |
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09225152 |
Jan 4, 1998 |
6180407 |
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09634039 |
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08379614 |
Jul 20, 1995 |
5910438 |
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PCT/BE93/00051 |
Aug 2, 1993 |
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09225152 |
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Current U.S.
Class: |
435/471 |
Current CPC
Class: |
C12N 15/70 20130101 |
Class at
Publication: |
435/471 |
International
Class: |
C12N 15/74 20060101
C12N015/74 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 31, 1992 |
BE |
BE 09200696 |
Claims
1. A method of cloning a DNA fragment in a prokaryotic cell, said
method comprising: providing a cloning vector comprising at least
one promoter nucleotide sequence and at least one nucleotide
sequence comprising at least one cloning site localized at the
level of a nucleotide sequence for a gene encoding CcdB, said CcdB
being cytotoxic; inserting said DNA fragment into said at least one
cloning site in said cloning vector such that said DNA fragment
interferes with the cytotoxic activity of said CcdB; transferring
the cloning vector with the DNA fragment into the prokaryotic cell;
and growing the prokaryotic cell to produce multiple copies of the
DNA fragment.
2. The method of claim 1, wherein said at least one nucleotide
sequence comprises a plurality of cloning sites.
3. The method of claim 1, wherein said at least one cloning site is
a unique cloning site.
4. The method of claim 1, wherein said at least one cloning site is
an artificially created cloning site.
5. The method of claim 1, wherein said at least one promoter
nucleotide sequence comprises a repressible promoter.
6. The method of claim 5, wherein said repressible promoter is a
Lac operon promoter.
7. The method of claim 1, wherein said cloning vector is a
recombinant virus or recombinant plasmid.
8. The method of claim 7, wherein said recombinant plasmid
comprises a pUC origin of replication.
9. The method of claim 8, wherein said pUC origin of replication is
the origin of replication from pUC18 or pUC19.
Description
RELATED APPLICATIONS
[0001] This application is a continuation application which claims
priority under 35 U.S.C. .sctn. 120 to U.S. patent application Ser.
No. 09/634,039, entitled CLONING AND/OR SEQUENCING VECTOR, filed
Aug. 8, 2000, which is a continuation of U.S. patent application
Ser. No. 09/225,152, entitled CLONING AND/OR SEQUENCING VECTOR,
filed Jan. 4, 1999 and issued as U.S. Pat. No. 6,180,407, which is
a continuation-in-part of U.S. patent application Ser. No.
08/379,614, entitled CLONING AND/OR SEQUENCING VECTOR, filed Jul.
20, 1995 and issued as U.S. Pat. No. 5,910,438, which is the U.S.
National Phase under 35 U.S.C. .sctn. 371 of International
Application PCT/BE93/00051, entitled CLONING AND/OR SEQUENCING
VECTOR, filed Aug. 2, 1993 and published in English as PCT
Publication No. WO94/03616, which claims priority to Belgian
Application No. BE 9200696, filed Jul. 31, 1992.
SEQUENCE LISTING
[0002] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled VANMA10-ICP1C3-SEQ.TXT, created Aug. 20, 2007, which
is 10 Kb in size. The information in the electronic format of the
Sequence Listing is incorporated herein by reference in its
entirety.
SUBJECT-MATTER OF THE INVENTION
[0003] The invention relates to a cloning and/or sequencing vector
which enables recombinant clones to be selected directly.
[0004] The invention also relates to the procaryote cell which is
transformed by this vector and to the procaryote 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
[0005] 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, 33 pp. 103 to 119) (1985)).
[0006] The multiple cloning sites (MCS--multiple cloning sites) of
these vectors are located in the coding sequence of the LacZ
gene.
[0007] Discrimination between the transformed cells which harbour a
recombinant vector and the cells which harbour 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 cleavage sites, Nature,
272, pp. 375-377 (1978)).
[0008] However, this "blue screen" technique suffers from the
disadvantage of using a screening procedure (discrimination) rather
than a procedure for selecting the clones.
[0009] 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.
[0010] 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
harbouring a non-recombinant vector produce a functional
beta-galactosidase and, by hydrolysing 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 harbouring a recombinant vector have a white
color.
[0011] 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.
[0012] On the other hand, various cloning vectors permitting direct
selection (positive selection) of recombinant strains have been
described in the scientific literature.
[0013] Pierce et al (Proc. Natl. Acad. Sci., vol 89. No. 6, 1992,
pp. 2056-2060) describe a vector which comprises the lethal gene
sacB from Bacillus amylolique-faciens, integrated into a plasmid
derived from the bacteriophage P1 and under the control of a
specific E. coli promoter.
