Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~ZS3'~6~S
X-5773A -1-
Chimeric Cloning Vectors For Use In
Streptomyces And E. coli
The present invention relates to selectable
novel recombinant DNA cloning vectors comprising a
functional origin of replication-containing restriction
fragment of plasmid SCP2 or SCP~* and a functional
origin of replication-containing and antibiotic re-
sistance-conferring restriction fragment of a plasmid
that is functional in E. coli. The invention also
relates to transformants and a method for detecting
transformants of the aforementioned vectors.
The vectors to which this invention relates
are particularly useful because they are small, versatile,
and can be transformed and selected in Streptomyces or
: 15 _. coli. Since over hal of the clinically importan-t
antibiotics are produced by Streptomyces strains, it is
desirable to develop cloning systems and vectors that
are applicable to that industrially important group.
Such vectors allow for the cloning of genes into Strepto-
myces both for increasing the yields oE known anti-
b.iotics as well as for the production of new anti
biotics and antibiotic derivatives~
The method of the present invention provides
for the convenient selection of transformantsO Since
transformation is a very low ~requency event, such a
runctional test is a practical necessity for deter-
mining which cell(s), of among the millions of cel~s,
has ac~ ired the plasmid DWA. This is important
because DNA sequences that are non~selectable can be
inserted into the vectors and, upon transformation,
X-5773A -~
cells containing the vector and the particular DNA
sequence of interest can be isolated by appropriate
phenotypic selection.
For purposes of the present invention as
disclosed and claimed herein, the following terms ~re
as defined below.
Plasmid pLRl or pLR4 3.4kb ~amHI Res~riction
Fragment - the same 3.4kb BamHI neomycin resistance-
conferring fragment contained in plasmid pIJ2.
AmpR - the ampicillin resistant pheno~yp~
pS _ the ampicillin sensitive phenotyp~.
Tet - the tetracycline resistant phenotype.
TetS - the tetracycline sensitive phenotype.
CMR - the chloramphenicol resistant phenotype.
CMS - the chloramphenicol sensitive phenotype.
Neo ~ the neomycin resistant phenotype.
Neo - the neomycin sensitive phenotype.
ThioR - the thiostrepton resistant phenotype.
ThioS - the thiostrepto,n sensitive phenotype.
Neo , NeoS, Thio , and ThioS refer only to
results of tests in Streptomyces as used in this dis-
`closure. AmpR, AmpS, TetR, TetS, CmR and CmS refer
only to results of tests in ~. coli as used in this
disclosure.
~5 The present invention specifically relates
to recombinant DNA cloning vectors comprising:
a) a functional origin of replication-containing
restriction fragment of plasmid SCP2 or
SCP2*,
3~76~
X-5773A -3~
b) a restriction fragment comprising an Eo coli
origin of replication,
c) one or more DNA segments that confer rasis-
tance to at least one antibiotic when trans-
formed into a cell of E. coli, said cell
being sensi~ive to the antibiotic for which
resistance is conferred, and
d) one or more DNA segments that independently
confer either or both of the Streptomyces
tra function or resistance to at least one
antibiotic when transformed into a cell of
Streptomyces, said cell being sensitive to
the antibiotic for which resistance .is
conferred.
The recombinant DNA cloning vectors are made
by a process which comprises ligating a functional
origin of replication-containing restric-tion fragmen-t
of plasmid SCP2 or SCP2* and one or more DN~ sequences
comprising:
a) a restriction fragment comprising an E~ coli
origin of replication,
b) one or more DNA segments that confer resis-
tance to at least one antibio*ic when trans-
formed into a cell of E. coli, said cell
being sensitive to the antibiotic for which
resistance is conferred, and
c) one or more DNA segments that independently
; confer either or both of the Streptomyces
tra function or resistance to at least one
; 30 antibiotic when transformed into a cell of
i2~3t,lt;~5
X-5773A -4- -
S~reptomyces, said cell being sensitive to
the antibiotic for which resistance is con-
~erred.
The invention further comprises trans~ormants
~nd a method for detecting transformants of the afore-
mentioned vectors comprising:
l) mixing Streptomyces cells, under transform
ing conditions, with a recombinant DNA clon-
ing vector, said vector comprising
a) an origin of replication and P gene~
containing restriction ragment of
plasmid SCP2 or SCP2*, and
b) a non-lethal DNA sequence cloned into
the EcoRI restriction sit.e of said P
~ gene, and
2) growing said Streptomyces cells on a lawn of
an indicator Streptomyces strain and select-
ing colonies that show the M pock phenotype~
The vectors of the present invention are
best constructed by ligating an origin of replication-
containing and Streptomyces tra function-conferring
restriction fragment of plasmid SCP2 or SCP2* into an
E. coli origin of replication-containing and antibiotic
resistance-conferring restriction ragment of an Eo
coli plasmid. Plasmids SCP2 and SCP2*, from which
origins of replication are constructed, are each ~31kb
and show similar restriction patternsO Plasmid SCP2~
arose as a spontaneous mutant of plasmid SCP2 and codes
for a selectable colony pock morphology. Although the
pock is distinguishable from that of plasmid SCP2, in
other ways plasmids SCP2 and SCP2* are virtually iden
tical.
t~6~3~
X-5773A 4a-
In the drawings appended to this specification:
Figure 1 is a detailed restriction site map of
plasmids SCP2 and SCP2*~
Figure 2 is a restriction site map of each of
5 plasmids pJL120 and pJL121;
Figure 3 is a restriction site map of each of
plasmids pJL180 and pJL181;
Figure 4 is a restriction site map of plasmid
pJL125 and is also a restriction site and functional map
10 of plasmid pJLl90; and
Figure 5 is a restriction site map of plasmids
pJL195 and pJL114.
.
~ .
~ ,,`,
s
X-5773A _5
Since the present disclosure teaches that
the Streptomyces tra function and the origin of repli-
-
cation of plasmids SCP2 and SCP2* are within their
respective ~5.4kb EcoRI-SalI restriction fragments, a
variety of different origin of replication-containing
and Streptomyces tra function-conferring fragments can
be generated. This is accomplished by digestion with
restriction enzymes that cut outside the ~5.4kb EcoRI-
SalI region. A detailed restriction site map of plasmid
SCP2* (and thus also pla~mid SCP2) is presented in
Figure 1 of ~he accompanying drawings.
Plasmids SCP2 and SCP2* can be conventionally
isolated respectively rom the Streptomyces coelicolor
A3(2) and Stxeptomyces coelicolor MllO strains deposited
and made part of the permanent stock culture collection
of the Northern Regional Research Laboratory, Peoria,
Illinois. Streptomyces coelicolor A312) is avai'able
to the public as a preferred source and stock reservoir
of plasmid SCP2 under the accession number 150420
Streptomyces coelicolor MllO is available to the public
as a preferred source and stock reservoir of plasmid
SCP2* under the accession number 15041.
Many ra function-conferring and origin of
replication-containing restriction fragments of plasmids
SCP2 and SCP2* can be constructed. Those specifically
exemplified, for illustrative purposes, include the
~5.4kb EcoRI-SalI, the ~6.Okb SalI, the ~19kb EcoRI~
, .
HindIII, and the ~31kb EcoRI restric-tion fragmen~s o
plasmid SCP2* and the ~31kb BglII restriction fragment
of plasmid SCP2. The aforementioned plasmid SCP2* and
SCP2 fragments were respectively ligated to an oxigin
~2~'7~S
X-5773A -6-
of replication-containing and antibiotic resistance-
conerring fragment of E. coli plasmids pBR325 and
pBR322. Those skilled in the art will recognize that
although not required; it is convenient for both the
DNA segment that confers antibiotic resistance in E.
coli and the E. coli origin of replication to comprise
a restriction fragment of the same E. coli plasmid.
Thus, for convenience and ease of construc-
tion, the ~31kb EcoRI fragment of plasmid SCP2* and the
~6kb EcoRI fragment of plasmid pBR325 were ligated to
form illustrative plasmids pJL120 and pJL121. Re-
combinant plasmids of two orientations result because
the fragments can be ligated in either direction.
Similarly, ligation of the SCP2* ~6.Okh SalI Eragment
15 and the 36kb SalI fragment of pBR325 results in the
illustrative plasmids pJL180 and pJL181; ligation of
the SCP2* ~5.4kb EcoRI-SalI fragment and the ~4.8kb
EcoRI-SalI fragment of pBR325 results in the illus-
trative plasmid pJL125; and ligation of the SCP2 BamHI
20 digest and the ~4.4kb BamHI fragment of plasmid pBR322
results in the illustrative plasmid pJL114.
All of the aforementioned vectors are readily
selectable in each of E. coli and Streptomyces. For
example, in E. coli, plasmids pJL120 and pJL121 confer
ampicillin and tetracycline resistance; plasmids pJL180
and pJL181 conEer ampicillin and chloramphenicol re-
sistance; and plasmids pJL125 and pJL114 confer only
ampicillin resistance. Therefore, the vectors are
conventionally selectable in the Eo coli host system by
adding the appropriate antibiotic to the culture medium~
76~
X-5773A -7-
The aforementioned vectors also produce the
'pock' phenotype and therefore are conventionally
selectable in Streptomyces. The 'pock' phenotype is an
assayable trait and known phenomenon (Bibb and Hopwood,
1981, J. Gen. Microbiol. 126:427) associated with
lethal zygosis and the tra function (tra - genes coding
for sexual transmissability) of StreptGmyces sex factors.
Three distinct 'pock' morphologies are associated with
transformants, when plated on an appropxiate indicator
strain, of plasmids SCP2, SCP2*, and SCP2 and SCP2*
derivatives. The colony morphology identified with the
wild-type SCP2 and the mutant SCP2* are respectively
designated herein as P and P*. A third and heretofore
unknown pock morphology results from cloning into the
J15 EcoRI restriction site of SCP2 or SCP~*. Such an
insertion inactivates the P gene and unexpectedly
results in a morphologically distinguishable minipock
phenotype, designated herein as M, when transformants
are appropriately plated. "Minipock" is a pock of
significantly smaller size than pocks caused ~y either
SCP2 or SCP2*.
The present invention thus provides a novel
method for detecting transformants comprising:
1) mixing Streptomyces cells, under transforming
_
conditions, with a recombinant DNA cloning
vector, said vector comprising
a) an origin of replication and P gene-
con~aining restriction fragment of
plasmid SCP2 or SCP2*, and
b) a non-lethal DNA sequence cloned into
the EcoRI restrictioll site of said P gene,
and
~2(~6~3S
X-5773A ~8-
2) growing said Streptomyces cells on a lawn of
an indicator Streptomyces strain and select~
ing colonies that show the M pock phenotype.
