CA1177420A - Broad host range small plasmid rings as cloning vehicles - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

The present invention relates to novel, broad bacterial host range small plasmid deoxyribonucleic acid rings which serve as cloning vehicles for DNA fragments, particularly those separated from other plasmid rings or from chromosomes, recombined with the small plasmid rings and to the processes for recombining the plasmid rings and to processes for transferring them between host bacteria. In particular, the present invention relates to the aggregate plasmid ring RP1/pRO1600, to pRO1600 and plasmid ring derivatives thereof, particularly including pRO1601; pRO1613 and pRO1614, all of which are carried for reference purposes in Pseudomonas aeruginosa ATCC 15692 (also known as strain PAOlc) and are on deposit at the Northern Regional Research Laboratories (NRRL) of the United States Department of Agriculture at Peoria, Illinois. The plasmid ring RPl (also known as R1822) is deposited in Pseudomonas aeruginosa NRRL-B-12123 (and is a known plasmid ring). The pRO1600 portion of the aggregate is a new plasmid ring. The novel small plasmid rings are particularly useful for recombinant genetic manipulation wherein the DNA fragments are introduced into the plasmid rings to produce useful, cloneable characteristics in the host bacterium, particularly chemical generating character-istics.

Description

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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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_-- _ 9 _ 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 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 a .

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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 .~ : ' ` .

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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.
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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

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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.
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~ 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 ;

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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;

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(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 .

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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~
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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 :. :

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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.

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 ~ ~ .

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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).

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Claims (5)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for producing recombinant deoxy-ribonucleic acid plasmids which comprises:
(a) providing a first plasmid as a cloning vehi-cle selected from pRO1600, pRO1601, pRO1613, pRO1614, pRO1615 and pRO1616 as carried in Pseudomonas aeruginosa NRRL-B-12124, NRRL-B-12125, NRRL-B-12126, NRRL-B-12127, NRRL-B-12149 and NRRL-B-12148, respectively;
(b) reacting the first plasmid and a second deoxyribonucleic acid source with at least one restriction endonuclease which cleaves the first plasmid and the second deoxyribonucleic acid source into linear deoxyribonucleic acid fragments; and (c) randomly recombining the linear deoxyribo-nucleic acid fragments using ligation to form recombinant plasmids which replicate during cell division when pro-vided in a bacterial cell, wherein deoxyribonucleic acid from the cloning vehicle controls replication of the re-combinant plasmid during cell division.

2. The process of Claim 1, wherein the second deoxyribonucleic acid source is a plasmid.

3. The process of Claim 1, wherein the second deoxyribonucleic acid source is a chromosome.

4. The process of Claim 1, wherein the re-striction endonuclease is PstI and ligation is accomplished using T4 ligase or derivatives thereof.

5. The process of Claim 1, wherein in addi-tion the recombinant plasmids are transformed using a plasmid receptive bacterium, and wherein the recombinant plasmids in the bacterium are selected for a particular chemical generating characteristic.