[0014] The promoter of this vector includes a region having several
specific cloning sites (cleavage site for a restriction
enzyme).
[0015] Since the gene sacB encodes levan sucrase, which catalyses
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.
[0016] 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.
[0017] 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 (characterised by an inactive lethal gene).
[0018] In this case, in order to ensure that the enzyme encoded by
the sacB gene does not kill the host cell, it is necessary to
incorporate a CI repressor, which regulates the expression of this
gene, into the cloning vector.
[0019] Furthermore, since sucrose is often incorporated into
bacterial culture media, it will be essential to prepare media
which are totally free of sucrose in order to carry out these
manipulations.
[0020] Henrich et al (Gene, vol 42, No. 3, 1986, pp. 345-349)
describe a vector which includes the E gene from the bacteriophage
.phi.174, the said E gene being incorporated into the plasmid pUH84
under the control of the Lac promoter.
[0021] In this case, the E gene includes six unique restriction
sites (located over the whole of the E gene sequence) and encodes
gpE, which causes lysis of the E. coli cell. In this case, positive
selection is effected when a foreign recombinant gene has been
inserted into one of the restriction sites.
[0022] However, this insertion of a foreign gene into a restriction
site located in the sequence of the E gene, encoding gpE, makes it
more difficult to sequence the foreign gene and/or amplify it by
PCR since, in this case, portions of useless sequences belonging to
the E gene encoding gpE are also sequenced, amplified and
characterised.
[0023] Kuhn et al (Gene, vol 42, No. 3, 1986, pp. 253-263) describe
a vector which includes a large gene encoding a restriction enzyme
which kills by cleaving the genome of the bacterium, the said gene
being incorporated into the plasmid pKG2 under the control of the
LacUV5 promoter.
[0024] The cloning vectors of the state of the art suffer from the
disadvantage of having to be maintained in a host strain which
includes the LacI.sup.q repressor in episomal form, or the CI
repressor, in order to inactivate the promoter and prevent
expression of the killer gene, leading to the death of the host
strain.
[0025] In addition, if it is desired to use this strain to produce
a large number of copies of the cloning vectors, the repressor will
not be adequate for preventing either a selective pressure which
modifies the cytotoxic activity of the vector or a "genetic
leakage", that is to say expression of certain copies of the vector
and death of the host strain.
[0026] Consequently, none of the documents of the state of the art
describes a cloning vector which can incorporate large nucleotide
fragments, which is easy to manipulate and which can be produced by
a micro-organism on an industrial scale; that is to say, which can
be produced in a large number of copies by a micro-organism without
bringing about the death of the latter.
OBJECTS OF THE INVENTION
[0027] The present invention aims to supply a novel cloning and/or
sequencing vector, and also its host strain, which are simple and
relatively inexpensive to construct and produce, and which enable
recombinant clones to be selected directly, without suffering from
the disadvantages of the above-mentioned state of the art.
[0028] A particular object of the present invention is to obtain a
vector which permits specific and certain selection of the
recombinant clones.
[0029] Another object of the present invention is directed towards
obtaining a vector which permits the sequencing, amplification
and/or characterisation, using the same primer, of any foreign DNA
fragment (whatever its size) in the recombinant clones.
[0030] An additional object of the present invention is directed
towards obtaining a vector which also permits simple extraction of
this foreign DNA fragment from the recombinant clone.
[0031] A final object of the present invention is directed towards
obtaining a host strain for the said vector which allows a large
number of copies of the said vector to be produced without bringing
about selective pressure which modifies the cytotoxic activity of
the said vector or causing the death of the host strain.
CHARACTERISTIC ELEMENTS OF THE INVENTION
[0032] The invention relates to a new cloning vector comprising at
least one promoter nucleotide sequence and at least one nucleotide
sequence comprising several unique cloning sites artificially
created, localized at the level of a nucleotide sequence for a gene
encoding a cytotoxic molecule.
[0033] The invention relates to a novel cloning and/or sequencing
vector which includes, incorporated into an autonomously
replicating vector, at least one promoter nucleotide sequence and
at least one nucleotide sequence encoding a fusion protein which is
active as a poison, the said nucleotide sequence being obtained by
fusing a coding nucleotide sequence which includes several unique
cloning sites and a nucleotide sequence which encodes a protein
poison.
[0034] Preferably, the autonomously replicating vector is a
recombinant virus or a recombinant plasmid such as a pUC
plasmid.
[0035] The promoter nucleotide sequence can comprise any promoter,
which permits expression of the nucleotide sequence encoding a
fusion protein which is active as a poison.