Only transformed Streptomyces cells will show
the M pock phenotype and therefore transformants can be
readily identified and selected. Those skilled in the
art will quickly recognize, from the above description
of the present pJL vectors, that plasmids pJL120,
pJL121, and pJL125 code for M phenotype, that plasmids
pJL180 and pJL181 code for P* phenotype, and that
plasmid pJL114 codes for P phenotype. Appropriate
indicator strains for expression of the pock phenotype
are known and include the various SCP2 and SCP2*
strains as illustrated in the Examples below. The
~resent vectors are thus selectable and extremely
useful in Streptomyces.
The aforementioned plasmids can also be
provided with a DNA segment that confers antibiotic
resistance in Streptomyces. Such derivatives, speci-
ically exemplified for illustrative purposes by plasmidspJL190 and pJL195, express an additional selectable
phenotype. Plasmid pJL190 was constructed by ligating
the neomycin resistance-conferring ~7.7kb EcoRI-HindIII
fragment of plasmid pLR4 to the ~19kb EcoRI-HindIII
2S fragment of plasmid pJL121. Plasmid pJL195 was con-
structed by ligating the pLR4 ~7.5kb EcoRI-partlal SalI
fragment to the ~5.4kb EcoRI-SalI fragment of plasmid
pJL125. The latter pJI,125 plasmid comprises the
largest (5.4kb) EcoRI-SalI fragment of plasmid pSCP2*
and was constructed by SalI deletion of plasmid pJL121.
Illustrative plasmi~s pJL190 and pJL195, in addition to
neomycin resista~ce, also express the M phenotype as
discussed above.
7~5
X-5773A _g_
Plasmid pLR4, the source of the neomycin
resistance conferring fragments, is ~7.7kb and is
constructed by ligating BamHI--treated plasmids pBR322
and pLRl. Plasmid pLRl is ~14.8kb and is constructed
by ligating HlndIII-treated plasmid pIJ2, disclosed in
Thompson et al., 1980, Nature 286.525, to HindIII-
treated plasmid pBR322. As is readily apparent to
those skilled in the art, both plasmïds pLR4 and pLRl
contain the same neomycin resistance gene and thus
either plasmid can be used for constructing the afore-
mentioned pJL neomycin resistant vectors.
An additional neomycin resistance-conferring
plasmid, designated as pJL192, was isolated as a
spontaneous mutant of plasmid pJLl90 resident in
Streptomyces ~riseofuscus. Plasmid pJLl92 specifies
resistance to elevated levels of neomycin and therefore
comprises a novel neomycin resistance gene which is
distinguishable Erom the resistance gene comprising
plasmids pJLl90, pJLl95, pIJ2, pLR4, and pLRl. In a
similar manner, an additional neomycin resistance;
conferring plasmid, designated as pJLl99, was isolated
as a spontaneous mutant of plasmid pJL195. Those
skilled in the art will recognize that the novel
neomycin resistance gene of plasmid pJLl92 or pJL19g
can be readily excised and ligated to other vectors.
The gene allows for improved and more efficient selec-
tion of transformants. As in the case of plasmids
pJL190 and pJLl95, transformants of plasmids pJLl92 and
pJL199 express the M phenotype when plated on an
appropriate indicator strain.
~Z()'7~5
X-5773A -10-
Plasmid pJL192 can be conventionally isolated
from E coli K12 C600~ -Mk-/pJL192, a strain deposited
and made part of the permanent stock culture collection
of the Northern Regional Research Laboratory, Peoria,
Illinois. It is available to the public as a stock
reservoir and preferred source of plasmid pJL192 under
the accession number B-15040.
A DN~ segment that confers resistance to
antibiotic thiostrepton, exemplified by the ~1.35kb
BamHI restriction fragment of plasmid pLR2, can also be
used with or substituted for the neomycin resistance~
conferring segment. Plasmid pLR2, the source of ~he
thiostrepton resistance conferrin~ fragment, is ~1807kb
and is constructed by ligating HindIII treated plasmid
pIJ6, disclosed in Thompson et al., 1980, Nature
286:525, to HindIII treated plasmid pBR322. Plasmid
pLR2 is functional in E. coli and there-Eore can be
amplified and isolated conveniently for subsequent
manipulation.
For convenience and ease of construction, the
thiostrepton resistance conferring ~1035kb BamHI
frasment of plasmid pLR2 was ligated into the Bam~lI
restriction site of plasmid pBR328 to form plasmid
pJI,193. The ~lkb ~clI restriction fragment of pJL193
contains the thiostrepton resistance-conferring DNA
segment. Therefore, ligation, as described in Examples
52-56, results in vectors that are within the scope of
the present invention.
Various plasmid SCP2 and SCP2* restriction
fragments can be used or purposes of constructing the
present invention provided that the origin of repli-
X-5773A -11-
cation contained in their respective ~5.4kb EcoRI~SalI
restriction fragments is present. Such additional
plasmid SCP2 and SCP2* restriction fragments include,
but are not limited to, the ~6kb SalI, ~15kb PstI, ~23kb
BglII, ~15kb BamHI, ~14kb EcoRI-PstI, ~13kb EcoRI--
BamHI, and ~15kb PstI-BamHI fragments. These fragments
contain the Streptomyces tra function and can be
ligated to a functional E. coli origin of replication-
containing and antibiotic resistance-conferring re-
striction fragment of an E. coli plasmidO Such E.
coli plasmids include, for example, plasmids pBR322,
pBR324, pBR325, pBR327, pBR328 and the like. There~
fore, the present invention is not limited to the use
of either plasmid pBR322 or pBR325 as exemplified in
several pJL constructions.
Although the neomycin and thiostrepton anti-
biotic resistance-conferring DNA segments exemplified
herein are respectively the ~7.7kb EcoRI-HindIII and
the ~7.5kb EcoRI-partial SalI fragments of plasmid
20 pLR4 and the pLR2 ~1.35 BamHI and the pJL193 ~lkb BclI
fragments~ those skilled in the art can construct and
use other DNA segments that also confer resistance to
neomycin or thiostrepton. Other neomycin resistance-
conferring DNA segments of plasmid pLRl include~ for
example, the ~3.4kb BamHI restriction fragment, the
~3.5kb PstI restriction fragment, and the larg~-r of the
SstI-KpnI subfragments of the ~3.4kb BamHI restriction
fragment. Other thiostrepton resistance~conferr:ing
segments include, for ~xample, the ~13kb PstI fragment
of plasmid pLR20 Still other DNA segments conferring
resistance to the same or to different antibiotics such
~l2~:~'7~3S
X-;7 73A --12--
as, for example, hygromycin, viomycin, tylosin, erythro-
mycin and the like, can also be constructed and used by
those skilled in the art. In addition, various func-
tional derivatives of the above described antibiotic
resistance-conferring DNA segments can be constructed
by adding, eliminaking, or substituting nucleotides in
accordance with the genetic code.
Ligation of the aforementioned derivatives,
or any of the other antibiotic resistance-conferring
DNA segments, to a vector comprising an E coli anti-
biotic resistance-conferring DNA segment, an E. coli
origin of replication-containing restriction fragment,
and also an origin of replication-containing restric-
tion fragment of plasmids SCP2 or SCP2*, ~esults in
plasmids that are within the scope of the present
invention. Therefore, an antibiotic resistance~con-
ferring DNA seyment can be used as a selectable marker
in place of the Streptomyces tra function and associated
pock phenotype. Thus, the present vectors are not
; 20 limited to the use of tra alone or in combination with
an antibiotic resistance-conferring DNA segment. In
addition, a particular antibiotic resistance-conferring
DN~ segment is not limited to a single ~osition on the
present chimeric plasmids but can be ligated or in-
2~ serted at varying sites provided that an origin of
replication or other critical plasmid controlled
physiological functions ~re not disrupted. Those
skilled in the art understand or can readily determlne
which sites are advantageous for ligation or ins~rtion
of a particular DNA segment.
.~ .
X-5773A -13-
The various restriction fragments of plasmids
SCP2, SCP2*~ pBR325, pBR322 and the like, and also the
various antibiotic resistance conerring DNA segmen~s
comprising the present vectors, can be modified to
facilitate ligation. For example, molecular linkexs
can be provided to some or all of the aforementioned
DNA fragments. Thus, specific sites for subsequent
ligation can be constructed conveniently. In addition,
the origin of replication-containing restriction
fragments can also be modiied by adding, eliminating,
or substituting certain nucleotides to alter charac-
teristics and to provide a variety of restriction sites
for ligation of DNA. Those skilled in the art under-
stand nucleotide chemistry and the genetic code and
thus which nucleotides are interchangeable and which
DNA modifications are desirable.for a specific purpose.
The recombinant DNA cloning vectors that
contain the SCP2 or SCP2* Streptomyces tra function are
self transmissable and thus readily transferred durlng
mating between transformed and non-transformed Strepto-
myces taxa~ This is advantageous because the present
vectors therefore can be transformed not only by proto-
plast transformation but also by conventional genetic
cros es. Consequently, the vectors are useful in
Streptomyces strains which are difficult to protoplast
thus greatly expanding the number of hosts in which
genetic manipulation and DNA cloning can be done~
More importantly, DNA-libraries constructed
in the present vectors can be conveniently and rapidly
screened for interesting genes by conventional replica-
plate mating proceduresO Without the tra function~ DNA
7~
X-5773 -14-
must be isolated from each of the thousands of clones
in the library and transformed into appropriate strains
to identify clones that contain desirable genes. Since
there are no broadly applicable phage vectors fox use
in Streptomyces, the present tra+ vectors fulfill the
general cloning and screening role analogous to that
of bacteriophage A in replica-plate transduction for
screening gene libraries in E coli. Desirable genes
can thus be readily identified by the replica-plate
mating procedure and then easily amplified by shuttling
into E. coli as described in Example 20C below.
