United States Patent (19)
`Cohen et al.
`
`54 PROCESS FOR PRODUCING
`BOLOGICALLY FUNCTIONAL
`MOLECULAR CHMERAS
`75 Inventors: Stanley N. Cohen, Portola Valley;
`Herbert W. Boyer, Mill Valley, both
`of Calif.
`
`73 Assignee:
`
`Board of Trustees of the Leland
`Stanford Jr. University, Stanford,
`Calif.
`21 Appl. No.: 1,021
`22 Filed:
`Jan. 4, 1979
`
`63
`
`Related U.S. Application Data
`Continuation-in-part of Ser. No. 959,288, Nov. 9, 1978,
`which is a continuation-in-part of Ser. No. 687,430,
`May 17, 1976, abandoned, which is a continuation-in
`part of Ser. No. 520,691, Nov. 4, 1974.
`51 Int. Cl. .............................................. C12P 21/00
`52 U.S. C. ...................................... 435/68; 435/172;
`435/231; 435/183; 435/317; 435/849; 435/820;
`435/91; 435/207; 260/112.5 S; 260/27R; 435/212
`58) Field of Search .............. 195/1, 28 N, 28 R, 112,
`195/78, 79; 435/68, 172, 231, 183
`References Cited
`U.S. PATENT DOCUMENTS
`5/1974 Chakrabarty ...................... 195/28 R
`OTHER PUBLICATIONS
`Morrow et al., Proc. Nat. Acad. Sci. USA, vol. 69, pp.
`3365-3369, Nov. 1972.
`Morrow et al., Proc. Nat. Acad. Sci. USA, vol. 71, pp.
`1743-1747, May 1974.
`Hershfield et al., Proc. Nat. Acad. Sci. USA, vol. 71,
`pp. 3455 et seq. (1974).
`Jackson et al., Proc. Nat. Acad. Sci. USA, vol. 69, pp.
`2904-2909, Oct. 1972.
`
`(56)
`
`3,813,316
`
`11)
`45)
`
`4,237,224
`Dec. 2, 1980
`
`Mertz et al., Proc. Nat. Acad. Sci. USA, vol. 69, pp.
`3370-3374, Nov. 1972.
`Cohen, et al., Proc. Nat. Acad. Sci. USA, vol. 70, pp.
`1293-1297, May 1973.
`Cohen et al., Proc. Nat. Acad. Sci. USA, vol. 70, pp.
`3240-3244, Nov. 1973.
`Chang et al., Proc. Nat. Acad. Sci., USA, vol. 71, pp.
`1030-1034, Apr. 1974.
`Ullrich et al., Science vol. 196, pp. 1313-1319, Jun.
`1977.
`Singer et al., Science vol. 181, p. 1114 (1973).
`Itakura et al., Science vol. 198, pp. 1056-1063 Dec.
`1977.
`Komaroffet al., Proc. Nat. Acad. Sci. USA, vol. 75, pp.
`3727-3731, Aug. 1978.
`Chemical and Engineering News, p. 4, May 30, 1977.
`Chemical and Engineering News, p. 6, Sep. 11, 1978.
`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 to which is inserted 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.
`14 Claims, No Drawings
`
`Page 1
`
`KASHIV EXHIBIT 1013
`IPR2019-00791
`
`

`

`4,237,224
`1
`PROCESS FOR PRODUCING BIOLOGICALLY
`FUNCTIONAL MOLECULAR CHIMERAs
`The invention was supported by generous grants of 5
`NIH, NSF and the American Cancer Society.
`CROSS-REFERENCE TO RELATED .
`
`. .
`
`APPLICATIONS
`
`-
`
`2
`DESCRIPTION OF THE SPECIFIC
`EMBODIMENTS
`The process of this invention employs novel plas
`mids, which are formed by inserting DNAhaving one
`or more intact genes into a plasmid in such a location as
`to permit retention of an intact replicator locus and
`system (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 property. The
`plasmimid chimera is used to transform a susceptible and
`competent microorganism under conditions where
`transformation occurs. The microorganism is then
`grown under conditions which allow for separation 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.
