United States Patent (19)
`Cohen et al.
`
`Patent Number:
`11
`(45) Date of Patent:
`
`4,740,470
`Apr. 26, 1988
`
`73 Assignee:
`
`(54) BIOLOGICALLY FUNCTIONAL
`MOLECULAR CHMERAS
`Stanley N. Cohen, Menlo Park;
`75 Inventors:
`Herbert W. Boyer, Mill Valley, both
`of Calif.
`The Board of Trustees of the Leland
`Stanford, Jr. University, Stanford,
`Calif.
`The portion of the term of this patent
`subsequent to Dec. 2, 1997 has been
`disclaimed.
`21 Appl. No.: 602,294
`22 Filed:
`Apr. 20, 1984
`
`*
`
`Notice:
`
`63
`
`(56)
`
`Related U.S. Application Data
`Continuation of Ser. No. 959,288, Nov. 9, 1978, Pat.
`No. 4,468,464, which is a continuation of Ser. No.
`687,430, May 17, 1976, abandoned, which is a continua
`tion-in-part of Ser. No. 520,691, Nov. 4, 1974, aban
`doned.
`51) Int. Cl." ....................... C12N 15/00; C12N 5/00;
`C12N 1/20; C12N 1/00; C12P 19/34
`52 U.S.C. ................................... 435/1723; 435/91;
`435/320, 435/240.2; 435/253; 935/27; 935/31;
`935/32
`58 Field of Search ...................... 435/172.3, 91, 317,
`435/253,240, 320, 240.2; 935/27, 31, 32
`References Cited
`PUBLICATIONS
`Maniatis, Cell Biology, vol. 3, pp. 563-608 (1980).
`Mentz et al., PNAS USA, vol. 69, pp. 3370-3374, Nov.
`1972.
`Jackson et al., PNAS USA, vol. 69, pp. 2904–2909, Oct.
`1972.
`Keeton, Biological Sciences, Pub. by Norton & Co.,
`Inc., New York, pp. 88-90 (1967).
`Hicks et al., Nature, vol. 269, pp. 265-267 (1977).
`Hinnen et al., PNAS USA, vol. 75, pp. 1929-1933
`(1978).
`Buggs, Nature, vol. 275, pp. 104-109 (1978).
`"The Integrated State of Viral DNA in SV40-Trans
`formed Cells', J. Sambrook et al., PNAS, (1968), 60:
`1288-1295.
`"Bacterial Conjugation', Curtiss, Ann. Rey. Microbiol,
`(1969), 23: 69-135.
`"Transduction of Lysogeny in Escherichia coli'', Jacob,
`Virology, (1955), 1:207-220.
`"Kinetics of Renaturation of DNA', Wetmur & David
`son, J. Molec. Biol, (1968), 31: 349-370.
`"Size Distribution of Circular DNA from petite-mutant
`Yeast Lacking poNA', Clark-Walker, Eur, J. Bio
`chem., (1973), 32: 263-267.
`"Isolation of Circular DNA from a Mitochondrial
`
`Fraction from Yeast', Clark-Walker, PNAS, (1972), 69:
`388-392.
`"A Map of the Restriction Targets in Yeast 2 Micron
`Plasmid DNA Cloned on Bacteriophage Lambda”,
`Beggs et al., Molec. gen. Geneti, (1976), 148: 287-294.
`"Bidirectional Replication of Simian Virus 40 DNA",
`Danna and Nathans, PNAS, (1972), 69: 3097-3100.
`"Virus-Specific RNA in Cells Productively Infected or
`Transformed by Polyoma Virus', Benjamin, J. Molec.
`Biol, (1966), 16:359-373.
`"Transduction in Escherichia coli K-12', Morse et al.,
`Genetics, (1956), 41: 142-156.
`"Construction of Biologically Functional Bacterial
`Plasmids in Vitro', Cohen et al., PNAS, (1973), 70:
`3240-3244.
`"Nonchromosomal Antibiotic Resistance in Bacteria:
`Genetic Transformation of Escherichia coli by R-Fac
`tor DNA', Cohen et al., PNAS, (1972), 69: 2110-21 14.