The vectors of the present invention are
broadly applicable and are transformed into host cells
OL many Streptomyces taxa, particularly restrictionless
strains of economically important taxa that produce
antibiotics such as aminoglycoside, macroiide, ~-lactam,
polyether, and glycopeptide antibiotics. Such re-
strictionless strains are readily selected and isolated
from Streptomyces taxa by conventional procedures well
-
known in the art ~Lomovskaya et al., 1980, Microbiological
Reviews 44~206). Host cells of restrictionless strains
lack restriction enzymes and therefore do not cut or
degrade plasmid DNA upon transformation. For purposes
of the present application, host cells containing
restriction enzymes that do not cut any of the restric-
tion ites of the present vectors are also considered
restrictionless.
Preferred host cells of restrictionless
strains of Streptomyces taxa that produce aminoglyco-
side anti~iotics and in which the present vectors areespecially useful and are transformed, include restric-
s
X-;773A -15-
tionless cells of, for example: S. kanamyceticus
(kanamycins), S. chrestomyceti ~s (aminosidine), S.
griseoflavus (antibiotic MA 1267), S. microspor us
(antibiotic SF-767), S. ribosidificus (antibiotic
SF733), S. flavopersicus (spectinomycin), S. spectabilis
(actinospectacin), S. rimosus fo.rma paromomycinus
(paromomycins, catenulin), S. fradiae var. italicus
(aminosidine), S. bluensis var. bluensis (bluensomycin),
S. catenulae (catenulin), S. olivoreticuli var. cellu-
lophilu~ (destomycin A), S. tenebrarius (tobramycin,
apramycin), S. lavendulae (neomycin), S. albogriseolus
(neomycins), S. albus var. m ~ (metamycin), S.
hyaroscopicus var. sagamiensis (spectinomycin), S.
bikiniensis (streptomycin), S. griseus (streptomycin),
l; S. erythrochromogenes var. narutoensis (streptomycin),
S. poolensis (streptomycin), S. galbus (streptomyci.n),
S. rameus (streptomycin), S~ olivaceus (streptomycin),
. .
S. mashuensis (streptomycin), S. hygroscopicus ~ar.
limoneus (validamycins), SO rimofaciens (destomycins),
S. hygroscopicus forma glebosus (glebomycin), S.
fradiae (hybrimycins neomycins), _. eurocidicus
(antibiotic Al6316-C), S. aquacanus (N methyl hygromycin
B), S. crystallinus (hygromycin A), S. noboritoensis
(hygromycin)~ S. hygroscopicus (hygromycins), S.
2~ atrofaciens (hygromycin), S. kasugaspinus (kasugamycins),
S. kasugaensis (kasugamycins), S. netropsis (antibiotic
_
LL-A~31), S. lividus (lividomycins), _. hofuensis
(seldomycin complex), and _. canus (ribosyl paromamine).
Preferred host cells of restric~ionless
strains of Streptomyces taxa that produc~ macrolide
antibiotics and in which the present vectors are
., .
~' d
37~
X-5773A -16-
especially useful and are transformed, include restric-
tionless c911s of, for example: S. caelestis (anti-
biotic M188), S. platensis (platenomycin), S. rochei
var. volubilis (antibiotic T2636), S. venezuelae
(methymycins), S. griseofuscus (bundlin), S. narbo-
nensis ~josamycin, narbomycin), S. fungicidicus
(antibiotic NA-181), S. griseofaciens (antibiotic
PA133A, B), S. roseocitreus (albocycline), S. bruneo-
grlseus (albocycline), S. roseochromogenes (albocycline~,
S cinerochromogenes (cineromycin B), S. albus (albo-
.
mycetin), S. felleus (argomycin, picromycin), SO
rochei (lankacidin, borrelidin), S. violaceoniger
(lankacidin), S. griseus (borrelidin), S. maizeus
(ingramycin), S. albus var. coilmyceticus (coleimycin),
S. mycarofaciens (acetyl-leukomycin, espinomycin), S.
hygroscopic_ (turimycin, relomycin, maridomycin,
tylosin, carbomycin), S. griseospiralis (relomycin), S.
lavendulae (aldgamycin), S. rimosus (neutramycin), S.
deltae (deltamycins), S. fungicidicus var. espino-
myceticus (espinomycins), S. ~urdicidicus (mydeca-
~ . .. _ . . .. .
mycin), S. eurocidicus ~methymycin), SO g eolus(griseomycin), S. flavochromogenes (amaromycin, shinco-
mycins), S. fimbriatus (amaromycin), S. fasciculus
(amaromycin), S. _ ~ (erythromycins), S. anti-
bioticus (oleandomycin), S. olivochromogenes (oleando-
mycin), S. spinichromo~enes var. suragaoensis (kujimycins),
S. kitasatoensis (leucomycin), S. narbonensis var.
josamyceticu~ (leucomycin A3, josamycin), S. albogr~seolus
(mikonomycin), SO bikiniensis (chalcomycin), S. irratus
(cirramycin), S. djakartensis (niddamycin), S~ eurythermus
(angolamycin), S. fradiae (tylosin~ lactenocin, macrocin),
~-5773A -17-
S. goshikiensis (bandamycin), S. griseoflavus (acumycin),
S. halstedii (carbomycin), S. ~endae (carbomycin) J S.
macrosporeus (carbomycin), S. thermotolerans (carbo-
_
mycin), S. albireticuli (carbomycin), and S. ambofaciens
(spiramycin).
Preerred host cells of restrictionlessstrains of Streptomyces taxa that produce ~-lactam
anti~iotics and in which the present vectors are
espec.ially useful and are transformed, include re-
strictionless cells of, for example: S. lipmanii(A16884, MM4550, ~M13902), S. clavuligerus (A16886B,
clavulanic acid), S. lactamdurans (cephamycin C), S.
griseus (cephamycin A, B)~ S. h groscopicus (deacetoxy-
cephalosporin C), S. wadayamensis (WS-3442-D), S.
chartreusis (SF 1623), S. heteromorphus and S. panayens1s
~C2081X); S. cinnamonensis, S. f.imbriatus, S. halstedii,
S. rochei and S. viridochromogenes (cephamycins A, B);
_~
S. cattleya (thienamycin); and S. ol _aceus, S. flavo-
virens, S. ~lavus, S. fulvoviridis, S. argenteolus~ and
S. sioyaensis (MM 4550 and MM 13902).
Preferred host cells of restrictionless
strains of Streptomyces taxa that produce polyether
antibiotics and in which the present vectoxs are
especially useful and are transformed, include re-
strictionless cells of, for example: S. albus (A204,A28695A and ~, salinomycin~, S. hygroscopicus (A218,
emericid, DE3936) , A120A, A28695A and B, etheromycin,
dianemycin), S. griseus (grisorixin~, S. onglobatus
(ionomycin), S. eurocidicus var~ asterocidicus ~laidlo-
mycin), _. lasallensis (lasalocid), S. ribosidificus
(lonomycin), S. cacaoi var. asoensis (lysocellin), S.
~LZ~9'~
X-5773A -18-
cinnamonensis (monensin), S. aureofaciens (narasin~, S.
gallinarius (RP 30504), S. longwoodensis (lysocellin),
S. flaveolus (CP38936), S. mutabilis ~S-11743a), and S.
violaceoniger (nigericin).
Preferred host cells of restrictionless
strains of Streptomyces taxa that produce glycopeptide
antibiotics and in which the present vectors are
especially useul and are transformed, include re-
strictionless cells of, for example: S. orientalis and
S. haranomachiensis (vancomycin); S. candidus ~A-35512,
avoparcin), and . eburosporeus (LL-AM 374).
Pre-ferred host cells of other Streptomyces
restrictionless strains in which the present vectors
are especially useful and can be transformed, include
restrictionless cells of, for example: S. granuloruber,
S. ros~osporus, S. lividans, S. e.spinosus, and S.
azure _.
In addition to the representative _repto-
myces host cells described above, the present vectors
are also useful and can be transformed into E. coli.
Thus, vectors of the present invention have wide
application and are useful and can be transformed into
host cells of a variety of organisms.
While all the embodiments of the present
2~ invention are useful, some of the present recombinant
DNA cloning vectors and transformants are preferredO
Accordingly~ preferred vectors are pJL114, ~JL121,
pJL125, pJL180, pJLl90, pJL192, pJL195, pJL197, pJLl99
and pHJL212 and preerred transformants are Strepto~
myces griseofuscus/pJL114, S. griseofu cus/pJL121, S.
griseofuscus/pJL125, S. ~ /pJL180, S.
griseofuscus/pJLl90, S. griseofuscus/pJL192, S.
Z1)7~
X-5773A . 19-
griseofuscus/pJL195, S.griseofuscus/pJLl99, S. griseo-
fuscus/pJL197, S. griseofuscusJpHJL212, E coli K12
C600Rk-M~-/pJL114, E. coli K12 C600Rk-Mk-jpJL121, E.
coli K12 C600Rk Mk-tpJL125, E. coli K12 C600Rk Mk-/pJL180,
E. coli K12 C600R M -/pJLlgO r E . coli K12 C600R -M -/
- k k - - k k
pJL192, E. coli K12 C600Rk-Mk-/pJL195, E. coli X12
C600Rk-Mk-/pJL139, E. coli X12 C600Rk-Mk~/pJL197, and
E. coli K12 C600Rk-Mk-/pHJL212. Moreover, of this
preferred group, plasmids pJLl90, pJL192, pJL195,
pJL197, pJLl99 and pJL and transfor~.ants S. griseo=
fuscus/pJLl90, S. griseofuscus/pJL192, S. griseo-
fuscus/pJL195, S. griseofuscus/pJL197, S. griseofuscus/
pJLl99, S. griseofuscus/pHJL21?, E~ coli K12 C600Rk-Mk-/
pJLl90, E. coli X12 C600Rk-Mk-/pJL192, E. coli K12
C600Rk-Mk-/pJ~95 and E. coli K12 C600Rk-Mk-/pJL197, E.
coli K12 C600Rk-Mk-/pJLl99 and E. coli K12 C600Rk-Mk-/
pHJL212 are most preferred~ Streptomyces
is a preferred host because it does not contain an
endogenous plasmid or synthesize an antibiotic.
Therefore, transformants of S. griseofuscus can be
screened for clones that express genes for antibiotic
synthesis.
Tne vectors of the present invention com-
prises origins of replication that are functional in E.