`Preparation of Plasmid Chimera
`In order to prepare the plasmid chimera, it is neces
`sary to have a DNA vector, such as a plasmid or phage,
`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. Of particular interest are
`those plasmids which have a phenotypical property,
`which allow for ready separation of transformants from
`the parent microorganism. The plasmid will be capable
`of replicating in a microorganism, particularly a bacte
`rium which is susceptible to transformation. Various
`unicellular microorganisms can be transformed, such as
`bacteria, fungii and algae. That is, those unicellular
`organisms which are capable of being grown in cultures
`of 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 Enterobacte
`riaceae, such as strains of Escherichia coli Salmonella;
`Bacillaceae, such as Bacillus subtilis Pneumococcus;
`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 1X 106 to 50x 106d, more usually from
`about 1 to 20x106d, and preferably, from about 1 to
`10x106d. The desirable plasmidsize is determined by a
`number of 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
`
`50
`
`genes.
`
`'' .
`
`.
`
`. .
`
`. .
`
`.
`
`This application is a continuatin-in-part of applicatin 10
`Ser. No. 959,288, filed Nov. 9, 1978, which is a continu
`ation of application Ser. No. 687,430 filed May 17, 1976,
`now abandoned, which was a continuation-in-part of
`application Ser. No. 520,691, filed Nov. 4, 1974, now
`abandoned.
`15
`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
`20
`plished by conjugation and/or transduction, it has not
`been previously possible to selectively introduce partic
`ular species of plasmid DNA into these bacterial hosts
`or other microorganisms. Since microorganisms that
`have been transformed with plasmid DNA contain au
`25
`tonomously replicating extrachromosomal DNA spe
`cies having the genetic and molecular characteristics of
`the parent plasmid, transformation has enabled the se
`lective cloning and amplification of particular plasmid
`30
`The ability of genes derived from totally different
`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,
`35
`genes specifying such metabolic or synthetic functions
`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.
`BRIEF DESCRIPTION OF THE PRIOR ART
`References which relate to the subject invention are
`Cohen, et al., Proc. Nat. Acad, Sci., USA, 69, 2110
`45
`(1972); ibid, 70, 1293 (1973); ibid, 70, 3240 (1973); ibid,
`71, 1030 (1974); Morrow, et al., Proc. Nat. Acad. Sci.,
`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
`55
`producing recombinant plasmids. A plasmid or viral
`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 bound together to form a "hybrid'
`plasmid molecule which is used to transform susceptible
`and compatible microorganisms. After transformation,
`the cells are grown and the transformants harvested.
`The newly functionalized microorganisms may then be
`employed to carry out their new function; for example,
`65
`by producing proteins which are the desired end prod
`uct, or metabolities of enzymic conversion, or be lysed
`and the desired nucleic acids or proteins recovered.
`
`Page 2
`
`

`

`4,237,224
`3
`4.
`complementary to the termini of the foreign gene(s) to
`DNA. Alternatively, one can chew back with an en
`allow fusion of the two DNA segments.
`zyme, such as a A-exonuclease, and it is found that there
`Another consideration for the recombinant plasmid is
`is a high probability that cohesive termini will be
`that it be compatible with the bacterium to be trans
`achieved in this manner.
`formed. Therefore, the original plasmid will usually be
`An alternative way to achieve a linear segment of the
`derived from a member of the family to which the bac
`plasmid with cohesive termini is to employ an endonu
`terium belongs.
`clease such as EcoRI. The endonuclease cleaves the
`The original plasmid should desirably have a pheno
`two strands at different adjacent sites providing cohe
`typical property which allows for the separation of
`sive termini directly.
`transformant bacteria from parent bacteria. Particularly
`With flush ended molecules, a T4 ligase may be em
`10
`useful is a gene, which provides for survival selection.