[0036] Preferably, this promoter nucleotide sequence consists of
the Lac operon promoter.
[0037] According to one preferred embodiment of the invention, the
unique cloning sites (MCS) of the nucleotide sequence which is
fused to the nucleotide sequence which encodes the protein poison
are absent from the remainder of the nucleotide sequence of the
vector according to the invention.
[0038] Advantageously, the nucleotide sequence of the gene which
encodes the protein poison comprises all or part of the nucleotide
sequence of the wild-type gene which encodes the protein CcdB or
the protein Kid.
[0039] Preferably, the nucleotide sequence of the gene which
encodes the protein poison lacks the cleavage site for the
restriction enzyme SmaI.
[0040] Another aspect of the invention relates to a procaryote cell
which is transformed with the cloning vector according to the
invention.
[0041] The invention also relates to a procaryote host cell for the
vector according to the invention which possesses a chromosomal
I.sup.q and an elevated transformation efficiency, and which
possesses a mutation conferring resistance to the poison activity
of the fusion protein, and/or which possesses a gene encoding a
protein which is an antipoison to the fusion protein.
[0042] Preferably, the procaryote host cell for the vector
according to the invention possesses a mutation in the gene
encoding subunit A, or in the gene encoding subunit B, of the
gyrase, and conferring resistance to the fusion protein, and/or a
gene which encodes the protein CcdA which is an antipoison to the
fusion protein comprising CcdB and/or encodes the protein K is
which is an antipoison of the fusion protein comprising Kid.
[0043] Preferentially, the procaryote cell is an Escherichia coli
cell which possesses a mutation which is responsible for replacing
arginine 462 with a cysteine in the amino acid sequence of the GyrA
polypeptide of the gyrase, thereby conferring resistance to the
fusion protein.
[0044] Preferably, this procaryote host cell also possesses the
LacI.sup.q mutation.
[0045] The present invention also relates to fragments of the
vector according to the invention, in particular primers for
sequencing and/or amplifying (for example by PCR) the foreign
nucleotide fragments inserted into the vector according to the
invention.
[0046] Preferably, these primers consist of sequences of from 10 to
30 nucleotides which hybridise to nucleotide sequences which are
situated on either side of the nucleotide sequence of the vector
according to the invention which contains several unique cloning
sites.
[0047] A final aspect of the invention relates to the use of the
vector according to the invention for selecting and sequencing
recombinant clones.
BRIEF DESCRIPTION OF THE FIGURES
[0048] FIG. 1 is a diagrammatic representation of a cloning vector
according to the present invention.
DESCRIPTION OF A PREFERRED EMBODIMENT OF THE INVENTION
[0049] According to the invention, the cloning and/or sequencing
vector 1 includes, incorporated into an autonomously replicating
vector 2, at least one promoter nucleotide sequence 3 and at least
one nucleotide sequence 4 which encodes a fusion protein which is
active as a poison, the said nucleotide sequence 4 being obtained
by fusing a coding nucleotide sequence 5 (or polylinker) which
encompasses several (multiple) unique cloning sites (MCS), and a
nucleotide sequence (6) which encodes a protein poison.
[0050] An autonomously replicating vector 2 is understood to mean
any nucleotide construct, such as a virus or a plasmid (preferably
a recombinant plasmid of the PUC series), which is capable of being
introduced into a micro-organism, of recombining therein and/or of
replicating therein.
[0051] FIG. 1 shows a diagrammatic representation of a cloning
vector according to the present invention, which vector is
constructed from a plasmid of the pUC series (pUC18 and pUC19),
which is described by Norrander et al (Construction of improved M13
vectors using oligo-deoxinucleotide-directed mutagenesis, Gene, 26,
pp. 101-106 (1983)) and by Yanisch-Perron et al (Improved M13 phage
cloning vectors and host strains-nucleotide sequences of the M13
mp18 and pUC19 vectors, Gene, 33, pp. 103-119 (1985)).
[0052] A coding nucleotide sequence 5 encompassing several
(multiple) unique cloning sites (MCS) is understood to mean a short
coding sequence (or polylinker) which comprises several cleavage
sites for restriction enzymes.
[0053] The advantage of having a polylinker in the vector according
to the invention is that different cloning sites are located on a
single short sequence, thereby permitting: [0054] rapid sequencing
and amplification, using the same primers, of any DNA fragment
which is inserted into this vector, [0055] rapid extraction of the
cloned fragment, facilitated by the proximity of the restriction
sites. Thus, in contrast to the state of the art, this proximity
avoids sequencing, amplifying and characterising useless fragments
from other sequences of the vector according to the invention.