~5 coli and Streptomyces and therefore provide flexib.ility
: in the choice of hosts. Consequently, cloned DNA
sequences can be shuttled into E. coli for construction
of new plasmids, physical analysis, and for mapping of
restriction sites and then shuttled back into Strepto-
myces for functional analysis and improvement of
strains. This is particularly advantageous because
. amplification and man.ipulation of plasmids can.be done
' ~
~2~
X-5773A ~20-
faster and more conveniently in E. coli than in Strepto-
myces. For example, the present vectors can be amplified
conventionally in E. coli K12 by growth with spectino-
mycin or chloramphenicolr This is not possible in the
Streptomyces host system. In addition, since all the
plasmid vectors contain resistance markers that are
expressed in E. coli K12, recombinant~ are easily
selected. Therefore, large amoun~s o~ plasmid DN~ can
be isolated conveniently and in a shorter time than
that required for doing similar procedures in ~
myces. Thus, after desired recombinant DNA procedures
are accomplished in the E. coli host system, the partic-
ular Strep omyces DNA can be removed, reconstructed to
plasmid form (if necessary~, and then transformed into
a Streptomyces host cell. Since the present vectors
are fully selectable in Streptomyces, identification of
recombinant clones can be done efficiently.
The recombinant DNA cloning vectors and
transformants of the present invention have broad
utility and help fill the need for suitable cloning
vehicles for use in Streptomyces and E. coli. More-
over, the ability of the present vectors to con~er a
pock phanotype or resistance to antibiotics also
provides a functional means for selecting transformants.
This is important because of the practical necessity
for determining and selecting the particular cells that
have acquired vector DNA. Additional DNA segments,
that lack functional tests for their presence, can also
be inserted into the prPsent vectors and then trans-
formants containing the non-selectable DNA can be
isolated by appropriate antibiotic or other phenotype
.
X-5773A -21-
selection. Such non-selectable DNA segments can be
inserted at any site, except within regions necessary
for plasmid function and replication, and include
genes that specify antibiotic modification enzymes and
regulatory genes of all types.
More particularly, a non-selectable DNA
segment that comprises a gene can be inserted into a
plasmid such as, for example, illustrative plasmid
pJL192, at the internal BamHI restriction site of the
~7.7kb EcoRI-HindIII resistance-conferring fragment.
Such an insertion inactivates the neomycin resistance
gene and thus allows for the easy identification of
Streptomyces transformants containing the recombinant
plasmid. This is done by first selecting ~or M pock
morphology and, secondarily, identifying those ~ trans
formants that are not resistant to neomycin. In a
similar manner, insertion of a DNA segment into illus~
trative plasmid pJLl80 at, for example, the unique PstI
restriction site, inactivates the ampicillin resistance
gene. Thus, E. coli transformants carrying this re-
combinant plasmid can also be identified easily by
first selecting for chloramphenicol resistance and,
secondarily, identifying those chloramphenicol resistant
transformants that are not resistant to ampicillin.
Therefore, the ability to select for antibiotic re-
sistance or other phenotypic markers in Streptomyces
and E. coli allows for the efficient isolation of the
extremely rare cells that contain the particular
non-selectable DNA of interest.
The functional test for antibiotic resis-
tance, as described above, can also be used to identify
:~2~'7~
X 5773A -22-
DNA segments that act as control elements and direct
expression of an individual antibiotic resistance gene.
Such segments, including but not limited to, promoters,
attenuators, repressors, inducers, ribosomal binding
sites, and the like, can be used to control the ex-
pression of other genes in cells of Streptomyces and _.
coli.
The antibiotic resistance-conferring vectors
of the prese~t invention are also useful for .insuring
that linked DNA segments are stably maintained in host
cells over many generations. These genes or DNA fxag-
ments, covalently linked to an antibiotic resis~ance
conferring fragment and propagated either in Strepto~
myces or E. coli, are maintained by exposing the trans-
_
formants to levels of antibiotic that are toxic tonon-transformed cells. Therefore, transformants that
lose the vector, and consequently any covalently linked
DNA, cannot grow and are eliminated from the culture.
Thus, the vectors oE the present invention can be
used to maintain any DNA sequence of interest.
The cloning vectors and transformants of the
present invention provide for the cloning of genes to
improve yields of various products that are currently
produced in Streptomyces and related cells. Exampl~s
of such products include, but are not limited to,
Streptomycin, Tylosin, Cephalosporins~ Actaplaninl
Nara~in, ~onensin, Apramycin, ~obramycin, Erythromycin,
and the like. The present invention also provides
selectable vectors that are useful for cloning, charac-
terizing, and reconstructing DNA sequences that codefor comm~rcially important proteins such as, for exam-
ple, human insulin, human proinsulin, human growth
~Z~t~6
X-5773A -23-
hormone, bovine growth hormone, glucagon, interferon,
and the like; for enzymatic functions in metabolic
pathways leading to commercially important processes
and compounds; or for control elements that improve
gene expression. These desired DNA sequences include,
but are not limited to, DNA that codes for enzymes that
catalyze synthesis of derivatized antibiotics suoh as,
for example, Streptomycin, Cephalosporin, Tylosin,
Actaplanin, Narasin, Monensin, Apramycln, Tobramycin,
and Erythromycin derivatives, or for enzymes that
mediate and increase bioproduction of antibiotics or
other products.
The capability of inserting, stabilizing, and
shuttling the aforementioned DNA segments into ~
myces and E. coli allows for easy recombinant genetic
manipulation for increasing the yield and availabili~y
of antibiotics that are produced by Streptomyces. In
addition, since the plasmid SCP2 or SCP2* origin of
replication codes for low copy number, almost any DNA
sequence, including those that are lethal when ex-
pressed from a high copy number plasmid, can be readily
cloned into the present vectors and shuttled between
Streptomyces and E. coli
_ .
Strep~omyces coe color A3(2) and S. coelicolor
MllO, as respective sources of plasmids SCP2 and SCP2*,
can be cultured in a number of ways using any of
several different media. Carbohydrate sources which
are prefexred in a culture medium include, for example,
molasses, glucose, dextrin, and glycerol, and nitrogen
sources include, for example, soy flour, amino acid
mixtures, and peptones. Nutrient inorganic salts are
also incorporated and include the customary salts
1L2~3"7~i~1S
X-5773A -24-
capable of yielding sodium/ potassium, ammonium, calcium,
phosphate, chloride, sulfate, and like ions. ~s i~
necessary for the growth and development of other
microorganisms, essential trace elem~nts are also
added. Such trace elements are commonly supplied as
impurities incidental to the addition of other con-
stituents of the medium.
Streptomyces coelicolor MllO and S. coeli~
color A3(2) are grown under aerobic culture conditions
over a relatively wide pH range of about 5 to 9 at
temperatures ranging from about 15 to 40C. For
production of plasmids SCP2 and SCP2* at highest copy
number, however, it is desirable to start with a
culture medium at a pH of about 7.2 and maintain a
culture temperature of about 30C. Culturing Stxepto-
myces coelicolor M110 and S. coelicolor A3(2) under the
aforementioned conditions, results in a reservoir of
cells from which plasmids SCP2 and SCP2* are respec-
tively isolated conveniently by techniques well known
in the art.
The following examples further illustrate and
detail the invention disclosed herein. Both an ex-
- planation of and the actual procedures for constructing
the invention are descxibed where appropriate.
Example 1
- Isolation of Plasmid SCP2*
. .
A. Culture of Streptomyces coelicolor MllO
_
A vegetative inoculum of S~reptomyces
coelicolor MllO (NRRL 15041) was conventionally pre-
pared by growing the strain under submerged aerobic
. .
X-5773~ -25-
conditions in 50 ml. of sterilized ~Trypticase~l soy
broth* at 35 q./l. in deionized water.
The 'Trypticase'l soy broth inoculum was incu-
bated for 48 hours at a ~emperature of 30C. The
50 ml. culture was then homogenized, transferred to
450 ml. of sterilized YEMESG** medium, and then in-
cubated for at least 40, but not more than 65 hours,
at 30C. The pH was not adjusted. After incubation,
the Streptomyces coelicolor M110 cells were ready for
-
harvest and subsequent isolation of plasmid DMAo
*'Trypticase'1 so~- ~roth is obtained from Difco
Laboratories, Detroit, Michigan.
YE~ESG comprises .3~ yeast extract, .5% peptone,
.3% malt extract, 1% dextrose, 34~ sucrose,
.1% MgCl2, and .1% glycine.
B. Plasmid Isolation
-
About 10 g. (wet wgt) of Streptomyces coeli-
color MllO cells were harvested by centrifugation (lO
2~ minutes, 4C., 10~000 rpm) and then about lO ml./g. wet
wgt cells of TES buffer (oOlM Tris(hydroxymethyl)amino-
ethane [tris], .OOlM EDTA, 25% sucrose, p~ 8) were added.
The cells were vortexed into suspension followed by
addition of 10 ml./g. wet wgt cells of .25M EDTA, pH 8
2~ and then 5 ml./g. wet wgt cells of lysozyme (lO mg./ml.
in TES). After the mixture was incubated at 37C. for
about 15 minutes, about 105 ml./g~ wet wgt cells o~ 20
- SDS (sodium lauryl sulfate (BDH Chemicals Ltd. Poole,
England), were added. The resultant mixture was allowed
to stand at room temperature for 30 minutes, and then
5M NaCl was added to give a final concentration of lM
1. Trademark for dehydrated peptone prePared from
i ~ casein by pancreatic digestion.
)'7~
X-5773A 26-
NaCl. After standing again at room temperature (15minutes), the mixture was placed on ice for 2 hours.
The lysate was centrifuged (20 minutes, 4C., 17,500 rpm)
and the supernatant was pooled and mixed with .64
volumes of isopropyl alcohol. The DNA precipitate was
collected by centrifugation (15 minutes, 4C., 10,000
rpm). The precipitate was air dried and then resus
pended in 1 ml./g. wet wgt cells of TE buffer (.OlM
` Tris, .OOlM EDTA). Centrifugation (20 hours, 20Co
10 50,000 rpm) using cesium chloride gradients with
propidium iodide was carried out to purify the plasmid
DNA. Following centrifugation, the desir~d plasmid
SCP2* DNA band was removed and the propidium iodide
extracted by conventional procedures. The CsCl DNA
solution was stored at -20C. Prior ~o use, the DNA
was desalted by either P~lO~(Bio Rad)'column exchange
with TE or by dialysis against TE. The D~A was precip--
itated with ethanol by conventional procedures and
redissolved in TE.
