 |
Description  |
|
|
BACKGROUND OF THE INVENTION
1. Field of the Invention
Although transfer of plasmids among strains of E. coli and other
Enterobacteriaceae has long been accomplished by conjugation and/or
transduction, it has not been previously possible to selectively introduce
particular species of plasmid DNA into these bacterial hosts or other
microorganisms. Since microorganisms that have been transformed with
plasmid DNA contain autonomously replicating extrachromosomal DNA species
having the genetic and molecular characteristics of the parent plasmid,
transformation has enabled the selective cloning and amplification of
particular plasmid genes.
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, genes specifying
such metabolic or synthetic functions as nitrogen fixation,
photosynthesis, antibiotic production, hormone synthesis, protein
synthesis, e.g. enzymes or antibodies, or the like--functions which are
indigenous to other classes of organisms--by linking the foreign 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 (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 genetically transforming
microorganisms, particularly bacteria, to provide diverse genotypical
capability and producing recombinant plasmids. A plasmid or viral DNA is
modified to form a linear segment having ligatable 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, by producing
proteins which are the desired end product, or metabolities of enzymic
conversion, or be lysed and the desired nucleic acids or proteins
recovered.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
The process of this invention employs novel plasmids, 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 plasmmid 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 necessary 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 bacterium 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 Enterobacteriaceae, 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 plasmids employed will have molecular weights in the
range of about 1.times.10.sup.6 to 50.times.10.sup.6 d, more usually from
about 1 to 20.times.10.sup.6 d, and preferably, from about 1 to
10.times.10.sup.6 d. The desirable plasmid size 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 provides for
a reasonable probability of recircularization with the foreign gene(s) to
form the recombinant plasmid chimera. Desirably, a restriction enzyme
should be available, which will cleave the plasmid without inactivating
the replicator locus and system associated with the replicator locus.
Also, means must be provided for providing ligatable termini for the
plasmid, which are complementary to the termini of the foreign gene(s) to
allow fusion of the two DNA segments.
Another consideration for the recombinant plasmid is that it be compatible
with the bacterium to be transformed. Therefore, the original plasmid will
usually be derived from a member of the family to which the bacterium
belongs.
The original plasmid should desirably have a phenotypical property which
allows for the separation of transformant bacteria from parent bacteria.
Particularly useful is a gene, which provides for survival selection.
Survival selection can be achieved by providing resistance to a growth
inhibiting substance or providing a growth factor capability to a
bacterium deficient in such capability.
Conveniently, genes are available, which provide for antibiotic or heavy
metal resistance or polypeptide resistance, e.g. colicin. Therefore, by
growing the bacteria on a medium containing a bacteriostatic or
bacteriocidal substance, such as an antibiotic, only the transformants
having the antibiotic resistance will survive. Illustrative antibiotics
include tetracycline, streptomycin, sulfa drugs, such as sulfonamide,
kanamycin, neomycin, penicillin, chloramphenicol, or the like.
Growth factors include the synthesis of amino acids, the isomerization of
substrates to forms which can be metabolized or the like. By growing the
bacteria on a medium which lacks the appropriate growth factor, only the
bacteria which have been transformed and have the growth factor capability
will clone.
One plasmid of interest derived from E. coli is referred to as pSC101 and
is described in Cohen, et al., Proc. Nat. Acad. Sci., USA, 70, 1293
(1972), (referred to in that article as Tc6-5). Further description of
this particular plasmid and its use is found in the other articles
previously referred to.
The plasmid pSC101 has a molecular weight of about 5.8.times.10.sup.6 d and
provides tetracycline resistance.
Another plasmid of interest is colicinogenic factor EI (ColE1), which has a
molecular weight of 4.2.times.10.sup.6 d, and is also derived from E.
coli. The plasmid has a single EcoRI substrate site and provides immunity
to colicin E1.
In preparing the plasmid for joining with the exogenous gene, a wide
variety of techniques can be provided, including the formation of or
introduction of cohesive termini. Flush ends can be joined. Alternatively,
the plasmid and gene may be cleaved in such a manner that the two chains
are cleaved at different sites to leave extensions at each end which serve
as cohesive termini. Cohesive termini may also be introduced by removing
nucleic acids from the opposite ends of the two chains or alternatively,
introducing nucleic acids at opposite ends of the two chains.
To illustrate, a plasmid can be cleaved with a restriction endonuclease or
other DNA cleaving enzyme. The restriction enzyme can provide square ends,
which are then modified to provide cohesive termini or can cleave in a
staggered manner at different, but adjacent, sites on the two strands, so
as to provide cohesive termini directly.
Where square ends are formed such as, for example, by HIN (Haemophilus
influenzae RII) or pancreatic DNAse, one can ligate the square ends or
alternatively one can modify the square ends by chewing back, adding
particular nucleic acids, or a combination of the two. For example, one
can employ appropriate transferases to add a nucleic acid to the 5' and 3'
ends of the DNA. Alternatively, one can chew back with an enzyme, such as
a .lambda.-exonuclease, and it is found that there is a high probability
that cohesive termini will be achieved in this manner.
An alternative way to achieve a linear segment of the plasmid with cohesive
termini is to employ an endonuclease such as EcoRI. The endonuclease
cleaves the two strands at different adjacent sites providing cohesive
termini directly.
With flush ended molecules, a T.sub.4 ligase may be employed for linking
the termini. See, for example, Scaramella and Khorana, J. Mol. Biol. 72:
427-444 (1972) and Scaramella, DNAS 69: 3389 (1972), whose disclosure is
incorporated herein by reference.
Another way to provide ligatable termini is to leave employing DNAse and
Mn.sup.++ as reported by Lai and Nathans, J. Mol. Biol, 89: 179 (1975).
The plasmid, which has the replicator locus, and serves as the vehicle for
introduction of a foreign gene into the bacterial cell, will hereafter be
referred to as "the plasmid vehicle."
It is not necessary to use plasmid, but any molecule capable of replication
in bacteria can be employed. Therefore, instead of plasmid, viruses may be
employed, which will be treated in substantially the same manner as the
plasmid, to provide the ligatable termini for joining to the foreign gene.
If production of cohesive termini is by restriction endonuclease cleavage,
the DNA containing the foreign gene(s) to be bound to the plasmid vehicle
will be cleaved in the same manner as the plasmid vehicle. If the cohesive
termini are produced by a different technique, an analogous technique will
normally be employed with the foreign gene. (By foreign gene is intended a
gene derived from a source other than the transformant strain.) In this
way, the foreign gene(s) will have ligatable termini, so as to be able to
covalently bonded to the termini of the plasmid vehicle. One can carry out
the cleavage or digest of the plasmids together in the same medium or
separately, combine the plasmids and recircularize the plasmids to form
the plasmid chimera in the absence of active restriction enzyme capable of
cleaving the plasmids.
Descriptions of methods of cleavage with restriction enzymes may be found
in the following articles: Greene, et al., Methods in Molecular Biology,
Vol. 9, ed. Wickner, R. B., (Marcel Dekker, Inc., New York), "DNA
Replication and Biosynthesis"; Mertz and Davis, 69, Proc. Nat. Acad. Sci.,
USA, 69, 3370 (1972);
The cleavage and non-covalent joining of the plasmid vehicle and the
foreign DNA can be readily carried out with a restriction endonuclease,
with the plasmid vehicle and foreign DNA in the same or different vessels.
Depending on the number of fragments, which are obtained from the DNA
endonuclease digestion, as well as the genetic properties of the various
fragments, digestion of the foreign DNA may be carried out separately and
the fragments separated by centrifugation in an appropriate gradient.
Where the desired DNA fragment has a phenotypical property, which allows
for the ready isolation of its transformant, a separation step can usually
be avoided.
Endonuclease digestion will normally be carried out at moderate
temperatures, normally in the range of 10.degree. to 40.degree. C. in an
appropriately buffered aqueous medium, usually at a pH of about 6.5 to
8.5. Weight percent of total DNA in the reaction mixture will generally be
about 1 to 20 weight percent. Time for the reaction will vary, generaly
being from 0.1 to 2 hours. The amount of endonuclease employed is normally
in excess of that required, normally being from about 1 to 5 units per 10
.mu.g of DNA.
Where cleavage into a plurality of DNA fragments results, the course of the
reaction can be readily followed by electrophoresis. Once the digestion
has gone to the desired degree, the endonuclease is inactivated by heating
above about 60.degree. C. for five minutes. The digestion mixture may be
worked up by dialysis, gradient separation, or the like, or used directly.
After preparation of the two double stranded DNA sequences, the foreign
gene and vector are combined for annealing and/or ligation to provide for
a functional recombinant DNA structure. With plasmids, the annealing
involves the hydrogen bonding together of the cohesive ends of the vector
and the foreign gene to form a circular plasmid which has cleavage sites.
The cleavage sites are then normally ligated to form the complete closed
and circularized plasmid.
The annealing, and as appropriate, recircularization can be performed in
whole or in part in vitro or in vivo. Preferably, the annealing is
performed in vitro. The annealing requires an appropriate buffered medium
containing the DNA fragments. The temperature employed initially for
annealing will be about 40.degree. to 70.degree. C., followed by a period
at lower temperature, generaly from about 10.degree. to 30.degree. C. The
molar ratio of the two segments will generally be in the range of about
1-5:5-1. The particular temperature for annealing will depend upon the
binding strength of the cohesive termi. While 0.5 hr to 2 or more days may
be employed for annealing, it is believed that a period of 0.5 to 6 hrs
may be sufficient. The time employed for the annealing will vary with the
temperature employed, the nature of the salt solution, as well as the
nature of the cohesive termini.
The ligation, when in vitro, can be achieved in conventional ways employing
DNA ligase. Ligation is conveniently carried out in an aqueous solution
(pH 6-8) at temperatures in the range of about 5.degree. to 40.degree. C.
The concentration of the DNA will generally be from about 10 to 100 g/ml.
A sufficient amount of the DNA ligase or other ligating agent e.g. T.sub.4
ligase, is employed to provide a convenient rate of reaction, generally
ranging from about 5 to 50 U/ml. A small amount of a protein e.g. albumin,
may be added at concentrations of about 10 to 200 g/ml. The ligation with
DNA ligase is carried out in the presence of magnesium at about 1-10 mM.
At the completion of the annealing or ligation, the solution may be chilled
and is ready for use in transformation.
It is not necessary to ligate the recircularized plasmid prior to
transformation, since it is found that this function can be performed by
the bacterial host. However, in some situations ligation prior to
transformation may be desirable.
The foreign DNA can be derived from a wide variety of sources. The DNA may
be derived from eukaryotic or prokaryotic cells, viruses, and
bacteriophage. The fragments employed will generally have molecular
weights in the range of about 0.5 to 20.times.10.sup.6 d, usually in the
range of 1 to 10.times.10.sup.6 d. The DNA fragment may include one or
more genes or one or more operons.
Desirably, if the plasmid vehicle does not have a phenotypical property
which allows for isolation of the transformants, the foreign DNA fragment
should have such property. Also, an intact promoter and base sequences
coding for initiation and termination sites should be present for gene
expression.
In accordance with the subject invention, plasmids may be prepared which
have replicons and genes which could be present in bacteria as a result of
normal mating of bacteria. However, the subject invention provides a
technique, whereby a replicon and gene can coexist in a plasmid, which is
capable of being introduced into a unicellular organism, which could not
exist in nature. The first type of plasmid which cannot exist in nature is
a plasmid which derives its replicon from one organism and the exogenous
gene from another organism, where the two organisms do not exchange
genetic information. In this situation, the two organisms will either be
eukaryotic or prokaryotic. Those organisms which are able to exchange
genetic information by mating are well known. Thus, prior to this
invention, plasmids having a replicon and one or more genes from two
sources which do not exchange genetic information would not have existed
in nature. This is true, even in the event of mutations, and induced
combinations of genes from different strains of the same species. For the
natural formation of plasmids formed from a replicon and genes from
different microorganisms it is necessary that the microorganisms be
capable of mating and exchanging genetic information.
In the situation, where the replicon comes from a eukaryotic or prokaryotic
cell, and at least one gene comes from the other type of cell, this
plasmid heretofore could not have existed in nature. Thus, the subject
invention provides new plasmids which cannot naturally occur and can be
used for transformation of unicellular organisms to introduce genes from
other unicellular organisms, where the replicon and gene could not
previously naturally coexist in a plasmid.
Besides naturally occurring genes, it is feasible to provide synthetic
genes, where fragments of DNA may be joined by various techniques known in
the art. Thus, the exogenous gene may be obtained from natural sources or
from synthetic sources.
The plasmid chimera contains a replicon which is compatible with a
bacterium susceptible of transformation and at least one foreign gene
which is directly or indirectly bonded through deoxynucleotides to the
replicon to form the circularized plasmid structure. As indicated
previously, the foreign gene normally provides a phenotypical property,
which is absent in the parent bacterium. The foreign gene may come from
another bacterial strain, species or family, or from a plant or animal
cell. The original plasmid chimera will have been formed by in vitro
covalent bonding between the replicon and foreign gene. Once the
originally formed plasmid chimera has been used to prepare transformants,
the plasmid chimera will be replicated by the bacterial cell and cloned in
vivo by growing the bacteria in an appropriate growth medium. The
bacterial cells may be lysed and the DNA isolated by conventional means or
the bacteria continually reproduced and allowed to express the genotypical
property of the foreign DNA.
Once a bacterium has been transformed, it is no longer necessary to repeat
the in vitro preparation of the plasmid chimera or isolate the plasmid
chimera from the transformant progeny. Bacterial cells can be repeatedly
multiplied which will express the genotypical property of the foreign
gene.
One method of distinguishing between a plasmid which originates in vivo
from a plasmid chimera which originates in vitro is the formation of
homoduplexes between an in vitro prepared plasmid chimera and the plasmid
formed in vivo. It will be an extremely rare event where a plasmid which
originates in vivo will be the same as a plasmid chimera and will form
homoduplexes with plasmid chimeras. For a discussion of homoduplexes, see
Sharp, Cohen and Davidson, J. Mol. Biol., 75, 235 (1973), and Sharp, et
al, ibid, 71, 471 (1972).
The plasmid derived from molecular cloning need not homoduplex with the in
vitro plasmid originally employed for transformation of the bacterium. The
bacterium may carry out modification processes, which will not affect the
portion of the replicon introduced which is necessary for replication nor
the portion of the exogenous DNA which contains the gene providing the
genotypical trait. Thus, nucleotides may be introduced or excised and, in
accordance with naturally occurring mating and transduction, additional
genes may be introduced. In addition, for one or more reasons, the
plasmids may be modified in vitro by techniques which are known in the
art. However, the plasmids obtained by molecular cloning will homoduplex
as to those parts which relate to the original replicon and the exogenous
gene.
II. Transformation
After the recombinant plasmid or plasmid chimera has been prepared, it may
then be used for the transformation 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 recircularization of the plasmid
vehicle. Therefore, a mixture is obtained of the original plasmid, the
recombinant plasmid, 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
transformation 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 bacterial 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 reduced temperatures
(approx. 5.degree.-15.degree. 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 Ca.sup.++ 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.degree. to 45.degree. C. for a short period of time;
namely from about 0.5 to 5 minutes. The transformed cells are then chilled
and may be transferred to a growth medium, whereby the transformed cells
having the foreign genotype may be isolated.
An alternative transformation technique may be found in Lederberg and
Cohen, I. Bacteriol., 119, 1072 (1974), whose disclosure is incorporated
herein by reference.
III. Replication and Transcription of the Plasmid
The bacterial cells, which are employed, will be of such species as to
allow replication of the plasmid vehicle. A number of different bacteria
which can be employed, have been indicated previously. Strains which lack
indigenous modification and restriction enzymes are particularly desirable
for the cloning of DNA derived from foreign sources.
The transformation of the bacterial cells will result in a mixture of
bacterial cells, the dominant proportion of which will not be transformed.
Of the fraction of cells which are transformed, some significant
proportion, but normally a minor proportion, will have been transformed by
recombinant plasmid. Therefore, only a very small fraction of the total
number of cells which are present will have the desired phenotypical
characteristics.
In order to enhance the ability to separate the desired bacterial clones,
the bacterial cells, which have beeen subjected to transformation, will
first be grown in a solution medium, so as to amplify the absolute number
of the desired cells. The bacterial cells may then be 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 indicated, 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 available cells having the resistant genotype
will survive. If the foreign gene does not provide a phenotypical
property, which allows for distinction between the cells transformed by
the plasmid vehicle and the cells transformed by the plasmid chimera, a
further step is necessary 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
transformants to determine which cells contain molecular chimeras. This is
accomplished by physically characterizing the DNA by electrophoresis,
gradient centrifugation 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 appropriate
restriction enzyme and analyze the resulting fragments for the presence of
the foreign gene. Other techniques have been indicated above.
Once the recombinant plasmid has been replicated in a cell and isolated,
the cells may be grown and multiplied and the recombinant plasmid employed
for transformation of the same or different bacterial strain.
The subject process provides a technique for introducing 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
bacterial cells capable of transformation. A plasmid must be available,
which can be cleaved to provide a linear segment having ligatable termini,
and an interact replicator 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 segment of DNA having one or more genes and the resulting
recombinant plasmid employed for transformation of the bacteria.
By introducing one or more exogeneous genes into a unicellular organism,
the organism will be able to produce polypeptides and proteins
("poly(amino acids)") which the organism could not previously produce. In
some instances the poly(amino acids) will have utility in themselves,
while in other situations, particularly with enzymes, the enzymatic
product(s) will either be useful in itself or useful to produce a
desirable product.
One group of poly(amino acids) which are directly useful are hormones.
Illustrative hormones include parathyroid hormone, growth hormone,
gonadotropins (FSH, luteinizing hormone, chorionogonadatropin, and
glycoproteins), insulin, ACTH, somatostatin, prolactin, placental
lactogen, melanocyte stimulating hormone, thyrotropin, parathyroid
hormone, calcitonin, enkephalin, and angiotensin.
Other poly(amino acids) of interest include serum proteins, fibrinogin,
prothrombin, thromboplastin, globulin e.g. gamma-globulins or antibodies,
heparin, antihemophilia protein, oxytocin, albumins, actin, myosin,
hemoglobin, ferritin, cytochrome, myoglobin, lactoglobulin, histones,
avidin, thyroglobulin, interferin, kinins and transcortin.
Where the genes or genes produce one or more enzymes, the enzymes may be
used for fulfilling a wide variety of functions. Included in these
functions are nitrogen fixation, production of amino acids, e.g.
polyiodothyronine, particularly thyroxine, vitamins, both water and fat
soluble vitamins, antimicrobial drugs, chemotheropeutic agents e.g.
antitumor drugs, polypeptides and proteins e.g. enzymes from apoenzymes
and hormones from prohormones, diagnostic reagents, energy producing
combinations e.g. photosynthesis and hydrogen production, prostaglandins,
steroids, cardiac glycosides, coenzymes, and the like.
The enzymes may be individually useful as agents separate from the cell for
commercial applications, e.g. in detergents, synthetic transformations,
diagnostic agents and the like. Enzymes are classified by the I.U.B. under
the classifications as I. Oxidoreductases; II. Transferases; III.
Hydrolases; IV. Lyases; V. Isomerases; and VI. Ligases.
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 Centrigrade. All percents not
otherwise indicated are percents by weight.)
EXAMPLE A
A. Preparation of pSC101 Plasmid
Covalently closed R6-5 DNA was sheared with a Virtis stainless steel
microshaft in a one milliliter cup. The R6-5 DNA was sheared at 2,000
r.p.m. for 30 minutes in TEN buffer solution (0.02 M Tris-HCl (pH 8.0)-1
mM EDTA (pH 8.0)-0.02 M NaCl), while chilled at 0.degree.-4.degree..
The sheared DNA sample was subjected to sucrose gradient sedimentation at
39,500 r.p.m. in a Spinco SW 50.1 rotor at 20.degree.. A 0.12 mil fraction
was collected on a 2.3 cm diameter circle of Whatman No. 3 filter paper,
dried for 20 minutes and precipitated by immersion of the disc in cold 5%
trichloroacetic acid, containing 100 .mu.g/ml thymidine. The precipitate
was filtered and then washed once with 5% trichloroacetic acid, twice with
99% ethanol and dried. pSC101 was the 27S species having a calculated
molecular weight of 5.8.times.10.sup.6 d.
B. Generalized Transformation Procedure
E. coli strain C600 was grown at 37.degree. in H1 medium to an optical
density of 0.85 at 590 nm. At this point the cells were chilled quickly,
sedimented and washed once in 0.5 volume 10 nM NaCl. After centrifugation,
the bacteria was resuspended in half the original volume of chilled 0.03 M
calcium chloride, kept at 0.degree. for 20 minutes, sedimented, and then
resuspended in 0.1 of the original volume of 0.03 M of calcium chloride
solution. Chilled DNA samples in TEN buffer were supplemented with 0.1 M
calcium chloride to a final concentration of 0.03 M.
0.2 ml of competent cells treated with calcium chloride was added to 0.1 ml
of DNA solution with chilled pipets and an additional incubation was done
for 60 minutes at 0.degree.. The bacteria were then subjected to a heat
pulse at 42.degree. for two minutes, chilled, and then either placed
directly onto nutrient agar containing appropriate antibiotics or, where
indicated, diluted 10 times in L-broth and incubated at 37.degree. before
plating. The cell survival is greater than 50% after calcium chloride
treatment and heat pulse. Drug resistance was assayed on nutrient agar
plates with the antibiotics indicated in specific experiments.
EXAMPLE I: Construction of Biologically Functional Bacterial Plasmids in
vitro
A. Covalently closed R6-5 plasmid DNA was cleaved by incubation at
37.degree. for 15 minutes in a 0.2 ml reaction mixture containing DNA (40
.mu.g/ml, 100 mM Tris.HCl (pH 7.4)), 5 mM MgCl.sub.2, 50 mM NaCl, and
excess (2 U) EcoRI endonuclease in 1 .mu.l volume. An additional
incubation at 60.degree. for 5 minutes was employed to inactivate the
endonuclease.
The resulting mixture of plasmid fragments was employed for transformation
of E. coli strain C600 in accordance with the procedure previously
described. A single clone was examined further which was selected for
resistance to kanamycin and was also found to carry resistance to neomycin
and sulfonamide, but not to tetracycline, chloramphenicol, or streptomycin
after transformation of E. coli by EcoRI generated DNA fragments of R6-5.
Closed circular DNA obtakined from this isolate (plasmid designation
pSC102) by CsCl-ethidium bromide gradient centrifugation had an S value of
39.5 in neutral surcrose gradients.
Treatment of pSC102 plasmid DNA with EcoRI resistriction endonuclease in
accordance with the above-described procedure resulted in the formation of
3 fragments that were separable by electrophoresis in agarose gels. Intact
pSC102 plasmid DNA and pSC101 plasmid DNA, which had been separately
purified by dye-buoyant density centrifugation, were treated with EcoRI
endonuclease followed by annealing at 0.degree.-2.degree. for about six
hours. The mixture was then subjected to ligation with pSC101 and pSC102
in a ratio of 1:1 respectively, by ligating for 6 hours at 14.degree. in
0.2 ml reaction mixtures containing 5 mM MgCl.sub.2, 0.1 mM NAD, 100
.mu.g/ml of bovine-serum albumin (BSA), 10 mM ammonium sulphate (pH 7.0),
and 18 U/ml of DNA ligase. (J. Mertz and Davis, Proc. Nat. Acad. Sci.,
USA, 69, 3370 (1972); and Modrich, et al., J. Biol. Chem., 248, 7495
(1973). Ligated mixtures were incubated at 37.degree. for 5 minutes and
then chilled in ice water. Aliquots containing 3.3-6.5 .mu.g/ml of total
DNA were used directly for transformation.
Transformation of E. coli strain C600 was carried out as previously
described. For comparison purposes, transformation was also carried out
with a mixture of pSC101 and pSC102 plasmid DNA, which had been subjected
to EcoRI endonuclease, but not DNA ligase. The antibiotics used for
selection were tetracycline (10 .mu.g/ml) and kanamycin (25 .mu.g/ml). The
results are reported as transformants per microgram of DNA. The following
table indicates the results.
TABLE I
______________________________________
Transformation of E. coli C600 by a mixture
of pSC101 and pSC102 DNA
Transformation frequency for
antibiotic resistence markers
Treatment Tetracycline +
of DNA Tetracycline
Kanamycin kanamycin
______________________________________
None 2 .times. 10.sup.5
1 .times. 10.sup.5
2 .times. 10.sup.2
EcoRI 1 .times. 10.sup.4
1.1 .times. 10.sup.3
7 .times. 10.sup.1
EcoRI+
DNA ligase
1.2 .times. 10.sup.4
1.3 .times. 10.sup.3
5.7 .times. 10.sup.2
______________________________________
Kanamycin resistance in the R65 plasmid is a result of the presence of the
enzyme kanamycin monophosphotransferase. The enzyme can be isolated from
the bacteria by known procedures and employed in an assay for kanamycin in
accordance with the procedure described in Smith, et al., New England J.
Medicine, 286, 583 (1972).
In the preparation for the enzyme extracts, the E. coli are grown in
ML-broth and harvested in a late logarithm phase of growth. The cells are
osmotically shocked (see Nossal, et al., J. Biol. Chem. 241, 3055 (1966),
washed twice at room temperature with 10 ml 0.01 M Tris and 0.03 M NaCl,
pH 7.3, and the pellet suspended in 10 ml 20% sucrose, 3.times.10.sup.3 M
EDTA and 0.033 M Tris (pH 7.5), stirred for 10 minues at room temperature
and centrifuged at 16,000 g for 5 minutes. The pellet is then suspended in
2 ml of cold 5.times.10.sup.-4 M MCl.sub.2, stirred for 10 minutes at
2.degree. and centrifuged at 26,000 g for 10 minutes to yield a
supernatant fluid referred to as the osmotic shockate. The solution should
be stored at -20.degree. or lower. (See Benveneste, et al., FEBS Leters,
14 293 (1971).
The osmotic shockate may then be used in accordance with the procedure of
Smith, et al., supra.
EXAMPLE II: Genome Construction between Bacterial Species in vitro:
Replication and Expression of Staphylococcus Plasmid Genes in E. coli
S. aureus strain 8325 contains the plasmid pI258, which expresses
resistance to penicillin, erythromycin, cadmium and mercury. (Lindberg, et
al., J. Bacteriol., 115, 139 (1973)). Covalently closed circular pSC101
and pI258 plasmid DNA were separately cleaved by incubation at 37.degree.
for 15 minutes in 0.2 ml reaction mixtures by EcoRI endonuclease in
accordance with the procedure described previously. Aliquots of the two
cleaved species were mixed in a ratio of 3 .mu.g of pI258:1 .mu.g of
pSC101 and annealed at 2.degree.-4.degree. for 48 hours. Subsequent
ligation was carried out for six hours at 14.degree. as described
previously and aliquots containing 3.3-6.5 .mu.g/ml of total DNA were used
directly in the transformation as described previously.
Other transformations were carried out employing the two plasmids
independently and a mixture of the two plasmids. Selection of
transformants was carried out at antibiotic concentrations for
tetracycline (Tc, 25 .mu.g/ml) or pencillin (Pc, 25 OU/ml). The
transformation was carried out with E. coli strain C600
r.sub.K.sup.-m.sub.K.sup.-. The following table indicates the results.
TABLE III
______________________________________
Transformation of C600 r.sub.K.sup.- m.sub.K.sup.- by pSC101
and pI258 Plasmid DNA
Transformants/.mu.g DNA
DNA Tc Pc
______________________________________
PSC101 closed circular
1 .times. 10.sup.6
<3
pI258 closed circular
<3.6 <3.6
pSC101 + pI258 untreated
9.1 .times. 10.sup.5
<5
pSC101 + pI258 EcoRI-treated
4.7 .times. 10.sup.3
10
______________________________________
The above table demonstrates that bacteria can be formed which have both
tetracycline resistance and penicillin resistance. Thus, one can provide
the phenotypical property penicillin resistance in bacteria from DNA,
which is indigenous to another biological organism. One can thus use E.
coli for the production of the enzyme, which imparts penicillin resistance
to bacteria, and assay for pe | | |