[0056] Nucleotide sequence 6 encoding a protein poison is
understood to mean any (wild-type) nucleotide structure encoding a
protein which displays an activity which is naturally poisonous and
specific for one or more vital functions of a host cell.
[0057] A protein poison is also characterised by the existence of
an antidote or antipoison, such as the proteins CcdB and CcdA, the
protein Kid and its antagonist K is, the protein PemK and its
antagonist PemI, the protein Doc and its antagonist Phd, the
protein HoK and its antagonist Sok, and other poison molecules
which are, or are not, of plasmid origin.
[0058] In this case, the nucleotide sequence 6 encoding a protein
poison consists of the wild-type gene CcdB, which encodes the
protein CcdB (control of cell death), obtained from the ccd locus
of the F plasmid (SEQ ID NO: 1 and SEQ ID NO:4).
[0059] The ccd locus of the F plasmid comprises the two wild-type
genes ccdA and ccdB, also termed H and G, or letA and letD, which
respectively encode proteins of 72 and 101 amino acids (Bex et al,
Mini-F encoded proteins; identification of a new 10.5 kilodalton
species. EMBO J.2, 1853-1861 (1983); Miki et al, Control of cell
division by sex factor F in Escherichia coli. I. The 42.84-43.6 F
segment couples cell division of the host bacteria with replication
of plasmid DNA, J. Mol. Bio., 174, 605-625, (1984)).
[0060] In Escherichia coli, the CcdB protein of the F plasmid is a
cytotoxin whose lethal activity is counteracted by the protein CcdA
(Karoui et al, Ham22, a mini-F mutation which is lethal to host
cell and promotes recA-dependent induction of lambdoid prophage.
EMBO J.2, 1863-1868 (1983); Ogura and Hiraga Mini-F plasmid gene
that couple host cell division to plasmid proliferation, Proc.
Natl. Acad. Sci. USA, 80, 4784-4788 (1983); Miki et al, Control of
cell division by sex factor F in Escherichia coli. Identification
of genes for inhibitor protein and trigger protein on the
42.84-43.6F segment, J. Mol. Biol. 174, 627-646 (1984b)).
[0061] The molecular mechanism by which protein CcdB exerts its
lethal activity has been elucidated; protein CcdB is poisonous to
DNA topoisomerase II.
[0062] The type II DNA topoisomerases are essential and ubiquitous
enzymes which alter the topology of the DNA by transiently
introducing a double-stranded break into the DNA. During the stage
of break-religation, topoisomerase II forms an intermediate complex
with its DNA substrate in which the enzyme is attached covalently
to the 5' end of the cleaved DNA, This transitory intermediate, in
which topoisomerase II is linked covalently to the DNA, has been
termed the "cleavable complex" (Wang, DNA topoisomerases. Annu.
Rev. Biochem. 54, 665-97, 1985; Maxwell & Gellert, Mechanistic
aspects of DNA topoisomerases. Advan. Protein Chem. 38, 69-107,
1986; Liu, DNA topoisomerase poisons as antitumor drugs, Annu. Rev.
Biochem. 58. 351-375, 1989).
[0063] Both in eucaryotes and in procaryotes, the cleavable
topoisomerase II-DNA complex is the target of powerful therapeutic
agents, including the antibiotics of the "quinolone" family, which
act on the gyrase (bacterial topoisomerase II), and anticancer
agents (acridines and epipodophyllotoxins), which act on the
mammalian topoisomerase II. The therapeutic efficacy of the
topoisomerase poisons is correlated with their ability to stabilise
the cleavable complex.
[0064] DNA topoisomerase II is an essential enzyme in all living
entities and is very conserved in the evolution of the species. The
CcdB protein thus displays an activity which is potentially
cytotoxic for a wide variety of procaryote species.
[0065] The small size of the wild-type ccdB gene allows it to be
inserted into plasmids without increasing their size excessively
and consequently allows large fragments of foreign DNA to be
included therein, Furthermore, given its small size, the wild-type
ccdB gene of the F plasmid contains very few restriction sites; it
is, therefore, simpler to preserve the uniqueness of the multiple
cloning sites (MCS) which are added to it.
[0066] Unexpectedly, the inventors observed that the in-phase
fusion of the nucleotide sequence 6, encoding protein CcdB, with
the coding nucleotide sequence (polylinker 5), comprising several
(multiple) unique cloning sites (MCS) gave a nucleotide sequence 4
which encodes a fusion protein which is active as a poison and
which makes it possible, as a consequence, to produce vectors for
the direct selection of recombinant plasmids (killer
selection).