~3 Example 2
Cons~ruction of Plasmid pLRl
A. EindIII Digestion o~ Plasmid pIJ2
?5 Abvut 20 ~1. (20 ~g.) of plasmid pIJ2 DNA,
disclosed in Thompson et al., 1980, Nature 286:525,
5 ~1. BSA(Bovine Serum albumin, 1 mg./mlO), 19 ~1.
water, 1 ~1. of HindIII (containing 3 New England Bio
Labs units) restriction enzyme*, and 5 ~1. reaction
mix** were incubated at 37C. for 2 hours. The reac
tion was terminated by the addition of ahout 50 ~1~ of
2. Trademark
i~ ?
s
X-5773A -27~
4M ammonium acetate and 200 ~1. of 95% ethanolO The
resultant DNA precipitate was washed twice in 70~
ethanol, dried in acuo, suspended in ao ~1. of TE
buffer, and frozen at -20C. for storage.
Restriction and other enzymes can be obtained from
the following sources~
New England Bio Labs., Inc.
32 Tozer Road
Beverly, Massachusetts 01915
Boehringer-Mannheim Biochemicals
7941 Castleway Drive
Indianapolis, Indiana 46250
Bethesda Research Laboratories (BRL)
Box 6010
Rockville, Maxyland 20850
Research Products
Miles Laboratories, Inc.
Elkhart, Indiana 46515
Reaction mix for HindIII restriction enæyme was pre
pared with the following composition:
60OmM NaCl
lOOmM Tris-EICl, pH7.9
70mM MgCl~
lO~M Dithiothreitol
B. HindIII Digestion of Plasmid pBR322
.. . . ...
About 8 ~1. (4 ~g.) of plasmid pBR322 DNA,
5 ~1 reaction mix, 5 ~1. BSA (1 mg./ml.), 31 ~1.
water, and 1 ~1. of H _ III restriction enzyme were
incubated at 7C. for 2 hours. After the reaction was
3~
~L~24~7~
X-5773A ~28-
terrninated by incubating at 60C. for 10 minutes, about
50 ~1. of ammonium acetate and 200 ~1. of 95% ethanol
were added. The resultant DNA precipitate was washed
twice in 70~ ethanol, dried ln vacuo, and suspended in
45 ~1. of water.
C. Ligation of HindIII Digested Plasmids pIJ2 and pBR322
.
About 20 ~1. of HindIII treated plasmid pIJ2
(from Example 2A), 20 ~1. of HindIII treated plasmid
pBR322 (from Example 2B), 5 ~l o BSA (1 mg./ml.), 1 ~1.
of T4 DNA ligase , and 5 ~1. ligation mix** were
incubated at 16C. for 4 hours. The reaction was ter-
minated by the addition of about 50 ~1. 4M ammonium
acetate and 200 ~1. of 95gO ethanol. The resultant DNA
precipitate was washed twice in 70% ethanol, dried ln
vacuo, and suspended in TE buffer. The suspended D~A
constituted the desired plasmid pLRl.
*
T4 DNA ligase can be obtained from the following
source:
New England Bio Labs., Inc.
32 Tozer ~d.
Beverly, Massachusetts 01915
**
Ligation mix was prepared with the following composi-
tion:
500mM Tris-HCl, pH7.8
200~ Dithiothreitol
lO~)mM MgC12
lOm2~ ATP
o~
X-5773~ -29-
Example 3
Construction of E. coli K12 HBlOlJpLRl
About 10 ml. of E. coli K12
HB101 cells (Bolivar et al., 1977, Gene 2:75-93) were
pelleted by centrifugation and then suspended in about
10 ml. of .OlM sodium chloride~ Next, the cells were
pelleted again, resuspended in about 10 ml. o .03M
calcium chloride, incubated on ice for 20 minutes,
pelleted a third time, and finall~, resuspended in
1025 ml. of .03M calcium chloride. The resultant cell
suspension was competent for subsequent transformation.
; Plasmid pLRl in TE buffer (prepared in
Example 2C) was ethanol precipitated, suspended in
15 150 ~1. of 30mM calciu~ chloride solution, and gently
mixed in a test tube with about 200 ~1. o competent E.
coli K12 HB101 cells. The resultant mixture was in
cubated on ice for about 45 minutes and then at 42C.
for about 1 minute. Next, about 3 ml. of L-broth
20 (Bertani, 1951, J. Bacteriology 62:2g3) containing
50 ~g./ml. of ampicillin was added. The mixture was
incubated with shaking at 37C. for 1 hour and then
plated on L-agar (Miller, 1972, Experiments in Molecular
Genetics, Cold Spring Harbor Labs, Cold Spring Harbor,
New York) containing ampicillin. Surviving coloni s
were selected and tested for the expected phenotype
(AmpR, TetS), and constituted the desired E coli K12
HB101/pLRl transformants.
~3'7~
,,
X~5773A -30-
Example 4
_ ,_
Construction of Plasmid pLR4
A. Partial BamHI Digestion of Plasmid pLRl
About 10 ~1. (10 ~g.) of plasmid pLRl, 5 ~1. BSA
(1 mg./ml.), 29 ~1. water, 1 ~1. of BamHI ~diluted 1:4
with water) restriction enzyme, and 5 ~1. reaction
mix* were incubated at 37C. for 15 minutes. The reac~
tion was terminated by the addition of about 50 ~lo of
10 4M ammonium acetate and 200 ~1. of 95~ ethanol. The
resultant DNA precipitate was washed twice in 70%
ethanol, dried in vacuo, and suspended in 20 ~1. water.
*
Reaction mix for BamHI restriction enzyme was pre-
pared with t~e following composition:
1.5M NaCl H
60mM Tris-HCl, p 7.9
6OmM MgC12
B. BamHI Digestion of Plasmid pBR322
; 20 The desired digestion was carried out in su~-
stantial accordance with the teaching of Example 2B
except that BamHI restriction enzyme and reaction mix
were used in place of ~indIII restriction enzyme and
reaction mix. The digested plasmid pBR322 was suspended
2 5 in 2 9 ~1 . o f water.
C. Ligation of Partial BamHI Diyest~d Plasmid pLRl
and BamHI Di~ested Plasmid pBR322
.. . _ . . . _
The desired ligation was carried out in substantial
accordance with the teaching of Example 2C. The resul-
tant ligated DNA was suspended in TE buffer and consti-
tuted the desired plasmid pLR4.
~Z~3~6~3~
X--5'773A -31-
Example 5
Construction of E. coli Xl2 HB101/pLR4
The desired construction was carried out in
substantial accordance with the teaching of Example 3
except that plasmid pLR4, rather than plasmid pLRl, was
used for transformation. Surviving colonies were se-
lected and tested for the expected phenotype (AmpR,
Tet ), and constituted the desired E. coli ~12 HBlOl/
pLR4 transformants.
Example 6
Construction of Plasmids pJLl20 and pJL121
A. EcoRI Digestion of Plasmid SCP2*
lS About lSO ~l. (5.7 ~g.) of plasmid SCP2* D~A,
l~l. water~ 2 ~l. of EcoRI (containlng 20 BRL units)
restriction enzyme, and 17 ~l. EcoRI reaction mix* were
incubated at 37C~ for 2.5 hours. The reaction was
; terminated by incubation at 65C. for 15 minu~es~
The reaction was conventionally analyzed by agarose gel
electrophoresis (AGE) to verify that restriction was
complete. The restricted DNA was stored at 4C. for
subsequent use.
.
_ ~ *
Reaction mix for EcoRI restriction enzyme was
- prepared with the following composition:
500~ NaCl
1000~`~1 Tris-HCl, pH7.5
lOOm,~ MgC12
-
.
X-5773A -32-
B. EcoRI Digestion of Plasmid pBR325
The desired digestion was carried out in
substantial accordance with the teaching of Example 6A
except that plasmid pBR325, rather than plasmid SCP2*,
was used. The resultant DNA was stored at 4~C. for
subsequent use.
C. Ligation of EcoRI Digested Plasmids SCP2* and pBR325
About 40 ~1. of EcoRI digested plasmid SCP2*
(from Example 6A), 10 ~1. of EcoRI digested plasmid
-
pBR325 ~'rom Example 6B), 10 ~1. of MgC12 (.lM), 10 ~1.
of ~NH4)2SO4 (.lM), 10 ~1. ATP (2~'1) .1 ~1. of T4 DNA
ligase, and 20 ~1. ligation mix* were incubated at 4C.
for 18 hours. The reaction was analyzed by AGE to
verify appropriate ligation. The suspended DNA con-
stituted the desired ~35.8kb plasmids pJL120 and pJL121.
; Recombinant plasmids of two orientations
result because the plasmid pBR325 Eco~I fragment can be
oriented in either direction. A restriction site map
of each of plasmids pJL120 and pJL121 was determined
(after isolation as disclosed in Example 7) and is
presented in Figure 2 of the accompanying drawingsO
~5 Ligation mix was prepared with the following composition:
50~ Tris-HC1~ pH 7.5
lOmM ~-mercaptoethanol
lmM EDTA
50 ~g./ml. BSA
X 5773A -33-
Example 7
Construction of E. coli K12 C600 ~ -Mk-/pJL120 and
E. coli K12 C600Rk-Mk-/pJ~121
A. Preparation of Frozen Competent E. coli K12
C600Rk-Mk-- -- --
- Fresh overnight cultures of E~ coli K12
C600Rk-Mk- (disclo~ed in Chang and Cohen, 1974, Proc.
Nat. Acad. Sci. 71:1030-1034) were subcultured 1:10 in
fresh L-broth ~disclosed in Miller, 1972, Experiments
in Molecular Genetics, Cold Spring Harbor Labs, Cold
Spring Harbor, New York) and grown at 37C. for 1 hour.
A total of 660 Klett Units of cells wera harvested,
washed with 2.5 ml. of lOO~M NaCl, suspended in 150mM
is CaC12 with 10% glycerol, and incubated at room tem-
perature for 20 minutes. The cells were harvested by
centrifugation, resuspended in .5 ml. of CaC12-glyc rol,
chilled on ice for 3-5 minutes and frozen. The sus-
pensions of cells were stored in liquid nitrogen until
use. Preservation and storage did not adversely a~ect
the viability or frequency of transformation by co~a-
lently closed circular DNA.