`"Calcium-Dependent Bacteriophage DNA Infection',
`Mandel et al., J. Mol. Biol, (1970) 53: 159-162.
`"Transfection of Escherichia coli Spheroplasts”, Hen
`ner et al., J. Virol, (1973), 12: 741-747.
`"Viable Molecular Hybrids of Bacteriophage Lambda
`and Eukaryotic DNA', Thomas et al., PNAS, (1974),
`71: 4579.4583.
`"Extrachromosomal Inheritance in Bacteria', Novick,
`Bac. Rey, (1969), 33: 210-263.
`Federal Register, vol. 44, No. 71, Wednesday Apr. 11,
`1979, Notices.
`Primary Examiner-Alvin E. Tanenholtz
`Attorney, Agent, or Firm-Bertram I. Rowland
`57
`ABSTRACT
`Method and compositions are provided for replication
`and expression of exogenous genes in microorganisms.
`Plasmids or virus DNA are cleaved to provide linear
`DNA having ligatable termini, which are bound to a
`gene having complementary termini, to provide a bio
`logically functional replicon with a desired phenotypi
`cal property. The replicon is inserted into a microor
`ganism cell by transformation. Isolation of the transfor
`mants provides cells for replication and expression of
`the DNA molecules present in the modified plasmid.
`The method provides a convenient and efficient way to
`introduce genetic capability into microorganisms for
`the production of nucleic acids and proteins, such as
`medically or commercially useful enzymes, which may
`have direct usefulness, or may find expression in the
`production of drugs, such as hormones, antibiotics, or
`the like, fixation of nitrogen, fermentation, utilization of
`specific feedstocks, or the like.
`The invention was supported by generous grants of
`NIH, NSF and the American Cancer Society.
`
`23 Claims, No Drawings
`
`Page 1
`
`KASHIV EXHIBIT 1015
`IPR2019-00791
`
`

`

`1.
`
`4,740,470
`
`BOLOGICALLY FUNCTIONAL MOLECULAR
`CHIMERAS
`
`5
`
`15
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`This application is a continuation of application Ser.
`No. 959,288, filed Nov. 9, 1978, now U.S. Pat. No.
`4,468,464; which is a continuation of application Ser. 10
`No. 687,430, filed May 17, 1976, now abandoned; which
`is a continuation-in-part of application Ser. No. 520,691,
`filed Nov. 4, 1974, now abandoned.
`BACKGROUND OF THE INVENTION
`1. Field of the Invention
`Although transfer of plasmids among strains of E. coli
`and other Enterobacteriaceae has long been accom
`plished by conjugation and/or transduction, it has not
`been previously possible to selectively introduce partic- 20
`ular species of plasmid DNA into these bacterial hosts
`or other microorganisms. Since microorganisms that
`have been transformed with plasmid DNA contain au
`tonomously replicating extrachromosomal DNA spe
`cies having the genetic and molecular characteristics of 25
`the parent plasmid, transformation has enabled the se
`lective cloning and amplification of particular plasmid
`genes.
`The ability of genes derived from totally different 30
`biological classes to replicate and be expressed in a
`particular microorganism permits the attainment of
`interspecies genetic recombination. Thus, it becomes
`practical to introduce into a particular microorganism,
`genes specifying such metabolic or synthetic functions 35
`as nitrogen fixation, photosynthesis, antibiotic produc
`tion, hormone synthesis, protein synthesis, e.g. enzymes
`or antibodies, or the like-functions which are indige
`nous to other classes of organisms-by linking the for
`eign genes to a particular plasmid or viral replicon.