[0067] The plasmids which have been obtained allow doubly digested
restriction fragments to be cloned in both orientations with
respect to the lac promoter. Insertion of a restriction fragment
into one of the unique cloning sites interrupts the genetic
information of the gene fusion, leading to the synthesis of a gene
fusion product which is not functional. Insertional inactivation of
the gene fusion ought always to take place when a termination codon
is introduced or when a change is made in the reading frame.
[0068] The cells which harbour an intact cloning vector of this
nature produce a poisonous fusion protein which is functional, and
die.
[0069] Insertion of a foreign DNA fragment into one of the unique
cloning sites of the gene fusion interferes with production of the
poison.
[0070] The cells which harbour a recombinant vector will be viable
while cells which harbour an intact vector will not be viable. This
killer selection, by simple culture on a solid medium, makes it
possible to eliminate cells which harbour a non-recombinant vector
(non-viable clones) and to select recombinant clones (viable
clones).
Example I
Construction of the Plasmid pKIL19
[0071] The ccdB gene was amplified by PCR using, as DNA template,
the plasmid pULB2208 (Bernard and Couturier, The 41
carboxy-terminal residues of the miniF plasmid CcdA protein are
sufficient to antagonise the killer activity of the CcdB protein,
Mol. Gen. Genet. 226, 297-304 (1991) as well as synthetic
oligonucleotides.
[0072] The synthetic oligonucleotide sequences were selected in
such a way as to create an EcoRI restriction site on either side of
the wild-type ccdB gene in order to be able to reclone this gene in
frame with the codons of the MC S19 multiple cloning site and to
eliminate the initiation codon of the native ccdB gene. The DNA
resulting from the PCR reaction was digested with the enzyme EcoRI
and cloned into the EcoRI site of the plasmid pUC19. The resulting
plasmid, in which the EcoRI fragment was integrated in the
orientation which permitted the ccdB gene, provided with the
additional codons corresponding to the MCS19 multiple cloning
sites, to be read from the Lac promoter, was termed pKIL2. Plasmid
pKIL2 is lethal for a wild-type bacterium (Ccdb.sup.S
sensitive).
[0073] pKIL2 also possesses two SmaI sites, one in the multiple
cloning sites and the other in the central region of the ccdB gene.
The latter was eliminated by site-directed mutagenesis. The
resulting plasmid pKIL3, having a unique SmaI site, still has two
EcoRI sites. The EcoRI site downstream of the ccdB gene was
eliminated by filling in its cohesive ends.
[0074] The resulting plasmid, pKIL19 (SEQ ID NO:2 and SEQ ID NO:5),
thus possesses a unique EcoRI restriction site within sequence 5,
which encompasses the multiple cloning site.
Example II
Construction of the Plasmid pKIL18
[0075] The ccdB gene was amplified by PCR using, as DNA template,
plasmid pKIL19 as well as synthetic oligonucleotides. The sequences
of the synthetic oligonucleotides were selected in such a way as to
create a HindIII site on either side of the ccdB gene in order to
be able to reclone this gene in frame with the codons of the MCS18
multiple cloning sites. The DNA resulting from the PCR reaction was
digested by the enzyme HindIII and cloned into the HindIII site of
the plasmid pUC18. The resulting plasmid, in which the HindIII
fragment was integrated in the orientation which permitted the ccdB
gene, provided with the additional codons corresponding to the MC
S18 multiple cloning sites, to be read from the Lac promoter, was
termed pKIL4. Plasmid pKIL4 is lethal for a Ccdb.sup.S-sensitive
bacterium.
[0076] The HindIII site downstream of the ccdB gene was eliminated
by filling in its cohesive ends. The resultant plasmid, pKIL18
((SEQ ID NO:4 and SEQ ID NO:6), possesses a unique HindIII
restriction site as well as a unique SmaI site (since constructed
from pKIL 19).
Example III
Construction of the Plasmid pKID18
[0077] ParD is a killer stability system of R1 plasmid located in
the proximity of the basic replicon. It is a small operon
containing two genes, Kid and K is, coding for a killer component
and its antagonist respectively (Bravo et al., Mol. Gen. Genet.,
Vol. 215, pp. 146-151 (1988)). This system is perfectly conserved
and functional in another incFII plasmid, R100 (pem system:
Tsuchimoto et al., J. of Bacteriol., Vol. 170, pp. 1461-1466
(1988)), PemA (identical to Kis) and PemB (identical to Kid).