B. Transformation
The competent cells were thawed in an ice
bath and mixed in a ratio of .1 ml. of cells to O05 ml.
of DNA (10 ~1. of the sample disclosed in Example 6C
and 40 ~1. of .lXSSC (.015M NaCl, ~0015M Sodium Citrate
at pH 7). The transformation mixture was chilled on
30 ice for 20 minutes, heat shocked at 42C. for 1 minute
and chilled on ice for 10 minutesD The samples were
:~p ~
X~5773A -34-
then diluted with .85 ml. of L broth, incubated at 37C.
for 1.5 hours, spread on L-agar containing ampicillin
(50 ~g./ml.) and tetracycline (12.5 ~g./ml~) and
incubated for 18 hours at 37C. The resultan~ colonies
were selected and tested for the expected phenotype
(Amp , Tet , CM ) and constituted the desired E. coli
K12 C600Rk-Mk-/pJL120 and E~ coli K12 C600Rk-Mk-/pJL121
transformants. The ampicillin and tetracycline re-
sistant colonies were isolated according to known
procedures, cultured, and then conventionally iden~
tified by restriction enzyme and AGE analysis of the
constitutive plasmids. The identified transformants
were then used for subsequent production and isolation
of plasmids pJL~20 and pJL121 acc~rding to known pro-
cedures.
Example 8
Construction of Plasmids pJL180 and pJL]al
A. SalI Digestion of Plasmid SCP2* and Isolation of
... _ . _ . . . .
2~ ~. okb S eI r~9m-nt
The desired digestion was carried out in
substantial accordance with the teaching of Example 6
except SalI restriction en7yme and reaction mix*,
rather than EcoRI restriction enzyme and reaction mix,
were used. The reaction was assayed by AGE ~o verify
completion and terminated by heatin~ at 65Co for 15
minutes. The resultant SalI restriction fragments were
separated by AGE and ~hen the separa~ed fragments werP
located in tAe gel by staining with ethidium bromide
and visualizing fluorescent bands wi~h an ultraviolet
~IL2(17~S
X 5773A -35-
light. The gel fragment containing the ~6.Okb fragment
of interest was excised from the gel and electroeluted
in.o TBE buffer (1.6~'Sigma 7~9'~uffer**, .093~ Na~EDTA,
.55~ boric acid). The gel-fragment in ~BE buffer was
placed in a dialysis bag and subjected to electrophoresis
at 100 V for 1 hour. The aqueous solution was collected
from the dialysis baa and passed over a DEAE cellulose
colum~*** (.5 ml.'h~atman DE;2~ that had been e~uilibrated
with equilibration buffer (.lM KCl, 10 ~ Tris HCl,
pH 7.8). The column was washed with 2.5 ml. of equili-
bration buffer and the DNA (about 5 ~g.) was eluted
with 1.5 ml. of elution buffer (lM NaCl, 10 mM Tris-
HCl, pH 7.8). The eluent was adjusted to about .35M
with respect to Na ion concentration, and then the DNA
was precipitated by adding 2 volumes (about 9 ml.) of
100~ ethanol followed by cooling to -20C. for 16
hours. The DNA precipitate was pelleted by cen~ri-
fugation, washed with 75% ethanol, dried, and dissolved
in TE buffer. Hereinafter, this conventional isolation
2~ technique is referred to as AGE~DE52~electroelution~
Reaction mix for SalI restriction enzyme was prepared
with the following composition:
1500 m~`~l NaCl
~; 80 ~ Tris-HCl, pH7.5
60 ~ ~gC12
~ ~ EDTA
**Sigma 7-9 buffer can be obtained from Sigma Chemical
Company, P.O. Box 14508, St. Louis, Missouri 63178
***DEAE cellulose (DE52) can be obtained from
What~an Inc., 9 Bridewell Place, Clifton,
New Jersey 07014.
S c.~ 3. Trademark
4. Trademark
~ z~t7~ ~ ~
X-5773A -36-
B. SalI Digestion of Plasmid pBR325
TAe desired digestion was caxried out in sub-
stantial accordance with the teaching of Example 8A
except that plasmid pBR325 was employed and fragments
were not separated by preparative AGE/DE52/electroelution.
The resultant ~NA was dissolved in TE buffer and stored
at 4C. for future use.
C. Ligation of SalI Digested Plasmid pBR325 and
~6.Okb SalI Fragment of Plasmid SCP2*
.
About 1.5 ~g. of the 6.Okb SalI fragment of
SCP2*, prepared in Example 8A, was mixed with .5 ~g.
of SalI digested pBR325, prepared in Example 8B.
The DNA mixture was precipitated by standard ethanol
precipita-tion and redissolved in 3 ~1. of distilled
water, 4 ~lo of .66M ATP, 2 ~1. of ligase-kinase
mixture (.25M Tris-HCl, pH 7.8, 50 mM MgC12, 25 mM
dithiothreitol and 25~ glycerol) and 1 ~1. of T4-DNA
ligase (1 unit). After incubation for 1 hour at
15C., the reac-tion mixture was diluted with 12 ~1. of
waterl 20 ~1. of .66M ATP, 8 ~1. of ligase-kinase
mixture and then incubated at 15C. for 18 hours. The
resultant ligated DNA was diluted 1:5 into .lXSSC
and constituted th~ desired ~12.Okb plasmids pJI,180 and
pJL181.
Recombinant plasmids of two orientations
result because the plasmid pBR325 SalI fragment can be
oriented in either direction. A restriction site map
of each of plasmids pJL180 and pJL181 is presented in
Figure 3 of the accompany~ng drawings.
. , .
:3L20~
X-5773A -37~
Example 9
Construction of E. coli K12 C600Rk-Mk-/pJL180 and s;
.
E~ coli X12 C6ooRk-Mk-/pJLl8l
The desired constructions were made in sub-
stantial accordance with the teaching of Example 7
except that the mixture of plasmid pJL180 and pJLl81
DNA (from Example 8C), rather than plasmid pJL120 and
pJL121, were used. The resultant transformant colonies
were selected and tested for the expected phenotype
(AmpR, TetS, CMR), and constituted the desired E. coli
Kl2 C600Rk-l~k-/pJLl80 and E. coli Kl2 C600Rk-M~/pJLl8l
transformants. The ampicillin and chloramphenicol
resistant colonies were isolated according to known
procedures, cultured, and then con~entionally iden-
tified by restriction enzyme and.AGE analysis of theconstitutive plasmids. The identified transforman~s
can then be used for subsequent production and iso-
lation of plasmids pJLl80 and pJLl81 according to known
procedures.
Example lQ
Construction of Plasmid pJLl25
A. SalI Digestion of Plasmid pJLl21 and Isolation of
~10.2kb SalI Fragment
The desired dige~tion was carried out insubstantial accordance with the teaching of Example 8
except that the reaction was stopped before digestion
was complete and except that plasmid pJLl21, rather
than plas~id SCP2*, was used. The resultant SalI
X 5773A 38-
restriction fragments were not separated by preparative
AGE but precipitated by standard ethanol precipitation.
The restriction fragments were dissolved in TE buffer
and immediately ligated.
B. Ligation of ~10.2kb SalI Fragment oE Plasmid pJL121
. . . _
The desired ligation was carried out in
substantial accordance with the teaching of Example 8C
except that the SalI fragments of plasmid pJL121,
rather than the SalI fragment of plasmid SCP2* and
pBR325, were used. The resultant ligated DNA con-
stituted the desired plasmid pJL125 plus 12 other
plasmids that were subsequently isolated and shown to
contain additional SalI restriction fragmen-ts o~
pJL121. Plasmid pJL125, which was conventionally
isolated and contains an origin of replication from
plasmid pBR325 and also the ~5.4kb origin of replication-
containing EcoRI-SalI fragment of plasmid SCP2*, was
dissolved in TE buffer and stored at 4C. for future
use. A restriction site map of plasmid p3L125 is
presented in Figure 4 of the accompanying drawing. The
restriction site map was determined with plasmid from
transformed E. coli R12 C600Rk-Mk-.
Example 11
Construction of E. coli K12 C600R~-Mk-/pJL125
_ _
The desired construction was made in sub-
stantial accordance with the teaching of Example 7
except that plasmid pJL125, rather than plasmid~ pJL120
and pJL121, was used. The resultant colonies were
~20'~ 5
~-5773A -39-
selected and tested for the expected phenotype (AmpR,
TetS, CMS) and constituted the desired E. coli K12
C600Rk-Mk-/pJL125 transformants. The identity of the
transformants was further confirmed by AGE and restriction
analysis by the procedure of Eckardt, 1978, Plasmid
1:584 and by Klein et al~, 1980, Plasmid 3:88. The
transformants were then conventionally cultured for
subsequent production and isolation of plasmid pJL125
according to known procedures.
Example 12
Construction of Plasmid pJLl90
A. EcoRI-HindIII Digestion of Plasmid pJL121 and
Isolation of ~l9.Okb EcoRI-HindIII Fragment
About 200 ~1. (80 ~g.) of plasmid pJL121 VNA,
30 ~1. BSA (1 mg./ml.), 40 ~1. of HindIII (containing
200 BRL units) restriction enzyme, and 30 ~1. HindIII
reaction mix* wera incubated at 37C. for ahout 3 hours
and then at 65C~ for 10 minutes. The 300 ~lo reaction
; mixture was cooled to 4C., supplemented with 110 ~1.
of lOX HindIII~EcoRI diluent reaction mix** and 30 ~1.
EcoRI restriction enzyme (containing 300 BRL units),
and then incubated at 37C. for 3 hours, then at ~5C.
for 10 minutes followed by cooling to 4C. Tha re-
sultant ~l9.Okb EcoRI-HindIII restriction fragment was
conventionally isolated by AGE/DE52/electroelution.
The desired DNA was dissolvad in TE buffer and stored
at 4C. for future use.
7~
X-5773A ~40
-
~indIII reaction mix was prepared with the following
COmpOSitlOn:
60m.`~ Tris-HCl, pH 7.5
500~ NaCl
60~ MgCl2
**H_ndIII~EcoRI diluent was prepared with the following
composition:
382mM Tris-HCl, pH 7.5
50~1 NaCl
22mM MgCl2
B. EcoRI-HindIII Digestion of Plasmid pLR4 and
Isolation of ~7.7kb Eco~I-HindIII Fragment
. _ .
The desired digestion and isolation was
carried out in substantial accordance with the -teaching
of Example 12A ~xcept that plasmid pLR4, rather than
plasmid pJLl21, was used. The desired ~7.7kb f.ragment
was dissolved in TE buffer and stored at 4C. for
future use.
