Note: Descriptions are shown in the official language in which they were submitted.
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RAPID GROWING MICROORGANISMS
FOR BIOTECHNOLOGY APPLICATIONS
BACKGROUND OF THE INVENTION
Field of the Invention
The present application relates to the field of biotechnology and, in
particular, to the fields of cloning and protein expression.
Related Art
The fundamental process that sustains the ongoing biotechnology
revolution is the cloning of DNA molecules for their further analysis or use.
Cloning of DNA molecules has been practiced in the art for many years. A
typical cloning protocol will involve identifying a desired DNA molecule,
preparing a population of recombinant vectors by ligating the DNA molecule
with a vector in a mixture of DNA molecule, vector and an appropriate ligase
enzyme, transforming the population of recombinant vectors into a competent
microorganism, growing the microorganism for some period of time sufficient
to permit the formation of colonies, selecting colonies of microorganisms that
potentially contain the desired DNA molecule correctly ligated in the vector,
growing a sufficient quantity of each selected colony from which to isolate
the
recombinant vector, analyzing the isolated vector to ensure that the vector
contains the desired DNA molecule and then growing a sufficient quantity of
the microorganism that contains the correct recombinant vector to perform
whatever subsequent manipulations are required. For details of various
cloning procedures the reader may consult Sambrook, et al. 1989, Molecular
Cloning: A Laboratory Manual 2°d Ed. Cold Spring Harbor Laboratory
Press,
Cold Spring Harbor, NY, specifically incorporated herein by reference.
The typical cloning protocol outlined above thus includes at least three
steps that involve growing of a microorganism. Since these growing steps
generally require 12-16 hours and are usually performed as overnight
incubations, the rate limiting steps for experiments involving cloning of a
DNA fragment are the steps requiring growth of a microorganism. Although
there are many variations on the basic practice of cloning, virtually all
cloning
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methods require the insertion of the DNA molecule of interest into a
microorganism and growth of the microorganism and, therefore, the speed of
virtually every cloning methodology is limited by the rate of growth of the
microorganism used for cloning.
For most cloning applications, the microorganism of choice is
Escherichia coli (E. coli). Although numerous strains of E. coli are known,
most cloning applications use one or another derivative of E. coli K-12. These
derivatives suffer from the slow growth rate discussed above. Other known
strains of E. coli, such as E. coli W (ATCC9637), have a rapid growth rate
when compared to E. coli K-12; however, wild type strains of E. coli W and
other rapid growing strains are not suitable for biotechnology applications
for
several reasons. First, the genetics of the organism have not been determined
to the level of detail required by cloning applications. Thus, those skilled
in
the art would not know whether the genome of a microorganism contained the
appropriate modifications of a number of genes that would make the
microorganism suitable for biotechnology applications. For example,
microorganisms are generally recA+ which leads to the formation plasmid
multimers and makes the microorganism less suitable for applications that
involve the isolation of plasmid. Microorganisms typically contain numerous
protease genes and may degrade overexpressed proteins thereby decreasing
the yield of a desired protein product. Microorganisms typically contain a lac
operon that does not permit alpha complementation and, therefore, the
identification of recombinant vectors is more difficult. Further, many
microorganisms contain endogenous plasmids that complicate the plasmid
isolation steps necessary for cloning applications. Finally, microorganisms
might contain genes coding for nucleases that could cause the degradation of
exogenous plasmids.
For a large number of biotechnology applications, a crucial step in the
development of the application involves cloning one or more fragments of
DNA. Given the central role of cloning in the development of the
biotechnology industry, there has long existed in the art a need for reagents
that speed the process of cloning. In particular, there exists a need in the
art
for microorganisms that have a desirable genotype and a rapid growth rate and
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can be employed to speed the cloning process. The present invention meets
this long felt need.