[0078] The vectors pKID18 and pKID19 contain the Kid gene fused to
different polylinkers (MCS18 and MSC19 for pKID18 and pKID19
respectively). The Kid sequence was amplified by PCR from the
plasmid R1 drd19 using the primers
kid1--gaggaattcattgggaaagaggggaaatctg--(SEQ ID NO:7) and
kid2--gaggaattctcaagtcagaatagtggaca--(SEQ ID NO: 8). The generated
insert was cloned into the EcoRI site of pUC19 (Yanish-Perron et
al. (1985)). This insertion generates a fusion gene between the
MCS19 and Kid. The vector pKID18 was obtained as follows: the Kid
sequence was amplified by PCR from the plasmid R1 drd19 using the
primers kid3--gagaagcttattggaaagaggggaaatctg--(SEQ ID NO:9) and
kid4--gagaagctttcaagtcagaatagtggaca--(SEQ ID NO:10). The generated
insert was cloned into the HindIII site of pUC18 (Yanish-Perron et
al. (1985)). This insertion generates a fusion gene between the
MCS18 and Kid.
[0079] In induce conditions (induction of the pLac) that control
the fuse Kid transcription of this construct for the E. coli strain
(Top-10 F Invitrogen), this vector which contains the Kid gene
fused to different polylinkers has retained the poison activity of
the original Kid protein.
[0080] The regulation and the expression of this vector in a
specific cell which is not killed by the poison activity of the
fusion protein can be obtained by a control of the promoter
activity of said vector or can be obtained by the production of
said vector in a cell expressing the K is protein which is the
antidote of the Kid protein.
Example IV
Construction of the Strains Ccdb.sup.r and Ccdb.sup.S
[0081] In order to be able to maintain plasmids pKIL18 and pKIL19
within a bacterium, the latter has to be resistant to the lethal
effect of the fusion protein which is active as a poison,
Unexpectedly, the chromosomal mutation gyrA462 confers on the
strains total resistance to the poisonous effect of the fusion
protein.
[0082] Moreover, since plasmids pKIL 18 and pKIL 19 derive directly
from plasmids pUC18 and pUC19 and express the ccdB genes from the
Lac promoter, it is preferable to maintain these plasmids in a
LacI.sup.q strain. Thus, while, in our case, continuous
overexpression of these genes does not exert a selection pressure
in favour of certain mutations, the LacI.sup.q strain allows
expression from the Lac promoter to be reduced and conserves the
bacterial machinery, thereby guaranteeing a rapid generation time
(increased production of the vector by the strain).
[0083] The strain D1210 (Sadler et al Gene 8, pp. 279-300 (1980)),
derived from the strain HB101 LacI.sup.q, LacY.sup.+ (Maniatis et
al Molecular Cloning Laboratories Man. Cold Spring Harbour
Laboratory N.Y.), and characterised by a chromosomal I.sup.q and
increased transformation efficiency, was transformed with the
plasmid pCOS2.1. This plasmid, which confers resistance to
kanamycin, carries the recA gene from Erwinia chrysanthemi 3665 and
allows recombination in E. coli. A lysate of P1 phage was prepared
on a Ccdb.sup.R gyrA462, zei298::Tn10 strain and used to infect the
strain D1210/pCOS2.1. The transductants which were resistant to
tetracycline were selected and tested for their resistance or
sensitivity to the CcdB protein. One of the Ccdb.sup.R
transductants was then cured of plasmid pCOS2.1 and termed
KIB22.
[0084] Strain KI322 constitutes an ideal host strain for plasmids
pKIL18 and pKIL19 while strain D1210 constitutes the ideal host for
selecting recombinant plasmids.
[0085] Thus, strain KIB22 advantageously possesses an elevated
efficiency of DNA extraction (comparable to the yield of the pUC
plasmids) and, unexpectedly, resistance to the fusion protein which
is encoded by pKIL18 and pKIL19.
[0086] Consequently, it is possible to use this micro-organism to
produce the cloning vector according to the invention on an
industrial scale in numerous copies without causing the death of
the said micro-organism.
[0087] The selection is carried out simply by spreading the
bacteria on a medium containing IPTG
(Isopropyl-.beta.-D-thiogalactopyranoside) as well as
ampicillin.
[0088] Strain KIB22 was deposited with the Laboratorium voor
Microbiologie-Bacterienverzameling (LMG) [Microbiological
Laboratory--Bacterial Collection] of the Belgian Coordinated
Collections of Microorganisms (BCCM) under No. LMG P-12601.
[0089] The cloning vector pKIL19 was deposited with the
Laboratorium voor Moleculaire Biologie-Plasmiden Collectie (LMBP)
[Molecular Biological Laboratory--Plasmid Collection] of the
Belgian Coordinated Collections of Microorganisms (BCCM) under the
No. LMBP 2781.