C. Ligation of ~l9.Okb EcoRI-HlndIII Fragment of
. ~
Plasmid pJLl2l and ~7.7kb EcoRI-HindIII Fragment of
Plasmid pLR4-
The desired ligation was carried out in
substantial accordance with the teaching of Example 3C
except that the ~l9.Okb EcoRI-HindIII fragment of
plasmid pJLl2l and the ~707 EcoRI-HindIII fragment of
plasmid pI.R4, rather than the 6.Okb SalI fragment of
plasmid SCP2* and SalI digested pBR325, were used. The
resultant ligated DNA constituted the desired plasmid
~2~'7~
X-5773A -41-
pJLl90 which was then stored at 4C. for future use. A
restriction site and ~unctional map of plasmid pJLl90
is presented in Figure 4 of the accompanying drawings.
The restriction site map was determined from plasmid
transformed into E. coli K12 C600R ~
- k k
Example 13
Construction of E. coli K12 C600~k-~qk-~pJL190
The desired construction was made in sub-
stantial accordance with the teaching of Example 7
except that plasmid pJLl90, rather than plasmids pJL120
and pJL121, was used. The resultant colonies were
selected and tested for the expected phenotype ~AmpR,
TetS) and size by conventional means (as in Example 11)
15 and constituted the desired E. coli K12 C600Rk~Mk-/pJL190
transformants. The transformants were then conventionally
cultured for sub5equent production and isolation o
plasmid pJLl90 according to known procedures.
Example 14
Isolation of Plasmid pJL192
. . . _ _ . . _ _
Plasmid pJL192, which confers high resistance
to antibiotic neomycin (10 ~g./ml.), can be conven-
tionally isolated from E. coli K12 C690Rk~Mk-/pJL192, a
strain deposited and made part o~ the permanent stock
culture collection of the Northern Regional Research
Laboratory, Peoria, Illinois under the accession number
15040. The restxiction site map of plasmid pJL192
appears not to be distinguishable from the plasmid
pJLl90 map presented in Figure 4O
~ZO'76~
X-5773~ -42-
Example 15
Construction of Plasmid pJL195
-
A. EcoRI-SalI Digestion of Plasmid pJL125 and Isola~ion
of ~5.4kb EcoRI-SalI Fragment
, .
The desired digestion and isolation was
carried out in substantial accordance with the teaching
of Example 12A except that plasmid pJL125 and SalI
restriction enzyme and reaction mix, rather than
plasmid pJL121 and HindIII restriction enzyme and
reaction mix, were used. In addition, SalI~EcoRI
diluent* was used. The resultant ~5.4kb EcoRI-SalI
fragment was dissolved in TE buffer and stored at 4C.
for future use
SalI~EcoRI diluent was prepared w.ith the followiny
composition:
940~1 Tris-HCl, pH 7.5
55m~ gC12
. EcoRI-Partial SalI Digestion of Plasmid pLR4 and
Isolation of ~7.5kb EcoRI-Partial SalI Fragmen~
The SalI digestion was carried out in su~-
2~ stantial accordance with the teaching of Example lOA
except plasmid pLR4, rather than pJL121, was used~
Since only a partial SalI digestion was desired, the
: resultant mixture was incubated first at 37C. for 15
minutes and then at 6;C. for 10 minutes. Following
cooling to 4C., the resultant partial SalI ~7.7kb
linear ~ragment was conventionally isolated by AGE/
~LZ~'7~
X-5773A -43-
DE52/electroelution. The desired DNA was dissolved in
TE buffer and digested with EcoRI restriction enzyme
in substantial accordance with the teaching of Example 6A
except that the above fragment r rather than
plasmid SCP2*, was used. The desired ~,7.5kb
E RI-SalI fragment (the largest possible EcoRI~SalI
fragment) was isolated by AGE/DE52/electroelution,
dissolved in TE buffer, and th~n stored at 4C. ~or
future use.
13 C. Ligation of ~5.4kb EcoRI-SalI Fragment of Plasmid
pJL125 and ~7.5kb EcoRI-Partial SalI Fragment of
Plasmid pLR4
The desired ligation was carried out i.n
substantial accordance with the teaching of Example 8C
except that the ~5.4kb EcoRI-SalI fragment of plasmid
pJL125 and the ~7.Skb EcoRI-partial S I fragment of
; plasmid pLR4, ratner than the ~6.Okb SalI ~ragment of
plasmid SCP2* and SalI digested pBR325~ were us2d~ The
23 resultant ligated DNA constituted the desired plasmid
pJL195 and was stored at 4C. for future use. A re-
striction site map of plasmid pJL195 is presented in
Figure 5 of the accompanying drawings. The restriction
site map was determined with plasmid isolation from
25 E. coli K12 C600Rk--~k-.
,.,
6~
~Y-5773A -44-
Example 16
Construction of E. coli ~12 C600Rk-Mk-/pJL195
Th~ desired construction was made in sub-
stantial accordance with the teaching of Example 7
S except that plasmid pJL195, rather than plasmids pJL120
and pJL121, was used. The resultant colonies were
tested for the expected phenotype (AmpR, TetS) and
size (as in Example 11) and constituted the desired E.
coli K12 C600Rk Mk-/pJL195 transformants. The trans-
formants were then conventionally cultured for sub-
sequen-t production and isolation of plasmid pJL195
according to known procedures.
Example 17
Construction of Plasmid pJL114
A. Partial BamHI Digestion of Plasmid SCP2
The desired digestion was carried out in
substantial accordance with the teaching of Example 6A
; 20 except that plasmid SCP2 (isolated, in accordance with
the teaching of Example 1, from Streptomyces coelicolor
A3(2), a strain deposited and made part of the per-
manent stock culture collection of the Northern Regional
Research Laboratory under the accession number 1$042),
and BamHI restriction enzyme and reaction mix*, rather
~han plasmid SCP2* and EcoRI restriction enzyme and
reaction mix, were used. The desired DNA was stored
at 4C. for subsequent use~
... .
*
Reaction mix for BamHI restriction enzyme was prepared
with the following composition:
lOOO~M Tris~HCl, pH 7O4
lOOm-~ MgC12
~ ~2~
X-5773A -45-
B. Llgation of Bam}iI Digested Plasmld SCP2
and BamHI Digested Plasmid pBR322
The desired liga~ion was carried out in
substantial accordance with the teaching of Example 6C
e~cept that the BamHI digest of plasmid SCP2 ~prepared
in Example 17A) and BamHI-digested plasmid pBR322
(prepared in Exampl~ 4B5, rather ~han plasmids SCP2*
and pBR325, were used. The resultant DNA was stored
at 4C. and constituted the desired ~34.6kb plasmid
pJL114.
Example 18
Construction of E. coli K12 C600Rk-Mk-/pJL114
.
The desired cons~ruction was made in sub-
s~antial accordance with the teachin~ of Example 7
except that plasmid pJL114, rather than plasmids pJL120
and pJL121, was used. The resultant colonies were
selected and tested for the expected phenotype (AmpR,
2~ TetS), and constituted the desired ~. coli K12
C600Rk-~1k-/pJL114 ~ransformants. The ampicillin re~
sistant, tetracycline sensitive colonies were isolated
according to known procedures, cultured, and then
conventionally identified by restriction enzyme and
agarose gel electrophoretic analysis of the consti-
tutive plasmids.
It was revealed upon analysis that the BamHI
restriction enzyme had cut only one of the ~glII re-
striction sites of SCP2 during the digestion described
in ~xample 17A. Since this event is raxe and has not
been repeated, E. coli K12 C600Rk~ pJL114 has been
~)76~3~
X 5773A -46-
deposited and made part of the permanent stock cultur~
collection of the Northern Regional Research Laboratory,
Peoria, Illinois under the aGcession number B-15039. The
strain i5 available as a preferred source and stock
5 reservoir of plasmid pJL114. A restriction site map of
plasmid pJL114 is presented in Figure 5 of the accom-
panying drawings.
Example 19
Construction of Streptomyces griseofuscus~pJL120
1 0 - e
A. Growth of Cultures for Preparation of Protoplasts
.
A vegetative inoculum was conventionally
prepared by growing ~he s_rain under submerged con-
ditions for 20 hours at 30C. in TSB supplemen-ted with
.4% glycine. The culture was homogenized and inocu-
lated at a 1/20 dilution into the same medium and then
grown for 18 hours at 30C.
B. Transformation
Using ~bout 20 llg. of plasmid pJL120 DNA and
lX109 protoplasts. of Strept_myces grlseofuscus, a
strain deposited and made part of the permanent stock
culture collection of the American Type Culture Col-
lection, Rockville, Maryland, from which it is avail-
able to the public under the accession number ATCC
23916, the desired transformation was carried out in
substantial accordance with the teaching o:E International
Publication (of International Patent Application No.
PCT/BG79/00095) No. W079/01169, Example 2.
'~ ~ Sb
~L)7~
X~5773A -47-
C. Selection
To assay for transformation even a~ low
frequencies, two procedures were employed~
(1) Pock-assay:
Spores were harvested from the regeneration
plates containing confluent lawns of regenerated proto-
plasts as follows. About 10 ml. of sterile distilled
water were added to the plate and the surface of the
: 10 culture gently scraped with a loop to remo~e the spores.
The resulting spore suspension was centrifuged at
20,000 rpm for 10 minutes. The supernatant was dis-
carded and the remaining spore pellet resuspended in
.3 ml~ of 20% v/v glycerol. Serial dilutions of the
prepar,ation were made down to 10 5 by successive
transfer of .1 ml. of the spore suspension to .9 ml. of
20~ v/v glycerol. The spores can then be stored
at -20C. with little loss of viability. About
.1 ml. aliquots of some of the dilution series (e.g.
10 1, 10 2, 10 4) of each of the harvested plates were
then transferred to R2 medium (Hopwood and Wright,
1978, Molecular and General Genetics 162:30) plates
which had sufficient spores of the Streptomyces
: ~riseofuscus strain originally used in the transfor-
mation procedure to produce a confluent lawn. This
procedure can also be carried out with the substitution
of YMX agar (.5~ yeast extract .5% malt extract, .1%
dextrose and 2~ ayarl. Transformants can typically be
- detected after 3 days' growth at 30C. by the appearance
of "pocks", a property expressed by spores containing
the plasmid in expressible form within the lawnO The
~ .
., ~ . ~
12~3~76~i
X~5773A -48
transformants were recovered by conventionally picking
spores frorn the centre of the "pock" to an agar plate
of YMX medium (Hopwood 1967, Bacter.iological Review,
31:373).