BRIEF SUMMARY OF THE INVENTION
The present invention provides microorganisms for biotechnology
applications characterized by a rapid growth rate as compared to the
microorganisms currently used for these applications. In particular, the
present invention provides a rapid growing microorganism that preferably
lacks endogenous plasmids and is, therefore, suitable for cloning
applications.
Because the microorganisms of the present invention form colonies faster than
the microorganisms currently in use in cloning applications, the present
invention provides an improvement in cloning desired nucleic acid molecules,
allowing more rapid identification and isolation of recombinant vectors and
clones of interest.
The present invention thus provides a method of cloning that employs
a rapid growing microorganism. The method entails constructing a population
of recombinant vectors, transforming competent microorganisms capable of
rapid growth with the population of recombinant vectors, selecting a
transformed microorganism containing one or more recombinant vectors of
interest and/or isolating one or more recombinant vectors of interest from the
transformed microorganism. In one embodiment, the rapid growing
microorganism is of the genus Escherichia. In another embodiment, the rapid
growing microorganism is an E. coli. In a further embodiment, the rapid
growing microorganism is an E. coli strain W. In a preferred embodiment, the
rapid growing microorganism is an E. coli strain W lacking endogenous
plasmids. In other preferred embodiments, the rapid growing microorganism
is selected from a group consisting of BRL3781, BRL3784 and recA~
derivatives thereof. The cloning methods of the present invention may
optionally include a step of growing transformed microorganism at an elevated
temperature to increase the growth rate of the microorganism, for example, at
a temperature greater than 37°C. In a preferred embodiment, the
transformed
microorganisms may be grown at about 42°C.
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The present invention provides a method of producing a protein or
peptide which comprises constructing a recombinant vector containing a gene
encoding a protein or peptide of interest, transforming the vector into a
competent microorganism capable of rapid growth and culturing the
transformed microorganism under conditions that cause the transformed
microorganism to produce said peptide or protein. In a preferred embodiment,
the rapid growing microorganism is of the genus Escherichia. In another
preferred embodiment, the rapid growing microorganism is an E. coli. In
another preferred embodiment, the rapid growing microorganism is an E. coli
strain W. Other embodiments include a microorganism deleted in the lon
protease. In some preferred embodiments, the microorganism cart-ies a gene
encoding a T7 RNA polymerase (RNAP). In other preferred embodiments,
the T7 RNAP gene is under the control of a salt inducible promoter. In
another preferred embodiment, the rapid growing microorganism does not
contain endogenous plasmids.
The present invention also includes a method of producing a
microorganism for cloning comprising the steps of obtaining a rapid growing
microorganism containing endogenous plasmids and curing the
microorganism of endogenous plasmids. In a preferred embodiment, the rapid
growing microorganism is of the genus Escherichia. In another preferred
embodiment, the rapid growing microorganism is an E. coli. In another
preferred embodiment, the rapid growing microorganism is an E. coli strain
W. In a related aspect of the present invention, any desired modification or
mutation may be made in the microorganisms of the present invention
including, but not limited to, alteration of the genotype of the microorganism
to a recA- genotype such as recAllrecAl3 or recA deletions, a LacZ- genotype
that allows alpha complementation such as lacX74 lacZOIVI I S or other lacZ
deletion, a protease deficient genotype such as Olon and/or ompT-, an
endonuclease minus genotype such as endAl, a genotype suitable for M13
phage infection by including the F' episome, a restriction negative,
modification positive genotype such as hsdRl7(rK-, mK+), a restriction
negative, modification negative genotype such as hsdS20(rB-, mg-), a
methylase deficient genotype such as mcrA and/or mcrB and/or mrr, a
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genotype suitable for taking up large plasmids such as deoR, a genotype
containing suppressor mutations such as supE and/or supF. Other suitable
modifications are known to those skilled in the art and such modifications are
considered to be within the scope of the present invention.