[0090] These depositions were made in accordance with the
provisions of the Budapest Treaty regarding the International
Recognition of the Deposition of Microorganisms.
Sequence CWU 1
1
101306DNAArtificial SequenceccdB gene of plasmid F 1atg cag ttt aag
gtt tac acc tat aaa aga gag agc cgt tat cgt ctg 48Met Gln Phe Lys
Val Tyr Thr Tyr Lys Arg Glu Ser Arg Tyr Arg Leu1 5 10 15ttt gtg gat
gta cag agt gat att att gac acg ccc ggg cga cgg atg 96Phe Val Asp
Val Gln Ser Asp Ile Ile Asp Thr Pro Gly Arg Arg Met20 25 30gtg atc
ccc ctg gcc agt gca cgt ctg ctg tca gat aaa gtc tcc cgt 144Val Ile
Pro Leu Ala Ser Ala Arg Leu Leu Ser Asp Lys Val Ser Arg35 40 45gaa
ctt tac ccg gtg gtg cat atc ggg gat gaa agc tgg cgc atg atg 192Glu
Leu Tyr Pro Val Val His Ile Gly Asp Glu Ser Trp Arg Met Met50 55
60acc acc gat atg ggc agt gtg ccg gtc tcc gtt atc ggg gaa gaa gtg
240Thr Thr Asp Met Gly Ser Val Pro Val Ser Val Ile Gly Glu Glu
Val65 70 75 80gct gat ctc agc cac cgc gaa aat gac atc aaa aac gcc
att aac ctg 288Ala Asp Leu Ser His Arg Glu Asn Asp Ile Lys Asn Ala
Ile Asn Leu85 90 95atg ttc tgg gga ata taa 306Met Phe Trp Gly Ile
*1002420DNAArtificial SequenceccdB gene of pKIL 18 2atg acc atg att
acg aat tcg agc tcg gta ccc ggg gat cct cta gag 48Met Thr Met Ile
Thr Asn Ser Ser Ser Val Pro Gly Asp Pro Leu Glu1 5 10 15tcg acc tgc
agg cat gca agc ttg tct ttg cag ttt aag gtt tac acc 96Ser Thr Cys
Arg His Ala Ser Leu Ser Leu Gln Phe Lys Val Tyr Thr20 25 30tat aaa
aga gag agc cgt tat cgt ctg ttt gtg gat gta cag agt gat 144Tyr Lys
Arg Glu Ser Arg Tyr Arg Leu Phe Val Asp Val Gln Ser Asp35 40 45att
att gac acg ccc ggg cga cgg atg gtg atc ccc ctg gcc agt gca 192Ile
Ile Asp Thr Pro Gly Arg Arg Met Val Ile Pro Leu Ala Ser Ala50 55
60cgt ctg ctg tca gat aaa gtc tcc cgt gaa ctt tac ccg gtg gtg cat
240Arg Leu Leu Ser Asp Lys Val Ser Arg Glu Leu Tyr Pro Val Val
His65 70 75 80atc ggg gat gaa agc tgg cgc atg atg acc acc gat atg
gcc agt gtg 288Ile Gly Asp Glu Ser Trp Arg Met Met Thr Thr Asp Met
Ala Ser Val85 90 95ccg gtc tcc gtt atc ggg gaa gaa gtg gct gat ctc
agc cac cgc gaa 336Pro Val Ser Val Ile Gly Glu Glu Val Ala Asp Leu
Ser His Arg Glu100 105 110aat gac atc aaa aac gcc att aac ctg atg
ttc tgg gga ata taa 381Asn Asp Ile Lys Asn Ala Ile Asn Leu Met Phe
Trp Gly Ile *115 120 125atgtcaggct ccgttataca caagctagct tggcactgg
4203416DNAArtificial SequenceccdB gene of plasmid pKIL 19 3atg acc
atg att acg cca agc ttg cat gcc tgc agg tcg act cta gag 48Met Thr
Met Ile Thr Pro Ser Leu His Ala Cys Arg Ser Thr Leu Glu1 5 10 15gat
ccc cgg gta ccg agc tcg aat tca ttg cag ttt aag gtt tac acc 96Asp
Pro Arg Val Pro Ser Ser Asn Ser Leu Gln Phe Lys Val Tyr Thr20 25
30tat aaa aga gag agc cgt tat cgt ctg ttt gtg gat gta cag agt gat
144Tyr Lys Arg Glu Ser Arg Tyr Arg Leu Phe Val Asp Val Gln Ser