(2) Back transformation to E. coli K12
C600 k k
The spores are collected as in (1) above but
are used to inoculate 50 ml. of TSB supplemented with
1 .4% glycine. The culture is grown for 20 hours at
30C. and the cells are harvested followed by isola~ion
of DNA~ Isolation is as disclosed in Example lB except
that centrifugation with CsC1 and pxopidium iodide is
omitted. Subsequently, 50 ~1. of this DNA is used to
15 transform E,coli K12 C600Rk~Mk- as disclosed in
Example 7B. Plasmids in the trans~ormants are verified
and identified by conventional means as taught in
Example 11.
Example 20
Construction of Streptomyces _riseofuscus/pJL114,
,
S. qriseofuScus/pJL121, S. griseofuscus/pJL125,
-
S. griseofuscus/pJL180, and S. ~riseofuscus~pJL181~
The desired constructions were each indi-
vidually and respectively made, selected, and recovered
in substantial accordance with the teaching of Example
19 except that plasmids pJL114, pJL121, pJL125, pJL180,
and pJL181, rather than plasmid pJL120, were appro-
priately used for the individual construction.
:~;
6~5
X-5773A _49_
Example ~1
Construction of Streptomyces griseofuscus/pJL190
A. Transformation
The desired transformation was carried out in
substantial accordance with the teaching of Example l9B
except that plasmid pJLl90, rather than plasmid pJL120,
was used.
B. Selection
lQ
The desired transformants were selected for
neomycin resistance by overlaying the regenerating
protoplasts w.ith R2 medium top agar containing suf-
ficient neomycin to bring the final plate conce~tration
to 1 ~g./ml. The resultant Streptomyces riseofuscus/
pJLl90 transformants were then tested for the expected
; pock morphology in suhstantial accordance with the
procedure of Example 2OB.
Example 22
2~ Construction of Streptomyces griseofuscus~pJLlgS
The desired construction was made, selected,
and recovered in substantial accordance with the
teaching of Example 21 except that ~lasmid pJL195,
xather than plasmid pJLl90, was used~
. .
~`
, ~
~ ..J
7 f;~5
X-5773A -50
Example 23
Construction of Streptomyces griseofuscus/pJL192
. . . _ .
The desired construction was made, selected,
and recovered in substantial accordance with the
teaching of Example 22 except that plasmid pJL192 and,
in the selection procedure, top agar containing 5uf-
ficient neomycin to bring the final plate concentration
to 10 ~g./ml., rather than plasmid pJL195 and top agar
containing sufficient neomycin to bring the final plate
concentration to 1 ~g./ml., were used.
Example 24
Construction of Streptomyces fradiae~pJL120, S.
radiae/pJL114, S. fradiae/pJL121, S. fradiae/pJL125,
S. fradiae/pJL180, S. fradiae/pJL181, S. ~radiae/
_
pJLl90, S. fradiae/pJL195 r and S. _radlae~pJL192
The desired construc-tions are individually
~ and raspectively made, selected, and recovered in
substantial accordance with the respective teachings of
Examples 19, 20, 21, 22, and ~3 except that S-treptomyces
fradiae, rather than S. ~ , is used. In
addition, the TSB medium or protoplasting and growing
5. fradiae was modified and contained only .2% glycine.
~Z~'7~
X-$773A -51-
Example 25
Constructi~n of Streptomyces lividans/pJL120, S.
. _ _
lividans/pJL114, S. lividans/pJL121, S. lividans/
pJL125, S. lividans/pJL180, S. lividans/pJL181,
SO lividans/pJLl90, S. lividans/~JL195, and S. lividans/
pJL192
The desired constructions are individually
and respectively made, selected, and recovered in
substantial accordance with the respective teachings of
Examples 19, 20, 21, 22, and 23 except that Strepto-
myces lividans, rather than S. griseofuscus, is used.
In addition, the media for protoplasting and growing S.
lividans is as described in International Publication
(of International Patent Application No. PCT/BG79/00095)
No. WO79/01169, Example 2.
Example 26
Isolation of Plasmid pJL192 Mutant that Confers
High Resistance To Antib otic Neomycin
Streptomyces griseofuscus/pJL190 was isolated
as described in Example 21. Analysis of growth of
colonies on nutrient agar supplemented with different
concentrations of neomycin revealed that S. griseofuscus/
pJLl90 exhibited resistance to 1.0 ~g./ml. of neomycin.
S. griseofuscus was spread conventionally on nutrient
agar plates supplemented with 10 ~g./ml. of neomycin~
; A colony was discovered that exhibited growth at this
high level of neomycin. After repeated analysis
verified that the colony exhibited the aforementioned
S
~-5773A -52-
resistance, the colony was designated S. griseofuscus/
pJL192. The plasmid, pJL192 was shuttled into E. coli
K12 C600Rk-Mk- by back transformation as taught in
Example l9C. The restriction site map of pJL192
appears not to be distinguishable from pJLl90.
Example 27
Isolation of Plasmid pJLl99 Mutant tha~ Confers
High Resistance to Antibiotic Neomycin
... ... _ _ _ . .
The desired isolation is carried out in
substantial accordance with the teaching of Example 26
except that Streptomyces griseofuscus/pJL195 ~prepared
in Example 22), rather than S. griseofuscus/pJLl90,
was used. A colony that exhibited high resistance
to neomycin was designated Sn griseouscus/pJLl99.
The plasmid, pJLl99, was shuttled into E. coll K12
C600Rk-Mk- by back transformation as ~aught in Example
l9C. The restriction site map of pJLl99 appears not to
be distinguishable from pJL195.
Those skilled in the art will recognize that
plasmid pJLl99 can also be conventionally constructed
by substitu-ting the neomycin resistance-conferring
fragment of plasmid pJL192 (prepared in Examples 15 and
27) for the pLR4-derived neomycin resistance-conferring
fragment of plasmid pJLlg5. Such a substitution thus
also results in the desired plasmid pJLl99.
Example 28
Construction of Plasmid pLR2
~.~
A. HindIII Digestion of Plasmid pIJ6
About 20 ~1. (20 ~g.) of plasmid pIJ6 DNA,
disclosed in Thompson et al., lq80, Nature 286:525,
- `
7~
X 5773A -53-
5 ~l. BSA(Bovine Serum albumin, l mg./ml.), l9 ~l.wa~er, l ~l. of indIII (containing 3 New ~ngland Bio
Labs units) restriction enzyme*, and 5 ~l. reaction
mix** were incubated at 37C. for 2 hours. The reac-
tion was terminated by the addition of about 50 ~l. of4~1 a~monium acetate and 200 ~l. of 95% ethanol. The
resultant DNA precipita-te was washed twice in 70~
ethanol, dried _ vacuo, suspended in 20 ~l. of TE
buffex, and frozen at -20C. for storage.
lC B. HindIII D~sestion of Plasmid pBR322
About 8 ~l. (4 ~g.) of plasmid pBR322 DNA,
5 ~l. reaction mix, 5 ~l. BSA (l mg./ml.), 31 ~l.
water, and 1 ~l. of HindIII restriction enzyme were
incubated at 37C. for 2 hours. After the reaction was
i terminated by incubating at 60C. for lO minutes, about
50 ~1. of ammonium acetate and 200 ~l. of ~5~ e~hanol
were added. The resultant DNA precipitate was washed
twice in 70% ethanol, dried ln vacuo, and suspended in
45 ~l. of water.
C. Ligation of HindIII Digested Plasmids pIJ6 and pBR322
A~out ~0 ~l. of HindIII treated plasm:id pIJ6
(from ~xample 28A), 20 ~l. of HindIII treated plasmid
pBR322 (from Example 28B), 5 ~l. BSA (lmg./ml.), l ~l.
of T4 DNA ligase , and 5 ~l. ligation mix** were
incubated at 16C. for 4 hours. The reaction was ~er-
minated by the addition of about 50 ~l. 4M ammonium
acetate and 200 ~l. of 95% ethanol. The resultant DNA
precipitate was washed twice in 70~ ethanol, dried in
vacuo, and suspended in TE buffer. ~he suspended DNA
constituted the desired plasmid pLR2.
* See the footnotes on pages 27 and 28 of the
,- ** specification
3'~
X~5773A -54-
Example_23
Construction of E. coll K12 HB101/pLR2
~ .
About 10 ml. of E. coli K12
HB101 cells (Bolivar et al., 1977, Gene 2:75-93) wexe
S pelleted by centrifug~tion and then suspended in about
10 ml. of O~OlM sodium chloride. Next, the cells were
pelleted again, resuspended in about 10 ml. of 0.03M
calcium chloride, incubated on ice for 20 minutes~
pelleted a third time, and finally, resuspended in
10 1.25 ml. of 0.03.~1 calcium chloride. The resultant cell
suspension was competent for subsequent transformation.
Plasmid pLR2 in TE buffer (prepared in
Example 28C) was ethanol precipitated, suspended in
150 ~1. of 30m~ calcium chloride solution, and gently
mixed in a test tube with about 200 ~1. of competent E.
coli R12 HB101 c~lls. The resultant mixture was in~
cubated on ice for about 45 minutes and then at 42C.
for about 1 minute. Next, about 3 ml. of L-broth
(~ertani, 1951, J. Bacteriology 62:293) containing
23 50 ~g./ml. of ampicillin were added. The mixture was
incubated with shaking at 37C. ~or 1 hour and then
plated on L-agar (Miller, 1~72, Experiments in Molecular
Gene~ics, Cold Spring Harbor Labs, Cold Spring Harbor,
New York) containing ampicillin. Surviving colonies
were selected and tested for the expected phenotype
(Amp , Tet ), and constituted the desired Eo coli K12
~B101/pLR2 transformants.
Representative plasmids and transfoxmants
that can be constructed in accordance with the fore-
going teaching include the ollowing listed below
in Tables 1 and 2.
,
, ;~
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Table 2
Representative Transformants
1. Streptomyces R/pR wherein R is griseofuscus,
_
ambofaciens, fradiae, or lividans and RL is
- -
independently pJL122, pJL123, pJL124, pJL126
pJL176, pJL1200, pJL1201, pJI.1202, pJL1203,
pJL1204, pJL1205, pJL1206, pJL1706, pJL1800,
pJL1801, pJLl900, pJL1902, pJL1905, pJL196,
pJL197, pJL1907, pJL198, pHJL212 and pHJ213.
2. E. coli K12 R /pRl wherein R2 is C600Rk-Mk-,
294, C600 or RV308 and R is independently
as defined above.
~ 25
:
