Note: Descriptions are shown in the official language in which they were submitted.
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This is a divisional application of copending
, application serial no. 355,182, filed June 30, 1980.
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BROAD ~3OST RANGE S~lALL PLASMID RINGS
AS CLONING VEHICLES
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` SU~IARY OF THE INVENTION
The present invention relates to novel small plasmid
rings having a broad bacterial host range ana to the pro-
cesses for recombining the fragments with other DNA frag-
S ments in v vo or in vitro and~or for transferring theplasmids between host bacteria. In particular, the present
invention relates to plasmid rings which are derivatives of
the aggregate plasmid rings RPl/pRO1600 which occurred by
chance only once as a result of a mutation in Pseudomonas
10 aeruginosa ATCC 15692 during conjugation of the bacteria
containing the natural plasmid ring RPl (also known as
Rl822). The novel plasmids are referred to herein as
pRO16xy wherein x and y are integers.
PRIOR ART
The prior art commercial efforts involving recom-
binant genetic manlpulation of plasmids for producing
various chemicals have centered on ~scherichia c~Zi as a host ,
- organism. The reason is that the recombinant plasmids are
not compatible with other host organisms. However, E. coZi
is not the most aesirable organism to use for these purposes
because of concerns about its ability to grow in the intesti-
nal tract in mammals and also because of its ability to pro-
auce disease, It would be very desirable to be able to
use host bacteria besides E, co~i with plasmids having a
broad bacterial host range. The ~resent invention is con-
cerned with such plasmids. It is particularly concerned with
the use of organisms that will not survive at mammalian body
tem~eratures.
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he following are definitions used in reference
` to the present invention~
The phrase "bacterial conjugation" means the mat-
ing and genetic transfer between at least two bacterial
hosts, the donor beiny designated as "male" and the reci-
`- pient as "female".
The phrase "p~asmid aggregate" means an associa-
- tion between two plasmid rings wherein each plasmid main-
tains its structure as a ring and wherein each ring is
capable of separate recombinant genetic manipulation.
The term "transport" means any process whereby
- DNA is transferred from one bacterium to another.
The term "transductioni' means DNA plasmids or
fragments caused to be transported by bacterial viruses
from one bacterium tc another in vivo by natural processes.
The term "transformation" rneans the injection of
DNA stripped from a whole bacterial cell in vitro into a
host bacterium.
The term "transposition" means the movement of
genetic material from one portion of a DNA molecule to
another or from one DNA molecule to another.
The term "transposon" refers to the genetic material
transferred by transposition.
The phrases "DNA source" or 'DNA fragment" means
25 DNA from chromosomal or extrachromosomal cellular elements
(such as plasmids) derived from eucaryotic or procaryotic
cells andfor their parasites including viruses.
The state of the patent art is generally described
in U. S. Patent ~os. 3,813,316; 4,038,143; 4,080,261;
4,082,613 and 4,190,495. These patents describe prior art
processes related to the present invention.
Two elements have been identified as necessary
for the replication of a DNA molecule in vivo other than
the battery of en~ymes and proteins required for synthesis
of the DNA molecule ~er se. They are (1) a region on the
D~A molecule which is identified as the origin of DNA repli-
cation and which is recognized by a protein and also (2) the
gene for this protein which is found on the same DNA
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mo]ecule Given this min;mum specification for a DNA
~-- molecule capable of aUtOnOInOUS self-reproduction in a
bac~erial cell, additional pieces of DNA rnay be included
insofar as they do not interfere with the two functions
identified above. The combination of l and 2 above found
in nature contained within and maintained extrachromo-
somally by host bacterial species has been called plasmids
or Pxtrachromosomal elements (sacteriological ~eviews,
September, pp. 552-590 (1976)). Until recently, microbial
r~`10 geneticists have manipulated DNA fragments in vivo using
host bac~erial cell recombination mechanisms to recom-
bine DNA from the bacterial chromosome into plasmids or
to efect recombination between plasmids co-maintained in
the same host bacterium. This practice has allowed puri-
fication of various regions of a more complex genome byremoval of a portion of the complex genome to another
replicating unit. However, this practice does not normally
allow for the construction of plasmid-hybrids comprised
of DNA from disparate sources or transported across large
biological barriers from different organisms.
In recent years, however, techniques for the
in vitro manipulation and recombination of heterogeneous
fragments of DNA have allowed the construction of hybrid
DNA molecules. A brief summary of the in vitro process
quoted from "Recombinant Molecules: _pact on Science and
Society`' R. F. Beers and E. G. Bassett, eds., pp 9-20,
Raven Press, New York (1977)) follows.
"There are several important technical components
to in vitro recombinant (DNA) technology which
ultimately result in the insertion of DNA frag-
ments from any source into replicons (plasmids)
--- and their recovery as replicating elements
in bacteria. These components are:
l. the systematic dissection of the DNA molecules
of interest with restriction endonucleases (see
Roberts, in Recombinant Molecules, for a dis-
cussion of these en~ymes~;
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2. the rejoining of DNA fragments by ligation
to an appropriate cloning vehicle (or replicon);
3~ the transformation of a cell with the
~` recombinant DNA and selection of cells con-
taining the recombinant plasmid; and
4. the identification and characterization of
`~ the resulting "cloned" fragment of DNA"
This invention relates to all of the above, parti-
cularly item 2, namely, the requirement for an appropriate
cloning vehicle (plasmid) and the novel properties of the
plasmid cloning vector described herein contributing
to the utility of the transformation process described
above.
Biological properties of a plasmid potentially
useful for molecular DNA cloning have heen summarized
by D. R. Helinski et al, in "Recombinant Molecules:
Impact on Science and Society, p. 151, R. F. Beers and
E. G. Bassett, eds., Raven Press, New York (1977)).
"Since the initial demonstration of the utility
of a plasmid element for the cloning of genes
in ~s~her*chia coZi -- a variety of plasmid ele-
ments have been developed as cloning vehicles in
both ~. cozi and (for different plasmids) in
other bacteria. These newer cloning vehicles
possess the following plasmid properties
that are advantageous for the cloning of DNA:
1. stable maintenance in the host bacterial
cell;
2. non-self-transmissibility;
3. ease of genetic manipulation;
4. ease of isolation;
5. the capacity of joining with and repli-
~ cating foreign DNA of a broad size range;
`~ 6. ease of introduction of the in vitro
~`~ 35 generated hybrid plasmid into a bacterial cell."