Asp35 40 45att att gac acg ccg ggg cga cgg atg gtg atc ccc ctg gcc
agt gca 192Ile Ile Asp Thr Pro Gly Arg Arg Met Val Ile Pro Leu Ala
Ser Ala50 55 60cgt ctg ctg tca gat aaa gtc tcc cgt gaa ctt tac ccg
gtg gtg cat 240Arg Leu Leu Ser Asp Lys Val Ser Arg Glu Leu Tyr Pro
Val Val His65 70 75 80atc ggg gat gaa agc tgg cgc atg atg acc acc
gat atg gcc agt gtg 288Ile Gly Asp Glu Ser Trp Arg Met Met Thr Thr
Asp Met Ala Ser Val85 90 95ccg gtc tcc gtt atc ggg gaa gaa gtg gct
gat ctc agc cac cgc gaa 336Pro Val Ser Val Ile Gly Glu Glu Val Ala
Asp Leu Ser His Arg Glu100 105 110aat gac atc aaa aac gcc att aac
ctg atg ttc tgg gga ata taa 381Asn Asp Ile Lys Asn Ala Ile Asn Leu
Met Phe Trp Gly Ile *115 120 125atgtcaggct ccgttataca cgaattaatt
cagtg 4164101PRTArtificial SequenceccdB protein of plasmid F 4Met
Gln Phe Lys Val Tyr Thr Tyr Lys Arg Glu Ser Arg Tyr Arg Leu1 5 10
15Phe Val Asp Val Gln Ser Asp Ile Ile Asp Thr Pro Gly Arg Arg Met20
25 30Val Ile Pro Leu Ala Ser Ala Arg Leu Leu Ser Asp Lys Val Ser
Arg35 40 45Glu Leu Tyr Pro Val Val His Ile Gly Asp Glu Ser Trp Arg
Met Met50 55 60Thr Thr Asp Met Gly Ser Val Pro Val Ser Val Ile Gly
Glu Glu Val65 70 75 80Ala Asp Leu Ser His Arg Glu Asn Asp Ile Lys
Asn Ala Ile Asn Leu85 90 95Met Phe Trp Gly Ile1005126PRTArtificial
SequenceccdB protein of plasmid pKIL 18 5Met Thr Met Ile Thr Asn
Ser Ser Ser Val Pro Gly Asp Pro Leu Glu1 5 10 15Ser Thr Cys Arg His
Ala Ser Leu Ser Leu Gln Phe Lys Val Tyr Thr20 25 30Tyr Leu Arg Glu
Ser Arg Tyr Arg Leu Phe Val Asp Val Gln Ser Asp35 40 45Ile Ile Asp
Thr Pro Glu Arg Arg Met Val Ile Pro Leu Ala Ser Ala50 55 60Arg Leu
Leu Ser Asp Lys Val Ser Arg Glu Leu Tyr Pro Val Val His65 70 75
80Ile Gly Asp Glu Ser Trp Arg Met Met Thr Thr Asp Met Ala Ser Val85
90 95Pro Val Ser Val Ile Gly Glu Glu Val Ala Asp Leu Ser His Arg
Glu100 105 110Asn Asp Ile Leu Asn Ala Ile Asn Leu Met Phe Trp Gly
Ile115 120 1256126PRTArtificial SequenceccdB protein of plasmid
pKIL 19 6Met Thr Met Ile Thr Pro Ser Leu His Ala Cys Arg Ser Thr
Leu Glu1 5 10 15Asp Pro Arg Val Pro Ser Ser Asn Ser Leu Gln Phe Leu
Val Tyr Thr20 25 30Tyr Lys Arg Glu Ser Arg Tyr Arg Leu Phe Val Asp
Val Gln Ser Asp35 40 45Ile Ile Asp Thr Pro Gly Arg Arg Met Val Ile
Pro Leu Ala Ser Ala50 55 60Arg Leu Leu Ser Asp Lys Val Ser Arg Glu
Leu Tyr Pro Val Val His65 70 75 80Ile Gly Asp Glu Ser Tyr Arg Met
Met Thr Thr Asp Met Ala Ser Val85 90 95Pro Val Ser Val Ile Gly Glu
Glu Val Ala Asp Leu Ser His Arg Glu100 105 110Asn Asp Ile Lys Asn
Ala Ile Asn Leu Met Phe Trp Gly Ile115 120 125731DNAArtificial
Sequencekid1 primer 7gaggaattca ttgggaaaga ggggaaatct g
31829DNAArtificial Sequencekid2 primer 8gaggaattct caagtcagaa
tagtggaca 29930DNAArtificial Sequencekid3 primer 9gagaagctta
ttggaaagag gggaaatctg 301029DNAArtificial Sequencekid4 primer
10gagaagcttt caagtcagaa tagtggaca 29
* * * * *