`ployed for linking the termini. See, for example, Scara
`Survival selection can be achieved by providing resis
`mella and Khorana, J. Mol. Biol. 72: 427-444 (1972) and
`tance to a growth inhibiting substance or providing a
`Scaramella, DNAS 69: 3389 (1972), whose disclosure is
`growth factor capability to a bacterium deficient in such
`incorporated herein by reference.
`capability.
`Another way to provide ligatable termini is to leave
`Conveniently, genes are available, which provide for
`employing DNAse and Mn as reported by Lai and
`antibiotic or heavy metal resistance or polypeptide re
`Nathans, J. Mol. Biol, 89: 179 (1975).
`sistance, e.g. colicin. Therefore, by growing the bac
`The plasmid, which has the replicator locus, and
`teria on a medium containing a bacteriostatic or bacteri
`serves as the vehicle for introduction of a foreign gene
`ocidal substance, such as an antibiotic, only the transfor
`into the bacterial cell, will hereafter be referred to as
`20
`mants having the antibiotic resistance will survive. Il
`"the plasmid vehicle."
`lustrative antibiotics include tetracycline, streptomycin,
`It is not necessary to use plasmid, but any molecule
`sulfa drugs, such as sulfonamide, kanamycin, neomycin,
`capable of replication in bacteria can be employed.
`penicillin, chloramphenicol, or the like.
`Therefore, instead of plasmid, viruses may be em
`Growth factors include the synthesis of amino acids,
`ployed, which will be treated in substantially the same
`25
`manner as the plasmid, to provide the ligatable termini
`the isomerization of substrates to forms which can be
`metabolized or the like. By growing the bacteria on a
`for joining to the foreign gene.
`medium which lacks the appropriate growth factor,
`If production of cohesive termini is by restriction
`only the bacteria which have been transformed and
`endonuclease cleavage, the DNA containing the for
`have the growth factor capability will clone.
`eign gene(s) to be bound to the plasmid vehicle will be
`One plasmid of interest derived from E. coli is re
`cleaved in the same manner as the plasmid vehicle. If
`ferred to as pSC101 and is described in Cohen, et al.,
`the cohesive termini are produced by a different tech
`nique, an analogous technique will normally be en
`Proc. Nat. Acad. Sci., USA, 70, 1293 (1972), (referred
`ployed with the foreign gene. (By foreign gene is in
`to in that article as Tc6-5). Further description of this
`particular plasmid and its use is found in the other arti
`tended a gene derived from a source other than the
`cles previously referred to.
`transformant strain.) In this way, the foreign gene(s)
`The plasmid pSC101 has a molecular weight of about
`will have ligatable termini, so as to be able to covalently
`5.8x106d and provides tetracycline resistance.
`bonded to the termini of the plasmid vehicle. One can
`Another plasmid of interest is colicinogenic factor EI
`carry out the cleavage or digest of the plasmids to
`(ColE1), which has a molecular weight of 4.2X 10%d,
`gether in the same medium or separately, combine the
`and is also derived from E. coli. The plasmid has a single
`plasmids and recircularize the plasmids to form the
`EcoRI substrate site and provides immunity to colicin
`plasmid chimera in the absence of active restriction
`enzyme capable of cleaving the plasmids.
`E1.
`In preparing the plasmid for joining with the exoge
`Descriptions of methods of cleavage with restriction
`nous gene, a wide variety of techniques can be pro
`enzymes may be found in the following articles:
`vided, including the formation of or introduction of
`Greene, et al., Methods in Molecular Biology, Vol. 9, ed.
`cohesive termini. Flush ends can be joined. Alterna
`Wickner, R. B., (Marcel Dekker, Inc., New York),
`"DNA Replication and Biosynthesis'; Mertz and Da
`tively, the plasmid and gene may be cleaved in such a
`manner that the two chains are cleaved at different sites
`vis, 69, Proc. Nat. Acad. Sci., USA, 69, 3370 (1972);
`The cleavage and non-covalent joining of the plasmid
`to leave extensions at each end which serve as cohesive
`termini. Cohesive termini may also be introduced by
`vehicle and the foreign DNA can be readily carried out
`removing nucleic acids from the opposite ends of the
`with a restriction endonuclease, with the plasmid vehi
`two chains or alternatively, introducing nucleic acids at
`cle and foreign DNA in the same or different vessels.
`opposite ends of the two chains.
`Depending on the number of fragments, which are
`To illustrate, a plasmid can be cleaved with a restric
`obtained from the DNA endonuclease digestion, as well
`55
`tion endonuclease or other DNA cleaving enzyme. The
`as the genetic properties of the various fragments, diges
`restriction enzyme can provide square ends, which are
`tion of the foreign DNA may be carried out separately
`and the fragments separated by centrifugation in an
`then modified to provide cohesive termini or can cleave
`in a staggered manner at different, but adjacent, sites on
`appropriate gradient. Where the desired DNA fragment
`the two strands, so as to provide cohesive termini di
`has a phenotypical property, which allows for the ready
`rectly.
`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,
`ing particular nucleic acids, or a combination of the
`usually at a pH of about 6.5 to 8.5. Weight percent of
`two. For example, one can employ appropriate transfer
`total DNA in the reaction mixture will generally be
`ases to add a nucleic acid to the 5' and 3' ends of the
`about 1 to 20 weight percent. Time for the reaction will
`
`35
`
`Page 3
`
`

`

`10
`
`4,237,224
`5
`6
`vary, generally being from 0.1 to 2 hours. The amount of
`such property. Also, an intact promoter and base se
`endonuclease employed is normally in excess of that
`quences coding for initiation and termination sites
`required, normally being from about 1 to 5 units per 10
`should be present for gene expression.
`ug of DNA.
`In accordance with the subject invention, plasmids
`Where cleavage into a plurality of DNA fragments
`may be prepared which have replicons and genes which
`results, the course of the reaction can be readily fol
`could be present in bacteria as a result of normal mating
`lowed by electrophoresis. Once the digestion has gone
`of bacteria. However, the subject invention provides a
`to the desired degree, the endonuclease is inactivated by
`technique, whereby a replicon and gene can coexist in a
`heating above about 60° C. for five minutes. The diges
`plasmid, which is capable of being introduced into a
`tion mixture may be worked up by dialysis, gradient
`unicellular organism, which could not exist in nature.
`separation, or the like, or used directly.
`The first type of plasmid which cannot exist in nature is
`After preparation of the two double stranded DNA
`a plasmid which derives its replicon from one organism
`sequences, the foreign gene and vector are combined
`and the exogenous gene from another organism, where
`for annealing and/or ligation to provide for a functional
`the two organisms do not exchange genetic informa
`recombinant DNA structure. With plasmids, the an
`15
`tion. In this situation, the two organisms will either be
`nealing involves the hydrogen bonding together of the
`eukaryotic or prokaryotic. Those organisms which are
`cohesive ends of the vector and the foreign gene to
`able to exchange genetic information by mating are well
`form a circular plasmid which has cleavage sites. The
`known. Thus, prior to this invention, plasmids having a
`cleavage sites are then normally ligated to form the
`replicon and one or more genes from two sources
`complete closed and circularized plasmid.
`20
`which do not exchange genetic information would not
`The annealing, and as appropriate, recircularization
`have existed in nature. This is true, even in the event of
`can be performed in whole or in part in vitro or in vivo.
`mutations, and induced combinations of genes from
`Preferably, the annealing is performed in vitro. The
`different strains of the same species. For the natural
`annealing requires an appropriate buffered medium
`formation of plasmids formed from a replicon and genes
`containing the DNA fragments. The temperature em
`25
`from different microorganisms it is necessary that the
`ployed initially for annealing will be about 40' to 70° C.,
`microorganisms be capable of mating and exchanging
`followed by a period at lower temperature, generally
`genetic information.
`from about 10 to 30° C. The molar ratio of the two
`In the situation, where the replicon comes from a
`segments will generally be in the range of about 1-5:-
`eukaryotic or prokaryotic cell, and at least one gene
`5-1. The particular temperature for annealing will de
`30
`comes from the other type of cell, this plasmid hereto
`pend upon the binding strength of the cohesive termi.
`fore could not have existed in nature. Thus, the subject
`While 0.5 hr to 2 or more days may be employed for
`invention provides new plasmids which cannot natu
`annealing, it is believed that a period of 0.5 to 6 hrs may
`rally occur and can be used for transformation of unicel
`be sufficient. The time employed for the annealing will
`lular organisms to introduce genes from other unicellu
`vary with the temperature employed, the nature of the
`35
`lar organisms, where the replicon and gene could not
`salt solution, as well as the nature of the cohesive ter
`previously naturally coexist in a plasmid.
`11.
`Besides naturally occurring genes, it is feasible to
`The ligation, when in vitro, can be achieved in con
`provide synthetic genes, where fragments of DNA may
`ventional ways employing DNA ligase. Ligation is
`be joined by various techniques known in the art. Thus,
`conveniently carried out in an aqueous solution (pH
`the exogenous gene may be obtained from natural
`6-8) at temperatures in the range of about 5' to 40° C.
`sources or from synthetic sources.
`The concentration of the DNA will generally be from
`The plasmid chimera contains a replicon which is
`about 10 to 100 g/ml. A sufficient amount of the DNA
`ligase or other ligating agent e.g. T4 ligase, is employed
`compatible with a bacterium susceptible of transforma
`tion and at least one foreign gene which is directly or
`to provide a convenient rate of reaction, generally rang
`45
`indirectly bonded through deoxynucleotides to the re
`ing from about 5 to 50 U/ml. A small amount of a pro
`plicon to form the circularized plasmid structure. As
`tein e.g. albumin, may be added at concentrations of
`indicated previously, the foreign gene normally pro
`about 10 to 200 g/ml. The ligation with DNA ligase is
`vides a phenotypical property, which is absent in the
`carried out in the presence of magnesium at about 1-10
`parent bacterium. The foreign gene may come from
`mM.
`50
`another bacterial strain, species or family, or from a
`At the completion of the annealing or ligation, the
`plant or animal cell. The original plasmid chimera will
`solution may be chilled and is ready for use in transfor
`have been formed by in vitro covalent bonding between
`mation.
`the replicon and foreign gene. Once the originally
`It is not necessary to ligate the recircularized plasmid.
`formed plasmid chimera has been used to prepare trans
`prior to transformation, since it is found that this func
`55
`formants, the plasmid chimera will be replicated by the
`tion can be performed by the bacterial host. However,
`bacterial cell and cloned in vivo by growing the bac
`in some situations ligation prior to transformation may
`teria in an appropriate growth medium. The bacterial
`be desirable.
`cells may be lysed and the DNA isolated by conven
`The foreign DNA can be derived from a wide variety
`tional means or the bacteria continually reproduced and
`of sources. The DNA may be derived from eukaryotic
`or prokaryotic cells, viruses, and bacteriophage. The
`allowed to express the genotypical property of the for
`fragments employed will generally have molecular
`eign DNA.
`weights in the range of about 0.5 to 20x106d, usually in
`Once a bacterium has been transformed, it is no
`the range of 1 to 10x 106d. The DNA fragment may
`longer necessary to repeat the in vitro preparation of
`the plasmid chimera or isolate the plasmid chimera from
`include one or more genes or one or more operons.
`65
`Desirably, if the plasmid vehicle does not have a
`the transformant progeny. Bacterial cells can be repeat
`edly multiplied which will express the genotypical
`phenotypical property which allows for isolation of the
`property of the foreign gene.
`transformants, the foreign DNA fragment should have
`
`Page 4
`
`

`

`4,237,224
`8
`7
`An alternative transformation technique may be
`One method of distinguishing between a plasmid
`found in Lederberg and Cohen, I. Bacteriol., 119, 1072
`which originates in vivo from a plasmid chimera which
`(1974), whose disclosure is incorporated herein by ref.
`originates in vitro is the formation of homoduplexes
`between an in vitro prepared plasmid chimera and the
`erence.
`plasmid formed in vivo. It will be an extremely rare
`III. Replication and Transcription of the Plasmid
`event where a plasmid which originates in vivo will be
`The bacterial cells, which are employed, will be of
`the same as a plasmid chimera and will form homodu
`such species as to allow replication of the plasmid vehi
`plexes with plasmid chimeras. For a discussion of
`cle. A number of different bacteria which can be em
`homoduplexes, see Sharp, Cohen and Davidson, J. Mol.
`ployed, have been indicated previously. Strains which
`10
`Biol, 75, 235 (1973), and Sharp, et al, ibid, 71, 471
`lack indigenous modification and restriction enzymes
`(1972).
`are particularly desirable for the cloning of DNA de
`The plasmid derived from molecular cloning need
`rived from foreign sources.
`not homoduplex with the in vitro plasmid originally
`The transformation of the bacterial cells will result in
`employed for transformation of the bacterium. The
`a mixture of bacterial cells, the dominant proportion of
`15
`bacterium may carry out modification processes, which
`which will not be transformed. Of the fraction 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 recombinant plasmid. Therefore, only a very
`genotypical trait. Thus, nucleotides may be introduced
`small fraction of the total number of cells which are
`20
`or excised and, in accordance with naturally occurring
`present will have the desired phenotypical characteris
`mating and transduction, additional genes may be intro
`tics.
`duced. In addition, for one or more reasons, the plas
`In order to enhance the ability to separate the desired
`mids may be modified in vitro by techniques which are
`bacterial clones, the bacterial cells, which have beeen
`known in the art. However, the plasmids obtained by
`subjected to transformation, will first be grown in a
`25
`molecular cloning will homoduplex as to those parts
`solution medium, so as to amplify the absolute number
`which relate to the original replicon and the exogenous
`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
`
`II. 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
`35
`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
`40
`replication. When the mixture is employed for transfor
`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.
`45
`Various techniques exist for transformation of a bac
`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
`55
`duced temperatures (approx. 5-15 C.), sedimented,
`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 0.1 M. After a
`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
`a growth medium, whereby the transformed cells hav
`ing the foreign genotype may be isolated.
`
`Page 5
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`4,237,224
`10
`cells, etc. The process is generally applicable to bacte
`mM EDTA (pH 8.0)-0.02 M NaCl), while chilled at
`rial cells capable of transformation. A plasmid must be
`O-4.
`available, which can be cleaved to provide a linear
`The sheared DNA sample was subjected to sucrose
`segment having ligatable termini, and an interact re
`gradient sedimentation at 39,500 r.p.m. in a Spinco SW
`plicator locus and system, preferably a system including
`50.1 rotor at 20. A 0.12 mil fraction was collected on a
`a gene which provides a phenotypical property which
`2.3 cm diameter circle of Whatman No. 3 filter paper,
`allows for easy separation of the transformants. The
`dried for 20 minutes and precipitated by immersion of
`linear segment may then be annealed with a linear seg
`the disc in cold 5% trichloroacetic acid, containing 100
`ment of DNA having one or more genes and the result
`ug/ml thymidine. The precipitate was filtered and then
`ing recombinant plasmid employed for transformation
`washed once with 5% trichloroacetic acid, twice with
`of the bacteria.
`99% ethanol and dried. pSC101 was the 27S species
`By introducing one or more exogeneous genes into a
`having a calculated molecular weight of 5.8x 106 d.
`unicellular organism, the organism will be able to pro
`duce polypeptides and proteins ("poly(amino acids)')
`B. Generalized Transformation Procedure
`which the organism could not previously produce. In
`E. coli strain C600 was grown at 37 in H1 medium to
`some instances the poly(amino acids) will have utility in
`an optical density of 0.85 at 590 nm. At this point the
`themselves, while in other situations, particularly with
`cells were chilled quickly, sedimented and washed once
`enzymes, the enzymatic product(s) will either be useful
`in 0.5 volume 10 nM. NaCl. After centrifugation, the
`in itself or useful to produce a desirable product.
`bacteria was resuspended in half the original volume of
`One group of poly(amino acids