The foregoing specifications relating to character-
`~ istics considered desirable for the maximum utility of a
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- plasmid useful for recombinant DNA techology,- however,
do not include a consideration for enhanced utility of
~ a cloning plasmid with a broad bacterial host range.
r` Most bacterial plasmids described to date can
be ma;ntained only in bacterial species closely related
to the bacterium from which the plasmid has originally
been isolated. Because of this, the requirements asso-
ciated with the maintenance and duplication of the parti-
cular plasmid's DNA generally are specific to the plasmid
in question. There have been, however, exceptions to
this general observation. For example, Olsen (the inventor
herein) and Shipley ~Journal of Bacteriology, 113, No. 2,
pp. 772-780 (1973)) showed that a plasmid specifying multi-
ple antibiotic resistances, designated R1822 (and later
changed to RPl), was transferred to a variety of bacterial
species representative of related ana unrelated bacterial
genera by sexual conjugation. The origin of the strain
Pseudomonas aeruginosa 1822 from which RPl was later obtained
is set forth in Lowbury, E~ J. et al Lancet ii 448-452
20 (1969)o The bacterial host range of the plasmid RPl
includes Entel~obacteriaceae, soil saprophytic bacteria
(Pse?~domcrnas), Neisseria pe~fZ~ a, and photosynthetic bacteria.
Plasmid RPl, then, is an example of a broad host range
bacterial plasmid which freely transfers among unrelated
bacterial species~
The plasmid ring RP1 is relatively large. The large
size and _omposition of this plasmid ring makes the process
of bacterial transformation inefficient. It would be a
significant improvement in the art to provide a small
` 30 plasmid ring as a cloning vehicle and recombinants thereof
`~' which were easily and widely transportable particularly
by transformation~ from bacterial host to bacterial host.
The plasmids would be particularly useful if they included
a single phenotypic marker for antibiotic resistance for
identification purposes. It would also be an improvement
to provide processes for in vivo or in vitro recombination
of fragments of the small plasmid ring with other genetic
fragments.
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OsJECTS
- It is therefore an object of the present inven-
tion to provide novel plasmid fragmentation and recombina-
tion and/or transport processes. Further it is an object
to provide novel recombinant plasmid rings derived from
small plasmids which act as cloning vehicles when combined
in the recombinant plasmid rings and wherein the plasmid
rings have a broad bacterial host transfer range. It is
particularly an object of the present invention to provide
recombinant plasmid rings which have useful chemical
generating properties or some other useful characteristic
in the host bacterium. These and other objects will become
increasingly apparent by reference to the following des-
cription and the drawings.
In the Drawings
....... . _
Figure 1 shows slab agarose gel electrophoresis
patterns for RPl of the prior art (A), E. coli V517 elec-
trophoresis size standard (D) and for RPl/pRO1600 (B? and
other plasmids (C, E, F) of the present invention. The
longer the pattern, the smaller the plasmid.
' Figure 2 shows restriction endonuclease PstI
and BglI maps in megadaltons (daltons x 106) for the
preferred plasmids of the present invention, particularly
plasmids pRO1601, pRO1613, pRO161~L and pRO1600. Numerical
values in parenthesis represent molecular size in daltons
x 10 for restriction endonuclease fragments. Numerical
values above or below the unbroken horizontal line are map
~` distance of the restriction endonuclease recognition site
in daltons x 106 from zero as defined by the single PstI
~' 30 restriction endonuclease-DNA cleavage site present in
plasmid pROl 600.
Figure 3, which is on the same sheet of drawings
as Figure 1, shows slab agarose gel electrophoresis pat-
terns for: pRO1614 (C); DNA from E. coli; V517 of the
prior art for reference purposes (B) and DNA from trans-
formant clones capable of growth without L-isoleucine or
L-valine (A), referred to as pRO1615, or L-methionene ~D),
referred to as pRO1616, as a result of the recombinant
modification of pRO1614.
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General Description
The present invention relates to a recombinant
deoxyribonucleic acid plasmid ring inclu~ing a first
plasmid fragment, the first plasmid being originally
derived from a plasmid aggregation with plasmid RPl and
measuring about 2 x 106 daltons or less in molecular size
and having a critical restriction endonuclease BglI diges-
tion fragment measuring 0.83 x 106 daltons in molecular
size which is indispensible for replication, covalently
combined with at least one second deoxyribonucleic acid
fragment which is a restriction endonuclease digestion
fragment ligated to the first plasmid in vitro or a
naturally occurring fragment inserted by a bacterium
_ vivo into the first plasmid as a recombinant plasmid
ring capable of being carried by Pseudomonas aeruginosa
PAO ~TCC 15692 ~PAOlc) and having a broad bacterial host
transmission range, wherein the second fragment contributes
a useful chemical characteristic to the recombinant plas-
mid ring and wherein the plasmid ring clones itself by
DNA replication during cell division of the host bacterium.
The present invention also relates to the bacterial
composition which comprises a deoxyribonucleic acid plas-
mid ring including a first plasmid fragment, the first
plasmid being originally derived as a plasmid aggregation
`~ 25 with plasmid RPl measuring about 2 x 106 daltons or less
in molecular size and having a critical restriction endo-
nuclease BglI digestion fragment measuring 0.83 x 10
daltons in molecular size which is indispensible for repli-
cation, covalently combined with at least one second
deoxyribonucleic acid fragment which is a restriction
endonuclease digestion fragment, ligated to the first plas-
mid in vitro or a naturally occurring fragment inserted
by a bacterium in vivo into the ~irst plasmid as a recom-
binant plasmid ring capable of being carried by Pseudomonas
aeruginosa PAO ATCC 15692 (PAOlc~ and having a broad bacteri-
al host transmission range, wherein the second fragment con-
tributes a useful chemical characteristic to the recombi-
nant plasmid ring and wherein the plasmid ring clones it-
self by DNA replication during cell division of the host
bacterium; and a host bacterium.