5 The present invention provides a method of transforming a competent
microorganism capable of rapid growth including the steps of obtaining a
recombinant vector and contacting a competent microorganism of the present
invention with the recombinant vector under conditions which cause the rapid
growing microorganism to be take up the recombinant vector. In a preferred
embodiment, the rapid growing microorganism is of the genus Escherichia. In
another preferred embodiment, the rapid growing microorganism is an E. coli.
In another preferred embodiment, the rapid growing microorganism is an E.
coli strain W. In another preferred embodiment, the rapid growing
microorganism is an E. coLi strain W lacking endogenous vectors. The
methods of the present invention may optionally include the step of growing
the transformed microorganism at elevated temperatures to increase the
growth rate of the microorganism, for example, at a temperature greater than
37°C. In a preferred embodiment, the transformed microorganisms may be
grown at about 42°C.
The present invention also includes kits comprising a carrier or
receptacle being compartmentalized to receive and hold therein at least one
container, wherein the container contains rapid growing microorganisms. The
kit optionally further comprises vectors suitable for cloning. In a preferred
embodiment, the kits may contain a vector suitable for recombinational
cloning. In a preferred embodiment, the rapid growing microorganisms may
be competent. In some preferred embodiments, the rapid growing
microorganisms may be chemically competent. In other preferred
embodiments, the rapid growing microorganisms may be electrocompetent. In
some preferred embodiments, the kits of the present invention may include
enzyme including, but not limited to, restriction enzymes, ligases, and/or
polymerases. In other preferred embodiments, the kits of the present invention
may include recombination proteins for recombinational cloning. The kits of
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the present invention may also comprise instructions or protocols for carrying
out the methods of the present invention.
The present invention includes compositions comprising rapid growing
microorganisms. In a preferred embodiment, the rapid growing
microorganism may be a competent microorganism. In some preferred
embodiments, the rapid growing microorganisms may be chemically
competent. In other preferred embodiments, the rapid growing
microorganisms may be electrocompetent. The compositions of the present
invention may optionally comprise at least one component selected from
IO buffers or buffering salts, one or more DNA fragments, one or more vectors,
one or more recombinant vectors, one or more recombination proteins and one
or more ligases. In a preferred embodiment, the compositions of the present
invention may comprise a rapid growing microorganism in a glycerol solution.
In other preferred embodiments, compositions of the present invention may
comprise rapid growing microorganisms in a buffer. In preferred
embodiments, the microorganisms of the present invention may be in a
competence buffer. In other preferred embodiments, the compositions of the
present invention may comprise a lyophilized rapid growing microorganism.
The present invention includes a method of making competent rapid
growing microorganisms comprising the steps of obtaining a rapid growing
microorganism, growing the rapid growing microorganism and treating the
rapid growing microorganism to make it competent. In some embodiments of
the present invention, treating the microorganisms may include the step of
contacting the microorganisms with a solution containing calcium chloride. In
other embodiments, treating may include the step of contacting the
microorganisms with water. Embodiments of the invention may include the
step of curing the rapid growing microorganism of endogenous plasmids. In a
preferred embodiment, the rapid growing microorganism is of the genus
Escherichia. In another preferred embodiment, the rapid growing
microorganism is an E. coli. In another preferred embodiment, the rapid
growing microorganism is an E. coli strain W.
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BRIEF DESCRIPTION OF THE DRAWING
Figure 1 is a restriction map of the 5.5 kb plasmid of ATCC9637.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
In the description that follows, a number of terms used in recombinant
DNA technology are utilized extensively. In order to provide a clear and more
consistent understanding of the specification and claims, including the scope
to be given such terms, the following definitions are provided.
Competent cells or competent microorganisms as used herein refers
to cells or microorganisms having the ability to take up and establish
IS exogenous DNA molecules. Competent cells include, but are not limited to,
cells made competent by chemical means, i.e. chemically competent cells, as
well as cells made competent for electroporation by suspension in a low ionic
strength buffer, i.e. electrocompetent cells.