`40
`2. Brief Description of the Prior Art
`References which relate to the subject invention are
`Cohen, et al., Proc. Nat. Acad, Sci., USA, 69, 2110
`(1972); ibid, 70, 1293 (1973); ibid, 70, 3240 (1973); ibid,
`71, 1030 (1974); Morrow, et al., Proc. Nat. Acad. Sci., 45
`71, 1743 (1974); Novick, Bacteriological Rev., 33, 210
`(1969); and Hershfeld, et al., Proc. Nat. Acad. Sci., in
`press; Jackson, et al., ibid, 69,2904 (1972);
`SUMMARY OF THE INVENTION
`Methods and compositions are provided for geneti
`cally transforming microorganisms, particularly bac
`teria, to provide diverse genotypical capability and
`producing recombinant plasmids. A plasmid or viral
`55
`DNA is modified to form a linear segment having liga
`table termini which is joined to DNA having at least
`one intact gene and complementary ligatable termini.
`The termini are then covalently bound to form a "hy
`brid” plasmid molecule which is used to transform sus- so
`ceptible and compatible microorganisms. After trans
`formation, the cells are grown and the transformants
`harvested. The newly functionalized microorganisms
`may then be employed to carry out their new function;
`for example, by producing proteins which are the de- 65
`sired end product, or metabolites of enzymic conver
`sion, or be lysed and the desired nucleic acids or prote
`ins recovered.
`
`50
`
`2
`DESCRIPTION OF THE SPECIFIC
`EMBODIMENTS
`The process of this invention employs novel plas
`mids, which are formed by covalently inserting DNA
`having one or more intact genes into a plasmid in such
`a location as to permit retention of an intact replicator
`locus and systems (replicon) to provide a recombinant
`plasmid molecule. The recombinant plasmid molecule
`will be referred to as a “hybrid” plasmid or plasmid
`"chimera.” The plasmid chimera contains genes that are
`capable of expressing at least one phenotypical prop
`erty. The plasmid chimera is used to transform a suscep
`tible and competent microorganism under conditions
`where transformation occurs. The microorganism is
`then grown under conditions which allow for separa
`tion and harvesting of transformants that contain the
`plasmid chimera.
`The process of this invention will be divided into the
`following stages:
`I. preparation of the recombinant plasmid or plasmid
`chimera;
`II. transformation or preparation of transformants;
`and
`III. replication and transcription of the recombinant
`plasmid in transformed bacteria.
`I. PREPARATION OF PLASMID CHIMERA
`In order to prepare the plasmid chimera, it is neces
`sary to have a plasmid, which can be cleaved to provide
`an intact replicator locus and system (replicon), where
`the linear segment has ligatable termini or is capable of
`being modified to introduce ligatable termini. A small
`number of such plasmids presently exist. Of particular
`interest are those plasmids which have a phenotypical
`property, which allow for ready separation of transfor
`mants from the patent microorganism. The plasmid will
`be capable of replicating in a microorganism, particu
`larly a bacterium, which is susceptible to transforma
`tion. Various unicellular microorganisms can be trans
`formed, such as bacteria, fungii and algae. That is, those
`unicellular organisms which are capable of being grown
`in cultures or fermentation. Since bacteria are for the
`most part the most convenient organisms to work with,
`bacteria will be hereinafter referred to as exemplary of
`the other unicellular organisms. Bacteria, which are
`susceptible to transformation, include members of the
`Enterobacteriaceae, such as strains of Escherichia coli
`Salmonella; Bacillaceae, such as Bacillus subtilis, Pneu
`mococcus; Streptococcus, and Haemophilus influenzae.
`A wide variety of plasmids may be employed of
`greatly varying molecular weight. Normally, the plas
`mids employed will have molecular weights in the
`range of about 1 x 106 to 50x 106d, more usually from
`about 1 to 20x106d, and preferably, from about 1 to
`10x106d, the desirable plasmid size is determined by a
`number for factors. First, the plasmid must be able to
`accommodate a replicator locus and one or more genes
`that are capable of allowing replication of the plasmid.
`Secondly, the plasmid should be of a size which pro
`vides for a reasonable probability of recircularization
`with the foreign gene(s) to form the recombinant plas
`mid chimera. Desirably, a restriction enzyme should be
`available, which will cleave the plasmid without inacti
`vating the replicator locus and system associated with
`the replicator locus. Also, means must be provided for
`providing ligatable termini for the plasmid, which are
`
`Page 2
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`4,740,470
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`3
`complementary to the termini of the foreign gene(s) to
`zyme, such as a A-exonuclease, and it is found that there
`is a high probability that cohesive termini will be
`allow fusion of the two DNA segments.