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... The present invention also relates to a process
for transporting DNA plasmids to bacterial hosts in vivo
using the processes of bacterial conj~gation, transformation
or transduction, the i.mprovement which comprises trans-
porting a aeoxyribonucleic acid plasmid ring, including
a first plasmid ring originally derived as a plasmid
aggrega~ion with plasmid RPl measuring about 2 x 106
daltons or less in molecular size and having a critical
. BglI restric~ion endonuclease digestion ragment measur-
ing 0.83 x 106 daltons in molecular size which is indis-
pensible for replication alone or with a fragment from the
first plasmid ring,covalently combined with at least one
`second deoxyribonucleic acid ~ragment which is a restriction
endonuclease digestion deoxyribonucleic acia fragment
ligated to the first plasmid or a naturally occurring frag-
ment inserted by a bacterium in vivo in the first plasmid
to form a recombinant plasmid ring, wherein the plasmids
are capable of being carried by Pseudomo~as aeruginosa PAO
ATCC 15692 (PAOlc~, wherein the plasmids have a broad
bacterial host range, and wherein the plasmid clones itself
by DNA replication during cell division of the host
bacterium.
~ The present invention relates to the process which
i comprises providing an aggregate of a first plasmid with
a second plasmid, wherein the second plasrnid has a trans-
poson which produces a use~ul chemical characteristic and
wherein the first plasmid was originally derived as an
aggregation with RPl and measures about 2 x 106 daltons or
less in molecular size and has a critical BglI endon~-
clease digestion fragment measuring 0.83 x 106 daltons inmolecular size indispensible for replication; providing
the aggregate plasmid in a plasmid receptive bacterial
cell; growing the receptive bacterial cell in a growth
medium with the aggregate plasmid to randomly produce a
recombined plasmid including the transposon and first
plasmid, wherein the recombined plasmid replicates upon
division o~ the bacterial cell; and selecting the bacterial
cells with the recombined plasmid with the transposon.
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Further, the present invention relates to the pro-
cess for producing recombinant deoxyribonucleic acid plas-
mids which comprises providing a first plasmid and a
second deoxyribonucleic acid source, wherein the first
plasmid was originally derived rom a plasmid agyregation
with plasmid ~Pl measures about 2 x 106 daltons or less in
molecular size and has a critical restriction endonuclease
sglI digestion segment measuring 0.83 x 106 dal~ons in
molecular size which is indispensible for replication;
reacting the plasmid and the second deoxyribonucleic acid
source with a~ l~ast one restriction endonuclease which
eleaves the first plasmid and the second deoxyribonucleie
aeid souree into linear DNA fragments; and randomly recom-
bining the linear deoxyribonucleie aeid fragments using
ligation to form recombinant plasmias which replicate in
a bacterial eell.
Finally, the present invention relates to a
deoxyribonucleie acid fragment for forming plasmids, the
fragment ~eing formed from a first plasmid originally
derived from a plasmid aggregation with plasmid RPl and
me~suring about 2 x 106 daltons or less in molecular
size, wherein the fragment has a critical restriction
endonuclease BglI digestion ~ragment measuring 0.83 x 10
daltons in molecular size whieh is indispensible for repli-
cation in a plasmid. BglI was isolated from BaciZ~usg~obi~gi . BglI produces 5' termini as follows:
5'-GGCCGAGGCGGCCTCGGCC-3'
3'-CCGGCT~CCGCCGGAGCCGG-5'
The plasmid content of bacteria ean be conveniently
and expeditiously estimated by employing the technique of
slab agarose gel electrophoresis with visualization of
the result on photographs of the resulting electrophero-
gram (for example, see Hansen and Olsen, Journal of
Bacteriology; 135, No. 1, pp. 227-238 (1978)). Thus DNA
was electrop~oresed as shown in ~igure 1 as follows: A,
DNA extracted from Pseudomonas aeruginosa NRRL 12123; B,
DNA from RPl/pRO1600; C, DNA from pRO1601; D, DNA from
Eschcrichia co~i V517, a multi-plasmid-containing strain
7~ J~
used as a size standard (Plasmid, 1, pp. 4~7-420 (1978)~;
E, D~A from pRO1613; F, DNA from p~O1614. rhis procedure
also allows an estimate of the molecular SiZe of any
plasmids present when suitable standards are incorporated
into the procedure.
When the plasmid RPl is caused to transfer from
one bacterium to another by the process called bacterial
conjugation (or sexual mating1, the recipient bacterium
that has newly acquired the plasmid normally contains a
plasmid that is indistinguishable on electropherograms
fro~ the plasmid present in the donor bacterium. In
Figu~e 1, file A, is depicted the usual appearance of
plasmid RPl when extracted from donor cell populations or
a recipient cell population subsequent to its transfer. On
one occasion another result was obtained. Analysis of a
culture derived from a single recipient cell of ATCC 15692
~strain PAO2) which had received plasmid RPl in a mating
experiment with ~seudomon~ aeruginosa PAO25 (another mutant
variation of ATCC 15692, the same as PAO2 as described above
except that it requires leucine and arginine for growth and
maintenance and on deposit at the University of Michigan)
produced a variant plasmid aggregate. The transconjugant
showed the presence of not only the parent plasmid, but also
a second and considerably smaller plasmid (Figure 1, file B).
This result has not been seen agaîn after many repetitions
of the process. The parental-size plasmid is shown at the
top of file B; the anomolous smaller plasmid appears at
the lower portion of the electropherogram depicted in
Figure 1. The size of the lower plasmid, estimated by com-
parison and calculation relative to the size standards infile D~ is 2 x lQ6 daltons compared to 38 x 106 daltons for
RPl. The appearance of the small plasmid, then, reflects
a novel event which, in general terms, may be considered a
; random mutational event ~hlch most likely occurred during
the transfer to the recipient ~acterium of the parental plas~
mid, ~Pl~ I fiave designated the small plasmid pRO1600 and the
aggregation with RPl as RPl/pRO16Q0.
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The utility of a small p]asmid for application in
recombinant DNA tcchnology derives, in part, from its
abilit~ to encode genetic in~ormation for a unique n~eta-
bolic trait not poisessed by the host bacterium. Accord-
ingly, the presence or absence of the plasmid, subsequentto genetic manipulations, can be determined on the basis
of the presence or absence of the metabolic trait in
question. ~reliminary analysis of the bacterial strain
shown in Figure 1, file B showed no unique metabolic trait
tphenotypic character~ associated with the presence of
plasmid pRO1600. I therefore applied standard bacterial
genetic techniques to add a distinctive phenotypic trait
to pRO1600 allowing its detection in later experiments
whereby I tested its ability to be transformed from
partially purified DNA solutions to recipient bacterial
strains. The distinctive phenotypic trait added was a
piece of DNA which encodes genetic information for resis-
tance to the antibiotic, carbenicillin. Accordingly, all
bacteria maintaining plasmid pRO1600 with this piece of
D~A will grow in the presence of carbenicillin unlike the
parental, plasmid-free, bacterial strain.
The genetic trait for carbenicillin resistance was
added to plasmid pRO1600 by the genetic process called
tr~nsposition. Some antibiotic resistance genes, called
transposons, are able to move from one location to another
on a piece of DNA or alternatively, able to move from one
DNA ~olecule to another within the bacterial cell by the
genetic process called transposition (S. Cohen, "Trans-
posable genetic elements and plasmid evolution" ~ature,
~63, pp~ 731-738 (1976~). These genetic elements accomp-
lish this process in the absence of bacterial host genetic
recombinational mechanisms. Plasmid RPl, shown in file A,
Figure 1, contains a transposon called Tnl which encodes
genetic information for resistance to carbenicillin and
related antibiotics (penicillin, ampicillin~. Transposon,
; Tnl is a transposable genetic element of 3.2 x 106 daltons
molecular size. Accordingly, DNA molecules that have been
transposed by Tnl would increase in size by 3.2 x 106
daltons.
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Transposition occurs randomly in populations of
bacterial cells which all contain a donor DNA molecule
which has the transposon. Thus, in the case o bacterial
strain Pse7~do~nonas a~ginosa RPl/pR01600, one would expect
a small proportion of the bacterial cells to contain
transposed-variant plasmids. For example, as shown in
file B, Figure 1, a few bacterial cells in the culture
would contain a DNA molecule larger by 3.2 x 106 daltons
- than the small DNA molecule shown at the bottom of the
electropherogram (the plasmid designated pR01600.) The
'~ relatively small number of these bacteria in the culture,
however, precludes their detection on agarose gels as sho~n
in Figur~ 1. However, t~ese transposed derivatives of plas-
mid pRO1600 can be detected and isolated by using the
genetic technique called bacterial transformation. sy
this process, DNA that has been extracted from cells called
donors is added to a plasmid-free recipient bacterial
cell culture. The admixture, in turn, is then grown on
bacteriological medium containing, in this instance, the
antibiotic carbenicillin. Under these conditions, only
bacterial cells that have taken up and replicated a
` ge~etic element which encodes genetic information for the
antibiotic carbenicillin will grow to dense populations.
When this experiment is done, two types of carbenicillin
resistant bacterial strains would be expected: those
which ~ave received all of the donor parent plasmid, RPl;
those which have only received pRO1600 combined with the
transposon which encodes carbenicillin resistance, Tnl,
which is now contained within its structure. The former
class of possible bacterial isolates can be distinguished
by the fact that these cells will be resistant to not only
carbenicillin, but also tetracycline and kanamycin, other
genes which are part of the structure of RPl. In the
case of the latter possibility; however, these bacterial
isolates would only demonstrate resistance to the antibiotic,
carbenicillin, since they have only received Tnl which
has jumped from plasmid RPl to plasmid pRO1600 co-maintainea
at the same time in a bacterial cell. Such a bacterial
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strain was obtained by the process described above and
its DNA, following extraction of its p]asmid and electro-
phoresis is shown in file C, Figure 1. This plasmid,
designated pRO1601, shows slower electrophoretic mobility
than plasmid pRO1600 reflecting its larger size. When
calculations are done using the standard DNA depicted in
file D, Figure 1, it is determined that pRO1601 is larger
than pRO1600 by 3.2 x 106 daltons molecular size. This
relationship, then, suggests that Tnl has been added to
pRO1600 and accordingly a unique metabolic trait (pheno-
typic mar~er) has been added to pRO1600 allowing its
detection by testing for resistance to the antibiotic,
carbenicillin.
DNA molecules are linear polymers comprised of
substituent molecules called purines and pyrimidines. The
exact linear sequence of joined purines or pyrimidines,
then, defines any region of a DNA molecule of interest.
Analagous regions of any DNA molecule can be detected
by their cleavage with unique enzymes specific in their
activity on those regions called Class II restriction
endonucleases (B. Allet, 'IFragments produced by cleavage
of lambda deoxyribonucleic acid with HaemophiZus parain~Zu-
enzae restriction enzyme ~ II" Biochemistry, 12, pp. 3972-
3977 (lq72)). The specific regions of a DNA molecule,
called recognition sequences, which serve as sites for
cleavage by any of more than 40 specific restriction enao-
nucleases will be distributed differently or absent when
DNA from different biological sources is compared. Con-
~ versely, related or identical DNA molecules will yield an
`; 30 identical set of fragments, subsequent to restriction
endonuclease digestion and analysis by the techniques of
~` slab agarose gel electrophoresis. Therefore new plasmids
can be produced subsequent to digestion as described
above. If the size of the D~'A molecule is small, and
the number of fragments generated by treatment withrestriction endonuclease enzyme is few, these fragments
can randomly associate in different order than originally
p~esent on the parent molecule. Following this random
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re-association, the fragments can be again covalently
joined when the re~associated complex is treated with a
second enzyme called DNA ligase and re~uired co-factors.
The foregoing pl-ocess, then, can be applied to the
analysis of structure of DNA molecules and production of
; new combinations of DNA sequences. By the foreyoing pro-
cess, then, plasmid structure can be altered by deleting
non-essential regions of DNA or the production of a DNA
molecule comprised of D~A from biologically unrelated
` 10 sources
The present invention particularly relates to
plasmid pRO1601, its modification and its utility for genetic
cloning and novelty by applying the rationale and pro-
cedures described in general terms above and commonly
referred to as recombinant DNA technology. The essential
features of this recombinant DNA technology have been
summarized previously (S. Cohen, Scientific American,
July, pp. 25-33 (1975)).
Analysis of plasmid pRO1601 DNA (restriction endo-
nuclease mapping) by conventional procedures referencedabove produced the structure depicted in Figure 2, part A.
;~ Inspection of this genetic map shows the presence of four
restriction endonuclease recognition sites for the restric-
tion endonuclease, PstI. Transposon, Tnl, which was added
~5 to pRO1600 to produce pRO1601 is known to contain 3 PstI
restriction endonuclease sites (J. Grinsted et al, Plasmid,
1, pp 34-37 (1977)). Therefore, the pRO1600 region of
the recombinant plasmid that has undergone transposition
by Tnl must contain a single PstI site. Also, since the
size of the PstI fragments associated with Tnl has been
estimated tsee above ref.~, the particular site for PstI
cleavage which is unique to pRO1600 can be identified.
This site has been chosen as the reference site for mapping
and appears at the left of the drawing shown in part A,
Figure 2. Other sites, representing regions of the
recombinant plasmid, pRO1601~ which are part of T_l, have
been juxtaposed in relation to the pRO1600 PstI site. Also
shown on this genetic map are specific recognition sites for
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~` the restriction endonucleases BglI. These were detérmined
by comparing other variants of Tnl-transposed plasmid
pRO1600 which differed from pRO1601 with respect to the
point of the insertion of the transposon Tnl into pRO1600.
For this,conventional procedures attendant to recombinant
D~A technology were used.
The PstI restriction endonuclease site drawn at
1.21 on panel A, Figure 2 which is kno~n to be part of the
region reflecting the addition of the transposon, Tnl, is
known to be in the middle of the genetic region which
encodes for resistance to carbenicillin. Accordingly,
insertion of extra DNA at this point wi]l destroy the
contiguity of the DN~ sequence and result in the loss of
the ability of this region of the DNA to speci~y the
1 5 enzyme associated with carbenicillin resistance. To demon-
strate the utility of pRO1601 for molecular cloning, a
piece of DNA was inserted into this site. This piece of
DNA, however, contains genetic information for resistance
to the antibiotic tetracycline. Consequently, if this
piece were incorporated into plasmid pRO1601 at this site,
ligated to form a closed circular DNA structure, trans-
formed into a recipient bacterium and cultured with selec-
tion for the ability to grow in the presence of tetra-
~ cycline, cloning of the inserted fragment would have
`; ~5 occurred.
The source of DNA for these experiments was yetanother plasmid called pBR322 (F. Bolivar et al, "Construc-
tion and Characterization of new cloning vehicles II. a
multipurpose cloning system" Gene, 2, pp~ 95-113 (1977)).
Plasmid pBR322, typical of cloning plasmids developed to
date, is unable to be maintained in bacteria not closely
related to the bacterium of its origin, Escherichia coZi.
Consequently, when this plasmid DNA is introduced into an
unrelated bacterium, Pseu~onas ~eluginosa, it will not con-
~ 35 fer the ability to grow in the presence of antibiotics for
v- which it encodes resistance, namely, resistance to car-
`` benicillin ana tetracycline. If, on the other hand, plas-
mid pBR322 DNA becomes part of the structure, by recombi-
nation, of a plasmid such as plasmid pRO1601, its genetic
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functions will be conferred to the recombinant plasmid
containing pRO1601-pBR322 hybrid. Plasmid pBR322, anala-
gous to plasmid p~O1601~ also contains a gene specifying
resistance to the antibiotic, carbenicillin. This gene
too has a site for restriction endonuclease PstI wi~hin
the region of its DNA associated with carbenicillin resis-
tance. To test the assumption that paxt of the carbeni-
cillin resistance gene of plasmid pRO1601 could be matched
up with part of the analogous gene from plasmid pBR322
- 10 resul~ingJ in this case, in the conservation of caxbeni-
cillin resistance for a hybrid molecule joined in one place
at this juncture, I cleaved both plasmid pRO1601 and plas-
mid pBR322 DNA with the restriction endonuclease PstI.
This produces two linear molecules from the parent circu-
lar structures which can randomly associate: one of ~ur
possible pieces from plasmid pRO1601 and a linear single
piece of DNA derived from plasmid pBR322. Following the
application of recombinant DNA technology, namely, plasmid
cleavage, ligation and transformation with selection for
the acquisition of resistance to tetracycline, two groups
of recombinant plasmids were obtained as judged later by
mapping with restriction endonucleases. These structures
are shown in Figure 2, panels B and C. They have been
designated, respectively, pRO1613 and pRO1614~
The structure determined for plasmid pRO1613 con-
forms to that expected from the following possibilities:
reassocation of plasmid pRO1601 PstI fragments depicted on
Figure 2, panel A as traversing the distance between 0
and 1.21 and between 1.21 and 2.93. However, theoretically,
this produces a plasmid having t~o PstI sites not one as
shown on Figure 2, Panel B, for the resultant plasmid,
pRO1613. Therefore, it is probable, based on precedent
from other systems, that the D~A at 0 and 2.93 on the
pRO1601 map has been altered by unknown factors during
the process which effected the deletion of a PstI site
but still allo~ed reformation of a closed ring structure
~` required for survival of the plasmid by a bacterium and
.~ : ' ` .
.
-17-
its progeny cells during growth and reproduction of the
bacterial culture. Plasmid pR01613 has potential utility
for the cloning of restriction endonuclease PstI gener~ted
fragments of DNA whose presence can be ascertained by the
embodiment within the cloned piece of DNA of a metabolic
- trait for which recombinant progeny can be selected.
The structure determined for plasmid pRO1614 con-
~orms to that expected for a hybrid-recombinant plasmid
which contains a part of the cloning vector plasmid,
pRO1601, and the cloned fragment, in this case, p]asmid
pBR322. Furthermore, the formation of this recombinant
plasmid, pRO1614, and its transformation into P~e~domonas
aeruginosa is accompanied by the occurrence of bacteria
which are now both carbenicillin resistant and tetracycline
resistant. Clearly, then, as hypothesized above, part of
the carbenicillin resistance gene has been derived from
plasmid pRO1601 (region to the left of the PstI site shown
` at 1.21) and part of the carbenicillin gene has been derived
from the inserted DNA fragment (region to the right
2~ of 1.21, Figure 2, Panel C,~ which is part of plasmid
pBR322. This result, then, has demonstrated the utility of
plasmid pRO1601 for cloning unrelated DNA (plasmid pBR322).
This result also demonstrates the derivation of related,
albeit size-reduced, derivative plasmids of greater utility
th~n the parent plasmid DNA molecule, pRO1601.
Additional information regarding the essential
features of plasmid pRO1601 required for maintenance by a
host bacterium is also provided here by these experiments:
the region on Figure 2t Panels A, B and C from 0.05 to 0.88
map units, measuring 0.83 megadaltons is common to all
plasmids shown. Accordingly, this is the region of plasmia
DNA present originally on plasmid pRO1600 which is essential
; for the replication and maintenance of the plasmid cloning
vector and its possible derivatives. This region, defined
by its restriction endonuclease restriction enzyme sites
at 0.05 and 0.88 map units is a critical embodiment of this
invention.
`~;
. . . . .
-18-
In addition to the foregoing, restric~ion éndo-
nuclease digest analysis of plasmids described in the
development of the invention allow deduction of the genetic
map of plasm;d pRO1600 independent of RPl. Its restriction
en~onuclease digest map is shown in panel D, Figure 2.
The present invention particularly relates to the
process which comprises the addition of chromosome DNA frag-
- ments produced by chromosome cleavage with a rcstrictiOn
endonuclease into a broad host range replicator plasmid
- 10 and its apertinent DNA which has also beèn cleaved by the
same restriction endonuclease. This ad~ixture of plasmid
and chromosome DNA fragments is then followed by ligase
treatment and transformation of recombined DNA to a sui~-
able bacterial host.
- The source of DNA for this series of cloning experi-
ments was plasmid pRO1614 and the Pseudomonas aeruginosa
strain PAO chromosome. The purpose of these experiments
was to demonstrate the utility of pRO1614 for cloning chro-
mosomal DNA fragments corresponding to genetic locations
on the chromosome associated with specific metabolic
functions contributing to the biosynthetic activities o
the bacterial cell. For this purpose a mutant of P.
aeruginosa designated strain PA0236 (D. Haas and B. Holloway,
Molecular and general genetics 144:251(1976)) was used as
the transformation-cloning recipient. Unlike the parent
bacterial P. aeruginosa strain, PAO lc (ATCC 15692), strain
PA0236 has been mutated to require the addition of the
amino acids L-isoleucine, L-valine and L-methionine to
growth medium to support cellular synthesis and growthO
These nutritional requirements for the amino acids,
-~ then, allow for the selection of recombinant clones which
contain chromosomal DNA fragments associated with synthesis
; of these compounds: bacteria which have received the appro-
priate cloned DNA fragment acquire the ability to grow in
the absence of the amino acid for which its biosynthesis
has mutated to a re~uirement. It also follows from the
application of this rationale that the recombinant plasmid,
in this instance pRO1614 plus the cloned chromosomal DNA
~`
: .
-19 -
fragment~ will be larger than the molecular size of plas-
- mid pR01614 re~lecting the ac~uisition of additional DNA
in the molecular cloning process. Within the DNA of plas-
mid psR322 cloned into the broad bacterial host range
replicator plasmid, pR01613, is con~ained the genetic
in~ormation which specifies resistance to the antibiotic,
tetracycline. Within this region of the cloned fragment
is also the recognition site for the res~riction endo-
nuclease, BamHl. It follo~s from this relationship, then,
lQ that interruption of the contiguity of this tetracycline
resistance gene by the in vitro insertion of a cloned DNA
fragment will destroy tetracycline resistance specified
~y this gene. Add;tionally, if the selection for cloned
DNA fragments is on the basis of the acquisition of a
specific metabolic trait, bacteria which have received the
appropriate recom~inant plasmid will grow in the absence
of the metabolite corresponding to the specific metabolic
trait in question. Such bacterial strains, were obtained
~y the process descri~ed a~ove and their DNA, following
2Q extraction of their plasmids are shown in Figure 3. In
Figure 3, file B contains reference DNA used as a
size standard, file C shows plasmid pR01614, file A shows
`; plasmid pR01614 with its DNA fragment now allowing growth
~: of bacterial strain PA0236 in the absence of the amino
25 acids isoleucine and L-valine, referred to as pR01615,
and file D shows plasmid pR01614 with its cloned DNA frag-
ment now allowing growth of ~acterial strain PA0236 in the
; absence of the amino acid L methionine, referred to as
pR01616. Clearly, the recombinant plasmids shown in files
3Q A and D are larger t~an pR01614. This is the expected
relationship associated with the acquisition of a hetero-
` logous DNA fragment following the application of recom~
; ~inant DNA technology.
~ ~ ... . . . .. . .. . . .
~ Specific Description
.. .
~` 35 1. Steps
The specific process for developing the genetic
cloning plasmid into the form of an artificial, chemically
aerived, non transmissi~le plasmid and its use for the
;
.
' '
7~
.. . . . . .
-20-
genetic cloning of a representative fragment of DNA is as
follows:
(1~ Recognition of the novelty and potential
usefulness of a mutant plasmid during routine analysis for
the presence of the progenitor plasmid, RPl, subsequent
to a genetic transfer experiment: (occurrence of pRO1600);
(2) The addition of a distinctive phenotypic
trait for resistance to the antibioti.c, carbenicillin, to
the small plasmid observed as an extra anamolous plasmid
(production of plasmid pRO1601);
(3) Physical mapping of plasmid pRO1601 with
restriction endonucleases:
~ 4) Size-reduction of plasmid pRO1601 (production
of plasmid pRO1613), genetic cloning o~ plasmid pBR322
15 into plasmid pRO1601 5production of plasmid pRO1614).
(5) Genetic cloning, using recombinant DNA techno-
logy, of bacterial chromosome DNA associated with growth
by strain PAO236 in the absence of L-isoleucine and
L-valine or L-methionine.
2. Strains Used
Bacterial strains used in the development and
characterization of plasmids for genetic cloning and
their saliant features are as follows:
( 1~ Pseudomonas Qeruginosa PAO2. This bacterial
25 strain is a mutant of P. aeruginosa lc (ATCC No. 15692)
which has been mutated to require the amino aci.d, serine,
for its growth and maintenance;
(2) P- aeruginosa PAO2(RPl). This strain was
derived from the foregoing bacterial strain PAO2 by the
. 30 addition of plasmid RPl by the-process of bacterial con-
jugation from PAO 2 (RPl) ~RRL-B-12123 previously
described;
(3) Pseu~o~snas putida PPO131. This bacterial
strain is a mutant of P. putida A.3.12 (A~CC no. 12633)
35 which has been mutated to require the amino acid, histidine,
for its gro~th and maintenance;
' ; ' '
'7~C)
.; :
-21-
(4) Ps~ domonas fZ~oresc~ns PFO141. This isolate
was derived from a wild-type strain isolated from nature.
The clone of this strain was influenced by its inability
- to grow and reproduce at temperatures exceeding 32C.
Accordingly this physiological trait effectively contains
growth of the bacterium and hence its plasmids to environ-
ments excluding mammilian enviromnents. P. fZuoræscens
PFO141 is a mutant of the wild-type strain which requires
the amino acid, histidine, for growth and maintenance.
(5) Eschel~ichia coZi ED8654. The strain was derived
from E. coli K12 and requires the amino acid, methionine,
for its growth and maintenance. It has also been mutated
; to not restrict or modify DNA.
(6) Azotobac~er vineZc~dii AVM100. This is a strain
15 isolated from nature.
(7) KZebsieZ~a pnew710n%ee KPO100. Thi s is a strain
isolated from nature.
(8) Pseud~nonas ae~uginosa PAO236. This bacterial
strain is a mutant of P. aeru~3inosa lc (ATCC No. 15692)
`20 which has been mutated to require the amino acids isoleu-
cine, valine and methionine and other amino acids.
~"All of the cultures are maintained in the culture
collection of the Department of Microbiology, University of
Michigan Medical School and are freely available to quali-
~`~25 fied recipients. Such cultures are also available from
other depositories.
3. Materials
The bacteriological medium used for routine mainten-
"ance and propagation of the above bacterial strains con-
tained the following inyredients:
Bacto-tryptone 5 g
Bacto yeast extract 3.75 g
Dextrose 1 g
KNO3 4 g
Distilled ~ater 1000 ml
When solid medium was used, Bacto-agar was added
(15 g) prior to sterilization. Medium was sterilized by
autoclaving at 121C for 15 minutes. Antibiotic ~as added
.
:
7 ~ ~" ~ ~
-22-
to the above medium after sterilizati.on and cooling to
50C. The plasmid DNA was added to recipient bacterial
cells and then selected for uptake and maintenance of the
plasmid which specified resistance to.an antibiotic. For
- S carbenicillin it was 0.5 mg per ml; for tetracycline it
was 0.025 mg per ml except for P. ael~uginosa PAO2. For
this bacterial strain, tetracycline was added at 0.05 mg
per ml medium.
4- T~peratu~es
Experiments utilizing P. putida or P f~uorescens
were carried out at 25~C. All other e~periments were
carried out at 37C.
Example 1
Addition of a selectable genetic marker to plasmid
15 pRO1600 (production of plasmid pRO1601).
For this example a partially purified DNA suspen-
sion derived from P. eeruginosa PAO2 tRPl) or from P.
. aeruginose (RPl/pRO1600) was added to a growing culture of
P. aeruginosa PAO2. Following experimental manipulations
.` 20 attendant to the generally known process of bacterial
transformation, the admixture was deposited on the surface
of solid medium which contained the antibiotic, carbeni-
cillin. This was incubated at 37C for 24 hours and the
number of bacterial colonies counted. The results of a
; 25 typical experiment are shown below in Table 1. These
colonies, selected for the ability to grow in the presence
- of carbenicillin, were then sub-propagated (grown out)
on nutrient medium containing either the antibiotic tetra-
cycline or the antibiotic kanamycin to score for the co-non-
~` 30 selected acquisition of phenotypic markers other than car-
: benicillin resistance which were present in the bacteria
;` used to prepare the transforming DNA.
7 7 ~ ~ ~3
T~I.E 1
Nonselccted markers which
Source of Were Acquired by 100 Trans-
Transforming Number of _ _ormant col nies
DNATrar,sformants Tetracycline Kanamycin
P. ae~ginosa
PAO2 (RPl~190 100~ 100%
P. ae~ginosa
- ~RPl~pRO/1600 397 92~ 92%
The results shown in Table 1 indicate that 8 percent
lQ o the transformants obtained from the P. ae ~ginosa PAO2
(XPl/pRO1600) DNA suspension acquired resistance to
` carbenicillin only and not resistance to tetracycline or
kanamycin. This result, then, suggests that pRO1600 with
a Tnl transposon has been transformed as suggested in
the foregoing background discussion. On the other hand,
transformants derived from the use of the P Qe~uginosa
PAO2~RPl~ DNA suspension, as expected, ~roduced no trans-
formants resistant to carbenicillin only, since this strain
` does not contain plasmid pRO1600 as a potential trans-
poson acceptor plasmid.
Example 2
Size-reduction of plasmid pRO1601 (production of
plasmid pRO1613~ and cloning of pBR322 DNA into plasmid
pRO1601 (production of plasmid pRO1614).
For this experiment, partially purified DNA solu-
tions identified in the Table 2 were treated with enzymes
~ listed. Transformants were selected using P. ae ~ ginosa
`~ PAO2 bacterial cells that had not previously received a
` `~ plasmid. The antibiotic resistance(s~ acquired by the
~ 30 bacteria as a consequence of their adrnixture with the
`~ various DNA preparations~ treated as indicated, are shown
on the right column of the Tahle~
~ `
,: ' o2'3 ~
.
-, .
-24-
TABLE 2
Plasmid Number of Transformant
DNA Used ~re~tment of ~NA Tralsformant_ _henotype _
pBR322 Nonc 0 Not Relevant
5 psRl6ol None 69,000 carbenicillin
resistant
pRO1601 PstI digestion + 70 carbenicillin
ligation resistant pRO1613
pRO1601 + PstI di~estion + 2 carbenicillin resistant,
1~ pBR322 ligation 4 carbenicillin and
tetracycline resistant
pRO1614
Also noteworthy from the above experiments is the failure
of plasmid pBR322, by itself to transform~ Therefore,
the expression of its tetracycline resistance, when cloned
into plasmid pRO1601 reflects its maintenance by virtue
of its association with critical unctions provided by
the vector, plasmid pRO1601O
Example 3
Demonstration of broad bacterial host range for plasmids
derived from pRO1600.
` To demonstrate the utility of derivatives of plas-
mid pRO1600 (pRO1613, pRO1614) with respect to the useful-
ness of this invention for conducting cloning experiments
using recombinant DNA technology in bacteria of disparate
`ecological niche and physiological properties, bacterial
transformation studies were done using bacteria other
than P. ~eruginosa PAO2 as the transformation-recipient
bacterium. The results of typical experiments are shown
.~ 30 below in Table 3. The procedure for producing and exacting
-the DNA is described in Hansen and Olsen referred to above.
For this work, approximately 0.5 micxograms of
DNA suspended in Q.025 ml buffer solution was added to
approximately 1 x 10 bacterial recipient cells in a volume
of Q 2 ml. The solution was heat cycled between 0 and 45C
uslng a procedure descrihed in Tr~ns ormation of Salmonella
typhinium by Plasmid Deo~ribonucl _ c Acid, J, Bact Vol
:. :
.
, .
-25-
119 pp 1072 to 1074 (1974) D. E. Lederburg and S. N. Cohen.
This admixture, following experimental manipulation
attendant to the known technique of bacterial transformation,
was deposited on the surface of solid medium which con-
tained antibiotic. ~his was incubated at 37C for 48hours at the appropriate temperature and the numher of
bacterial colonies counted. The results of these experi-
ments are shown in Table 3.
TABLE 3
10 Sourceof Transformation- Number of
; Transforming Recipient Bacterial Selective Transfor-
; DNA Strain Antibiotic mants*
. ~,
pRO1613 P. aeruginosa PAO2Carbenicillin i32,000
pRO1614 P. aeruginosa PAO2Tetracycline 38,000
pRO1614 P. putida PPO131 Tetracycline 960
pRO1614 P. f?,uo~escens PFO141Tetracycline 24,600
pRO1614 E. coZi ED8654 Tetracycline 5,500
pRO1614 K. pneulr~oniae KPM100Tetracycline 1,300
pRO1613 A. vinZandii AVM100Carbenicillin 176
*out of 108 potential recipient cells
The experiments shown above in Table 3, clearly
`; indicate the broad host range feature of plasmids derived
from plasmid pRO1600. Variation in the efficiency of the
transformation process with the bacterial strain used,
in all probability, is associated with the use of a trans-
formation process which is not optimal for the bacterial
strain in question. Accordingly, quantitative aspects
of these results do not limit the utility of plasmid
pRO1600-derivatives for genetic cloning experiments with
bacteria showing poor transformation efficiencies. In
these instances, improvements in the process of bacterial
transformation for the particular bacterial strain in
question should, in the future, enhance the efficiency of
transformation per se usin~ these bacterial strains.
Example 4
Demonstration of the molecular cloning of chromosomal DNA
from Pseudomonas aeruginosa using the recombinant plasmid,
pRO1614.
To demonstrate the utllity of the derived plasmid,
.
-2~;-
pR01614, with respect to the usefu]ness of this inventionfor conducting cloning experiments using recomhinant DNA
technology in bac~eria for the cloning of bacterial chromo-
some genes, a cloning experiment was done for the selection
and isolation of bac-terial genes associated ~;ith the bio-
synthesis of the amino acids L-isoleucine, L-valine and
L-methionine. The procedure for producing chromosomal
and plasmid D~A is described in l~ansen and Olsen referred
to above.
For this work, approximately 0.5 micrograms of
either chromosomal DNA extracted from PAO lc or plasmid
pRO1614 DNA were suspended in 0.025 ml buffer and digested
with the restriction endonuclease, BamHl. The separate
solutions were then mixed and treated with the enzyme T4
ligase and cofactors. This admixture, following experimental
manipulation attendant to the technique of bacterial
transformation, was deposited on the surface of solid
`~` medium which was supplemented with the nutrients required
for growth by Pseudomonas ~eruginosa for growth except in one
instance the amino acids L-isoleucine and L-valine and in
another instance, the amino acid L-methionine. A single
colony of bacterial growth appeared on each of the above
selective medium. (A frequency of one in 10 recipients).
~hen these recombinant DNA plasmids are extracted from
transformation recipient bacteria,the resulting DNA shows
high transformation frequencies when tested by retrans-
formation into yet another recipient. The frequency for
amino acid biosynthesis acquisition corresponds to the
~` frequency ~or the acquisition of carbenicillen resistance
in these experiments. These colonies were then purified
and grown up for the production of plasmid DNA.
Figure 3 shows slab agarose gel electrophoresis
portions for plasmid DNA from Pseudomonas ae~uginosa PA0236.
Bacterial cells were grown in nutrient broth medium and
plasmid DNA was extracted and processed as described in
reference to Figure 1. DNA was electrophoresed and samples
were as follows: A, DNA extracted from a transformant
~ ~ .
~'7'7'~ ¢D
clone capable o~ growth without L-isoleucine or L-valine
(pRO1615); B, DNA from Esche~ichia c~li V517, a multi~plas-
mid-containin~ strain used as a size standard; C, DNA from
Pseudomonas aeruginosa PAO2(pR01614); D, DNA extracted
from a transformant clone capable of growth without L-
methionine (pRO1616). The estimated molecular sizes for
the recombinant plasmids are 14 x 106 daltons for the plas-
mid in file A; 10.4 x 106 daltons for the plasmid in file D.
Recombinant clones for isoleucine and valine bio-
synthesis (file A) or methionine biosyn~hesis (file D) are
clearly larger than the plasmid pRO1614 cloning vector
(file C). This relationship reflects the acquisition,
using recombinant DNA technology of chromosomal DNA directing
the biosynthesis of the amino acids in ques~ion. The re-
combinant plasmid maintained carbenicillin resistance as
expected.
The novel plasmids of the present invention and
RPl have been deposited for reference purposes with the
Northern Regional Research Laboratory and are freely avail-
able upon request by number. The plasmids are also avail-
able from the University of Michigan, Ann Arbor, Michigan
by internal reference numbers:
Internal
Reference NR~L Reference
.
RPl Pseudomonas aeruginosa B-12123
pRORPl/1600 Pseudomonas aeruginosa B-12124
pRO1601 Pseudomonas aeruginosa B-12125
pRO1613 Pseudomonas aeruginosa B-12126
pRO1614 Pseudomonas aeruginosa B-12127
pRO1615 Pseudomonas aeruginosa B-12149
pRO1616 Pseudomonas aeruginosa B-12148
In various preEerred embodiments this application
relates to the plasmids defined above (by the internal
reference) carried in the respective hosts defined above
(by the NRRL reference).
- 27 -
mab/~'.`) -
: - . . . .
. . . .