Expression vector as used herein refers to a vector which is capable of
enhancing the expression of a gene or portion of a gene which has been cloned
into it, after transformation or transfection into a host cell. The cloned
gene is
usually placed under the control (i.e., operably linked to) certain control
sequences such as promoter sequences. Such promoters include but are not
limited to phage lambda PL promoter, and the E. coli lac, trp and tac
promoters, the T7 promoter and the baculovirus polyhedron promoter. Other
suitable promoters will be known to the skilled artisan.
Gene as used herein refers to a sequence of nucleotides that is
transcribed in a cell. The term includes sequences that code for proteins
and/or peptides as well as other sequences that do not code for such proteins
or
peptides. Examples of genes that do not code for proteins include, but are not
limited to, the genes for tRNA, rRNA and the like. A gene includes a promoter
sequence to control the transcription of the gene. A gene may also contain
other DNA sequence elements that regulate the amount or timing of
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transcription. Such sequences elements are seen to include, but are not
limited
to, enhancers and the like.
Cell or microorganism as used herein, and which terms may be used
interchangeably with each other and with the terms "host cell" and "host cell
strain," includes microorganisms that can be genetically engineered. Both
gram negative and gram positive prokaryotic cells may be used in accordance
with the present invention. Typical prokaryotic host cells that may be used in
accordance with the present invention include, but are not limited to,
microorganisms such as those of the genus Escherichia sp. (particularly E.
coli), Klebsiella sp., Streptomyces sp., Streptocococcus sp., Shigella sp.,
Staphylococcus sp., Erwinia sp., Klebsiella sp., Bacillus sp. (particularly B.
cereus, B. subtilis, and B. megaterium), Serratia sp., Pseudomonas sp.
(particularly P. aeruginosa and P. syringae) and Salmonella sp. (particularly
S. typhi or S. typhimurium). It will be understood, of course, that there are
many suitable strains and serotypes of each of the host cell species described
herein, any and all of which may be used in accordance with the invention.
Preferred as a host cell is E. coli, and particularly preferred are E. coli
strains
derived from E. coli W.
As used herein, a "derivative" of a specified microorganism is a
progeny or other recipient microorganism that contains genetic material
obtained directly or indirectly from the specified microorganism. Such a
derivative microorganism may, for example, be formed by removing genetic
material from a specified microorganism and subsequently introducing it into
another microorganism (i.e., the progeny or other recipient microorganism) by
any conventional methodology including, but not limited to, transformation,
conjugation, electroporation, transduction and the like. A derivative may be
formed by introducing one or more mutations into the genome of a
microorganism. The mutations may be insertions into the genome of the
microorganism. Alternatively, the mutations may be deletions of one or more
bases from the genome of the microorganism. In some instances, the
mutations may be the alteration of one or more bases in the genome of the
microorganism. In addition, one microorganism is a derivative of a parent
microorganism if it contains the genome of the parent microorganism but does
not contain the same extrachromosomal nucleic acid molecules. Thus, a strain
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produced by curing endogenous vectors from a parent strain is a derivative of
the parent strain. Examples of mutations or other genetic alterations which
may be incorporated into the microorganisms of the present invention include,
but are not limited to, mutations or alterations that create: a recA- genotype
such as recAllrecAl3 or recA deletions, a lacZ- genotype that allows alpha
complementation such as lacX74 lacZO1VI15 or other lacZ deletion, a protease
deficient genotype such as Olon and/or ompT-, an endonuclease minus
genotype such as endAl, a genotype suitable for M13 phage infection by
including the F' episome, a restriction negative, modification positive
genotype such as hsdRl7(rK , mK+), a restriction negative, modification
negative genotype such as hsdS20(rB-, ms-), a methylase deficient genotype
such as mcrA and/or mcrB and/or mrr, a genotype suitable for taking up large
plasmids such as deoR, a genotype containing suppressor mutations such as
supE and/or supF. Other suitable modifications are known to those skilled in
the art and such modifications are considered to be within the scope of the
present invention.
Insert or inserts as used herein refers to one or more desired nucleic
acid segments.