`Another consideration for the recombinant plasmid is
`achieved in this manner.
`An alternative way to achieve a linear segment of the
`that it be compatible with the bacterium to be trans
`plasmid with cohesive termini is to employ an endonu
`formed. Therefore, the original plasmid will usually be
`clease such as EcoRI. The endonuclease cleaves the
`derived from a member of the family to which the bac
`two strands at different adjacent sites providing cohe
`terium belongs.
`The original plasmid should desirably have a pheno
`sive termini directly.
`typical property which allows for the separation of
`With flush ended molecules, a T4 ligase may be em
`ployed for linking the termini. See, for example, Scara
`transformant bacteria from parent bacteria. Particularly
`useful is a gene, which provides for survival selection.
`mella and Khorana, J. Mol. Biol. 72: 427-444(1972) and
`Survival selection can be achieved by providing resis
`Scaramella, DNAS 69: 3389(1972), whose disclosure is
`tance to a growth inhibiting substance or providing a
`incorporated herein by reference.
`Another way to provide ligatable termini is to cleave
`growth factor capability to a bacterium deficient in such
`capability.
`employing DNAse and Mn+ as reported by Lai and
`Conveniently, genes are available, which provide for
`Nathans, J. Mol. Biol. 89: 179(1975).
`The plasmid, which has the replicator locus, and
`antibiotic or heavy metal resistance or polypeptide re
`serves as the vehicle for introduction of a foreign gene
`sistance, e.g. colicin. Therefore, by growing the bac
`teria on a medium containing a bacteriostatic or bacteri
`into the bacterial cell, will hereafter be referred to as
`“the plasmid vehicle.”
`ocidal substance, such as an antibiotic, only the transfor
`It is not necessary to use plasmid, but any molecule
`mants having the antibiotic resistance will survive. Il
`capable of replication in bacteria can be employed.
`lustrative antibiotics include tetracycline, streptomycin,
`sulfa drugs, such as sulfonamide, kanamycin, neomycin,
`Therefore, instead of plasmid, viruses may be em
`penicillin, chloramphenicol, or the like.
`ployed, which will be treated in substantially the same
`manner as the plasmid, to provide the ligatable termini
`Growth factors include the synthesis of amino acids,
`25
`for joining to the foreign gene.
`the isomerization of substrates to forms which can be
`If the production of cohesive termini is by restriction
`metabolized or the like. By growing the bacteria on a
`endonuclease cleavage, the DNA containing the for
`medium which lacks the appropriate growth factor,
`eign gene(s) to be bound to the plasmid vehicle will be
`only the bacteria which have been transformed and
`cleaved in the same manner as the plasmid vehicle. If
`have the growth factor capability will clone.
`30
`the cohesive termini are produced by a different tech
`One plasmid of interest derived from E. coli is re
`ferred to as pSC101 and is described in Cohen, et al.,
`nique, an analogous technique will normally be em
`ployed with the foreign gene. (By foreign gene is in
`Proc. Nat. Acad. Sci., USA, 70, 1293 (1972), (referred
`to in that article as Tc6-5). Further description of this
`tended a gene derived from a source other than the
`transformant strain.) In this way, the foreign gene(s)
`particular plasmid and its use is found in the other arti
`cles previously referred to.
`will have ligatable termini, so as to be able to be cova
`lently bonded to the termini of the plasmid vehicle. One
`The plasmid pSC101 has a molecular weight of about
`... 5.8x106d and provides tetracycline resistance.
`can carry out the cleavage or digest of the plasmids
`together in the same medium or separately, combine the
`Another plasmid of interest is colicinogenic factor EI
`...
`plasmids and recircularize the plasmids to form the
`(ColE1), which has a molecular weight of 4.2X106d,
`40
`plasmid chimera in the absence of active restriction
`and is also derived from E. coli. The plasmid has a single
`enzyme capable of cleaving the plasmids.
`EcoRI substrate site and provides immunity to colicin
`Descriptions of methods of cleavage with restriction
`E1.
`enzymes may be found in the following articles:
`In preparing the plasmid for ligation with the exoge
`nous gene, a wide variety of techniques can be pro
`Greene, et al., Methods in Molecular Biology, Vol. 9, ed.
`45
`vided, including the formation of or introduction of
`Wickner, R. B., (Marcel Dekker, Inc., New York),
`"DNA Replication and Biosynthesis'; Mertz and Da
`cohesive termini. Flush ends can be joined. Alterna
`tively, the plasmid and gene may be cleaved in such a
`vis, 69, Proc. Nat. Acad. Sci., USA, 69, 3370 (1972);
`The cleavage and non-covalent joining of the plasmid
`manner that the two chains are cleaved at different sites
`vehicle and the foreign DNA can be readily carried out
`to leave extensions at each and which serve as cohesive
`50
`with a restriction endonuclease, with the plasmid vehi
`termini. Cohesive termini may also be introduced by
`removing nucleic acids from the opposite ends of the
`cle and foreign DNA in the same or different vessels.
`two chains or alternatively, introducing nucleic acids at
`Depending on the number of fragments, which are
`opposite ends of the two chains.
`obtained from the DNA endonuclease digestion, as well
`as the genetic properties of the various fragments, diges
`To illustrate, a plasmid can be cleaved with a restric
`55
`tion endonuclease or other DNA cleaving enzyme. The
`tion of the foreign DNA may be carried out separately
`and the fragments separated by centrifugation in an
`restriction enzyme can provide square ends, which are
`appropriate gradient. Where the desired DNA fragment
`then modified to provide cohesive termini or can cleave
`has a phenotypical property, which allows for the ready
`at different, but adjacent, sites on the two strands, so as
`to provide cohesive termini directly.
`isolation of its transformant, a separation step can usu
`Where square ends are formed such as, for example,
`ally be avoided.
`by HIN (Haemophilus influenzae RII) or pancreatic
`Endonuclease digestion will normally be carried out
`DNAse, one can ligate the square ends or alternatively
`at moderate temperatures, normally in the range of 10
`one can modify the square ends by chewing back, add
`to 40 C. in an appropriately buffered aqueous medium,
`usually at a pH of about 6.5 to 8.5. Weight percent of
`ing particular nucleic acids, or a combination of the
`65
`two. For example, one can employ appropriate transfer
`total DNA in the reaction mixture will generally be
`about 1 to 20 weight percent. Time for the reaction will
`ases to add a nucleic acid to the 5' and 3' ends of the
`DNA. Alternatively, one can chew back with an en
`vary, generally being from 0.1 to 2 hours. The amount
`
`35
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`5
`of endonuclease employed is normally in excess of that
`tion. In this situation, the two organisms will either be
`eukaryotic or prokaryotic. Those organisms which are
`required, normally being from about 1 to 5 units per 10
`able to exchange genetic information by mating are well
`ug of DNA.
`Where cleavage into a plurality of DNA fragments
`known. Thus, prior to this invention, plasmids having a
`replicon and one or more genes from two sources
`results, the course of the reaction can be readily fol
`lowed by electrophoresis. Once the digestion has gone
`which do not exchange genetic information would not
`to the desired degree, the endonuclease is inactivated by
`have existed in nature. This is true, even in the event of
`heating above about 60° C. for five minutes. The diges
`mutations, and induced combinations of genes from
`tion mixture may then be worked up by dialysis, gradi
`different strains of the same species. For the natural
`ent separation, or the like, or used directly.
`formation of plasmids formed from a replicon and genes
`from different microorganisms it is necessary that the
`The plasmid vehicle and foreign DNA fragments are
`microorganisms be capable of mating and exchanging
`then allowed to combine to form hydrogen bonds and
`recircularize. This process is referred to as annealing
`genetic information.
`and DNA ligation. An appropriate buffered medium is
`In the situation, where the replicon comes from a
`employed containing the DNA fragments, DNA ligase,
`eukaryotic or prokaryotic cell, and at least one gene
`and appropriate cofactors. The temperature employed
`comes from the other type of cell, this plasmid hereto
`initially for annealing will be about -5 to 15 C. When
`fore could not have existed in nature. Thus, the subject
`DNA segments hydrogen bond, the DNA ligase will be
`invention provides new plasmids which cannot natu
`rally occur and can be used for transformation of unicel
`able to introduce a covalent bond between the two
`segments. Where the two ends of each of the segments
`lular organisms to introduce genes from other unicellu
`lar organisms, where the replicon and gene could not
`are hydrogen bonded to one another, they may be li
`previously naturally coexist in a plasmid.
`gated to form a circularized recombinant plasmid. The
`Besides naturally occurring genes, it is feasible to
`mole ratio of the two segments will generally be in the
`provide synthetic genes, where fragments of DNA may
`range of 1-5:5-1. The particular temperature for anneal
`ing will depend upon the binding strength of the cohe
`be joined by various techniques known in the art. Thus,
`25
`the exogenous gene may be obtained from natural
`sive termini. While 0.5 to 2 or more days have been
`employed for annealing, it is believed that only a short
`sources or from synthetic sources.
`The plasmid chimera contains a replicon which is
`period of 0.5 to 6 hours may be sufficient, since anneal
`compatible with a bacterium susceptible of transforma
`ing and ligation can occur under ligating conditions.
`The time employed for the annealing will vary with the
`tion and at least one foreign gene which is directly or
`indirectly bonded through deoxynucleotides to the re
`temperature employed, the nature of the salt solution, as
`plicon to form the circularized plasmid structure. As
`well as the nature of the sticky ends or cohesive termini.
`indicated previously, the foreign gene normally pro
`The foreign DNA can be derived from a wide variety
`vides a phenotypical property, which is absent in the
`of sources. The DNA may be derived from eukaryotic
`or prokaryotic cells, viruses, and bacteriophage. The
`parent bacterium. The foreign gene may come from
`35
`fragments employed will generally have molecular
`another bacterial strain, species or family, or from a
`weights in the range of about 0.5 to 20x106d, usually in
`plant or animal cell. The original plasmid chimera will
`the range of 1 to 10x106d. The DNA fragment may
`have been formed by in vitro covalent bonding between
`the replicon and foreign gene. Once the originally
`include one or more genes or one or more operons.
`Desirably, if the plasmid vehicle does not have a
`formed plasmid chimera has been used to prepare trans
`40
`phenotypical property which allows for isolation of the
`formants, the plasmid chimera will be replicated by the
`transformants, the foreign DNA fragment should have
`bacterial cell and cloned in vitro by growing the bac
`such property.
`teria in an appropriate growth medium. The bacterial
`The covalent joining can be achieved in conventional
`cells may be lysed and the DNA isolated by conven
`ways employing a DNA ligase. Ligation is conveniently
`tional means or the bacteria continually reproduced and
`45
`carried out in an aqueous solution (pH, 6-8) at tempera
`allowed to express the genotypical property of the for
`eign DNA.
`tures in the range of 5 to 40 C. The concentration of
`the DNA will generally be from about 10 to 100 ug/ml.
`Once a bacterium has been transformed, it is no
`longer necessary to repeat the in vitro preparation of
`A sufficient amount of the DNA ligase or other ligating
`agent, e.g. T4 ligase, is employed to provide a conve
`the plasmid chimera or isolate the plasmid chimera from
`50
`nient rate of reaction, generally ranging from 5 to 50
`the transformant progeny. Bacterial cells can be repeat
`edly multiplied which will express the genotypical
`U/ml. Small amounts of a protein, e.g. albumin, may be
`property of the foreign gene.
`added at concentrations of 10 to 200 g/ml. The liga
`tion with DNA ligase is carried out in the presence of
`One method of distinguishing between a plasmid
`Mg+ + at about 1-10 mM.
`which originates in vivo from a plasmid chimera which
`55
`At the completion of the ligation, the solution may be
`originates in vitro is the formation of honoduplexes
`chilled and is ready for use in transformation.
`between an in vitro prepared plasmid chimera and the
`In accordance with the subject invention, plasmids
`plasmid formed in vivo. It will be an extremely rear
`may be prepared which have replicons and genes which
`event where a plasmid which originates in vivo will be
`could be present in bacteria as a result of normal mating
`the same as a plasmid chimera and will form homodu
`60
`of bacteria. However, the subject invention provides a
`plexes with plasmid chimeras. For a discussion of
`technique, whereby a replicon and gene can coexist in a
`homoduplexes, see Sharp, Cohen and Davidson, J. Mol.
`plasmid, which is capable of being introduced into a
`Biol., 75, 235 (1973), and Sharp, et al, ibid, 71, 471,
`unicellular organism, which could not exist in nature.
`(1972).
`The first type of plasmid which cannot exist in nature is
`The plasmid derived from molecular cloning need
`a plasmid which derives its replicon from one organism
`not homoduplex with the in vitro plasmid originally
`and the exogenous gene from another organism, where
`employed for transformation of the bacterium. The
`the two organisms do not exchange genetic informa
`bacterium may carry out modification processes, which
`
`65
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`which will not be transformed. Of the function of cells
`will not affect the portion of the replicon introduced
`which are transformed, some significant proportion, but
`which is necessary for replication nor the portion of the
`normally a minor proportion, will have been trans
`exogenous DNA which contains the gene providing the
`formed by the recombinant plasmid. Therefore, only a
`genotypical trait. Thus, nucleotides may be introduced
`very small fraction of the total number of cells which
`or excised and, in accordance with naturally occurring
`are present will have the desired phenotypical charac
`mating and transduction, additional genes may be intro
`duced. In addition, for one or more reasons, the plas
`teristics.
`In order to enhance the ability to separate the desired
`mids may be modified in vitro by techniques which are
`known in the art. However, the plasmids obtained by
`bacterial clones, the bacterial cells, which have beeen
`subjected to transformation, will first be grown in a
`molecular cloning will homoduplex as to those parts
`which relate to the original replicon and the exogenous
`solution medium, so as to amplify the absolute number
`of the desired cells. The bacterial cells may then be
`gene.
`harvested and streaked on an appropriate agar medium.
`Where the recombinant plasmid has a phenotype,
`which allows for ready separation of the transformed
`cells from the parent cells, this will aid in the ready
`separation of the two types of cells. As previously indi
`cated, where the genotype provides resistance to a
`growth inhibiting material, such as an antibiotic or
`heavy metal, the cells can be grown on an agar medium
`containing the growth inhibiting substance. Only avail
`able cells having the resistant genotype will survive. If
`the foreign gene does not provide a phenotypical prop
`erty, which allows for distinction between the cells
`transformed by the plasmid vehicle and the cells trans
`formed by the plasmid chimera, a further step is neces
`sary to isolate the replicated plasmid chimera from the
`replicated plasmid vehicle. The steps include lysing of
`the cells and isolation and separation of the DNA by
`conventional means or random selection of transformed
`bacteria and characterization of DNA from such trans
`formants to determine which cells contain molecular
`chimeras. This is accomplished by physically character
`izing the DNA by electrophoresis, gradient centrifuga
`tion or electron microscopy.
`Cells from various clones may be harvested and the
`plasmid DNA isolated from these transformants. The
`plasmid DNA may then be analyzed in a variety of
`ways. One way is to treat the plasmid with an appropri
`ate restriction enzyme and analyze the resulting frag
`ments for the presence of the foreign gene. Other tech
`niques have been indicated above.
`Once the recombinant plasmid has been replicated in
`a cell and isolated, the cells may be grown and multi
`plied and the recombinant plasmid employed for trans
`formation of the same or different bacterial strain.
`The subject process provides a technique for intro
`ducing into a bacterial strain a foreign capability which
`is genetically mediated. A wide variety of genes may be
`employed as the foreign genes from a wide variety of
`sources. Any intact gene may be employed which can
`be bonded to the plasmid vehicle. The source of the
`gene can be other bacterial cells, mammalian cells, plant
`cells, etc. The process is generally applicable to bacte
`rial cells capable of transformation. A plasmid must be
`available, which can be cleaved to provide a linear
`segment having ligatable termini, and an intact replica
`tor locus and system, preferably a system including a
`gene which provides a phenotypical property which
`allows for easy separation of the transformants. The
`linear segment may then be annealed with a linear seg
`ment of DNA having one or more genes and the result
`ing recombinant plasmid employed for transformation
`of the bacteria.
`By introducing one or more exogenous genes into a
`unicellular organism, the organism will be able to pro
`duce polypeptides and proteins ("poly(amino acids)')
`which the organism could not previously produce. In
`
`I. TRANSFORMATION
`After the recombinant plasmid or plasmid chimera
`has been prepared, it may then be used for the transfor
`mation of bacteria. It should be noted that the annealing
`and ligation process not only results in the formation of
`the recombinant plasmid, but also in the recirculariza
`tion of the plasmid vehicle. Therefore, a mixture is
`obtained of the original plasmid, the recombinant plas
`mid, and the foreign DNA. Only the original plasmid
`and the DNA chimera consisting of the plasmid vehicle
`and linked foreign DNA will normally be capable of
`replication. When the mixture is employed for transfor
`25
`mation of the bacteria, replication of both the plasmid
`vehicle genotype and the foreign genotype will occur
`with both genotypes being replicated in those cells
`having the recombinant plasmid.
`Various techniques exist for transformation of a bac
`30
`terial cell with plasmid DNA. A technique, which is
`particularly useful with Escherichia coli, is described in
`Cohen, et al., ibid, 69, 2110 (1972). The bacterial cells
`are grown in an appropriate medium to a predetermined
`optical density. For example, with E. coli strain C600,
`the optical density was 0.85 at 590 nm. The cells are
`concentrated by chilling, sedimentation and washing
`- with a dilute salt solution. After centrifugation, the cells
`are resuspended in a calcium chloride solution at re
`duced temperatures (approx. 5-15 C.), sedimented,
`40
`resuspended in a smaller volume of a calcium chloride
`solution and the cells combined with the DNA in an
`appropriately buffered calcium chloride solution and
`incubated at reduced temperatures. The concentration
`of Cat - will generally be about 0.01 to 0.1M. After a
`45
`sufficient incubation period, generally from about
`0.5-3.0 hours, the bacteria are subjected to a heat pulse
`generally in the range of 35 to 45° C. for a short period
`of time; namely from about 0.5 to 5 minutes. The trans
`formed cells are then chilled and may be transferred to
`50
`a growth medium, whereby the transformed cells hav
`ing the foreign genotype may be isolated.
`An alternative transformation technique may be
`found in Lederberg and Cohen, J. Bacteriol., 119, 1072
`(1974), whose disclosure is incorporated herein by ref.
`erence.
`
`35
`
`55
`
`III. REPLICATION AND TRANSCRIPTION OF
`THE PLASMID
`The bacterial cells, which are employed, will be of
`60
`such species as to allow replication of the plasmid vehi
`cle. A number of different bacteria which can be em
`ployed, have been indicated previously. Strains which
`lack indigenous modification and restriction enzymes
`are particularly desirable for the cloning of DNA de
`65
`rived from foreign sources.
`The transformation of the bacterial cells will result in
`a mixture of bacterial cells, the dominant proportion of
`
`Page 5
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`5
`
`10
`
`EXPERIMENTAL
`. In order to demonstrate the subject invention, the
`following experiments were carried out with a variety
`of foreign genes.
`(All temperatures not otherwise indicated are Centri
`grade. All percents not otherwise indicated are percents
`45
`by weight.)
`
`4,740,470
`10
`9
`some instances the poly(amino acids) will have utility in
`in 0.5 volume 10mM NaCl. Af