Isolating as used herein means separating the desired material,
component, or composition at least partially from other materials,
contaminants, and the like which are not part of the material, component, or
composition that has been isolated. For example, "isolating a recombinant
vector" means treating a cell, tissue, organ or organism containing the
recombinant vector in such a way as to remove at least some of the other
nucleic acid molecules (e.g., large nucleic acid molecules) with which it may
be associated in the cell, tissue, organ or organism. As one of ordinary skill
will appreciate, however, a solution comprising an isolated recombinant vector
may comprise one or more buffer salts and/or a solvents, e.g., water or an
organic solvent such as acetone, ethanol, methanol, and the like, and yet the
nucleic acid molecule may still be considered an "isolated" nucleic acid
molecule with respect to its starting materials.
Plasmid as used herein refers to a stable extrachromosomal genetic
element.
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Promoter as used herein refers to a DNA sequence that controls the
transcription from another DNA sequence. A promoter is generally described
as the 5'-region of a gene and is customarily located proximal to the start
codon. The transcription of an adjacent DNA segment is initiated at the
5 promoter region. A repressible promoter's rate of transcription decreases in
response to a repressing agent. An inducible promoter's rate of transcription
increases in response to an inducing agent. A constitutive promoter's rate of
transcription is not specifically regulated, though it can vary under the
influence of general metabolic conditions.
10 Rapid growing microorganism as used herein refers to a
microorganism that grows more rapidly than E. coli K-12 derived strains
typically used in molecular biology applications. Rapid growing
microorganisms produce colonies of a defined size from individual cells faster
than non-rapid growing microorganisms. In general, a rapid growing
microorganism will have an increased growth rate, such as a growth rate that
is greater by 5%, 10%, 25%, 50%, 75%, 100%, 150%, 200% than the growth
rate of a reference microorganism. Greater increases in growth rate may be
included depending upon the microorganisms compared. A preferred
reference strain is a strain such as E. coli MM294 (ATCC33625). Other
suitable reference strains include DHSa and DH10B (Life Technologies,
Rockville, MD) and any other strain routinely used in cloning applications.
The invention also contemplates any microorganism which has an increased
growth rate, such as a growth rate that is greater by 5%, 10%, 25%, 50%,
75%, 100%, 150%, 200%, when compared to E. coli W more particularly the
specified E. coli W strains described herein. In the examples set forth below,
rapid growing microorganisms of the present invention were identified by a
comparison of the time necessary to grow a colony of 1 mm diameter on
antibiotic containing LB plates after transformation with a plasmid conferring
resistance to the antibiotic.
Rapid growing microorganisms of the present invention may also be
identified by a comparison of the doubling time of a putative rapid growing
microorganism to the doubling time of a reference strain. The rapid growing
microorganisms of the present invention have a faster doubling time than
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known strains. Those skilled in the art a capable of determining the doubling
time of a microorganism using standard techniques.
In determining whether a microorganism is a rapid growing
microorganism, the growth rate is preferably compared to a reference strain
having the same or similar genotype. For example, a putative rapid growing
microorganism that is recA- should be compared to a recA- reference strain.
Those skilled in the art will appreciate that a recA- microorganism may have a
slower growth rate than a comparable recA+ microorganism.
Recombinant microorganism as used herein refers to any
microorganism which contains a desired cloned gene in a recombinant vector,
cloning vector or any DNA molecule. The term "recombinant microorganism"
is also meant to include those host cells which have been genetically
engineered to contain the desired gene on the host chromosome or genome.
Recombinant vector as used herein includes any vector containing a
fragment of DNA that is not endogenous to the vector.
Vector as used herein refers to a nucleic acid molecule (preferably
DNA) that provides a useful biological or biochemical property to an Insert.
Examples include plasmids, phages, viruses, autonomously replicating
sequences (ARS), centromeres, transposons, and other sequences which are
able to replicate or be replicated in vitro or in a host cell, or to convey a
desired nucleic acid segment to a desired location within a host cell. A
vector
can have one or more restriction endonuclease recognition sites at which the
sequences can be cut in a determinable fashion without loss of an essential
biological function of the vector, and into which a nucleic acid fragment can
be spliced in order to bring about its replication and cloning. Vectors can
further provide primer sites, e.g., for PCR, transcriptional and/or
translational
initiation and/or regulation sites, recombinational signals, replicons,
selectable
markers, etc. Clearly, methods of inserting a desired nucleic acid fragment
which do not require the use of homologous recombination, transpositions or
restriction enzymes (such as, but not limited to, UDG cloning of PCR
fragments (U.S. Patent No. 5,334,575, entirely incorporated herein by
reference), T:A cloning, and the like) can also be applied to clone a fragment
into a cloning vector to be used according to the present invention. The
cloning vector can further contain one or more selectable markers suitable for
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use in the identification of cells transformed with the cloning vector.
The present invention may be used with vectors suitable for
recombinational cloning as disclosed in United States Patent No. 5, 888,732
which is specifically incorporated herein by reference. Vectors for this
purpose may comprise one or more engineered recombination sites. Vectors
suitable for recombinational cloning may be linear or circular. When linear, a
vector may include DNA segments separated by at least one recombination
site. When circular, a vector may include DNA segments separated by at least
two recombination sites. In one embodiment, a vector may comprise a first
IO DNA segment and a second DNA segment wherein the first or the second may
comprise a selectable marker. In other embodiments, a vector may comprise a
first DNA segment and a second DNA segment, the first or the second
segment comprising a toxic gene. In other embodiments, a vector may
comprise a first DNA segment and a second DNA segment, the first or the
second DNA segment comprising an inactive fragment of at least one
selectable marker, wherein the fragment of the selectable marker is capable of
reconstituting a functional selectable marker when recombined across the first
or second recombination site with another inactive fragment of a selectable
marker.
In accordance with the invention, any vector may be used. In
particular, vectors known in the art and those commercially available (and
variants or derivatives thereof) may be used in accordance with the invention.
Such vectors may be obtained from, for example, Vector Laboratories Inc.,
InVitrogen, Promega, Novagen, NEB, Clontech, Boehringer Mannheim,
Pharmacia, Epicenter, OriGenes Technologies Inc., Stratagene, Perkin Elmer,
Pharmingen, Life Technologies, Inc., and Research Genetics. Such vectors
may be used for cloning or subcloning nucleic acid molecules of interest and
therefore recombinant vectors containing inserts, nucleic acid fragments or
genes may also be used in accordance with the invention. General classes of
vectors of particular interest include prokaryotic and/or eukaryotic cloning
vectors, expression vectors, fusion vectors, two-hybrid or reverse two-hybrid
vectors, shuttle vectors for use in different hosts, mutagenesis vectors,
recombinational cloning transcription vectors, vectors for receiving large
inserts (yeast artificial chromosomes (YAC's), bacterial artificial
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chromosomes (BAC's) and P1 artificial chromosomes (PAC's)) and the like.
Other vectors of interest include viral origin vectors (M13 vectors, bacterial
phage ~, vectors, baculovirus vectors, adenovirus vectors, and retrovirus
vectors), high, low and adjustable copy number vectors, vectors which have
compatible replicons for use in combination in a single host (e.g., pACYC184
and pBR322) and eukaryotic episomal replication vectors (e.g., pCDMB). The
vectors contemplated by the invention include vectors containing inserted or
additional nucleic acid fragments or sequences (e.g., recombinant vectors) as
well as derivatives or variants of any of the vectors described herein.
Expression vectors useful in accordance with the present invention
include chromosomal, episomal and virus derived vectors, e.g., vectors
derived from bacterial plasmids or bacteriophages, and vectors derived from
combinations thereof, such as cosmids and phagemids, and will preferably
include at least one selectable marker (such as a tetracycline or ampicillin
resistance genes) and one or more promoters such as the phage lambda P,_,
promoter, and/or the E. coli lac, trp and tac promoters, the T7 promoter and
the baculovirus polyhedron promoter. Other suitable promoters will be known
to the skilled artisan.
Among vectors preferred for use in the present invention include
pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript
vectors, Bluescript vectors, pNHBA, pNHl6a, pNHl8A, pNH46A, available
from Stratagene; pcDNA3 available from InVitrogen; pGEX, pTrxfus,
pTrc99a, pET-5, pET-9, pKK223-3, pKK233-3, pDR540, pRTTS available
from Pharmacia; and pSPORTI, pSPORT2 and pSV~SPORTI, available from
Life Technologies, Inc. Other suitable vectors will be readily apparent to the
skilled artisan.
Other terms used in the fields of recombinant DNA technology and
molecular and cell biology as used herein will be generally understood by one
of ordinary skill in the applicable arts.
The present invention provides novel microorganisms for
biotechnology applications characterized by a more rapid growth rate than
those microorganisms currently in use in the art. Both gram negative and gram
positive prokaryotic cells may be used. The microorganisms of the present
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invention may be of any genus of microorganism known to those skilled in the
art. The preferred characteristics of the microorganism are a rapid growth
rate
and the capability to be transformed with and to maintain exogenously applied
DNA, in particular, to be transformed with and to maintain recombinant
vectors. In preferred embodiments, host cells that may be used in accordance
with the present invention include, but are not limited to, microorganisms
such
as those of the genera Escherichia sp. (particularly E. coli), Klebsiella sp.,
Streptomyces sp., Streptocococcus sp., Shigella sp., Staphylococcus sp.,
Erwinia sp., Klebsiella sp., Bacillus sp. (particularly B. cereus, B.
subtilis, and
B. megaterium), Serratia sp., Pseudomonas sp. (particularly P. aeruginosa and
P. syringae) and Salmonella sp. (particularly S. typhi or S. typhimurium). In
a preferred embodiment, the microorganisms of the present invention are of
the genus Escherichia. In other preferred embodiments, the microorganisms
of the present invention may be of the species E. coli. In a preferred
embodiment, the microorganisms of the present invention may be of the E.
coli strain W. In another preferred embodiment, the present invention
includes derivatives of E. coli W that do not contain endogenous vectors. In
other preferred embodiments, the microorganisms of the present invention
may be E. coli strains K, B or C.
The microorganisms of the present invention may be identified by
comparison to known bacterial strains. In general, comparison may be made
to one or another derivative of Escherichia coli K-12. A well known strain
that can serve as a reference strain is E. coli MM294 (ATCC33625). Other
suitable reference strains include E. coli strains specifically describe
herein.
Thus, the invention contemplates any microorganism which grow at the same
rate ar at a faster rate when compared to the E. coli W strains of the present
invention. Such comparison can be made by any means known to those
skilled in the art, including time to colony formation and/or doubling time.
The microorganisms of the present invention preferably form colonies
more rapidly than the strains to which they may be compared. In particular,
the microorganisms of the present invention will more rapidly form antibiotic
resistant colonies after transformation with a vector containing an antibiotic
resistance gene than the microorganisms of the prior art. To identify the
microorganisms of the present invention, a potential candidate microorganism
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and a reference strain are spread on suitable solid plates, preferably agar
media
plates known to those skilled in the art, in parallel. The selection and
preparation of a suitable solid plate are within the capabilities of those
skilled
in the art. A suitable plate may prepared using the medium recommended by
5 the American Type Culture Collection or other suitable media for cultivation
of the candidate microorganism. Alternatively, a comparison of the doubling
time in liquid media may be used.
The plates may optionally contain an antibiotic if, for example, a
competent reference strain is to be compared to a competent, putative rapid
10 growing microorganism. Both microorganisms can be transformed with a
vector that confers an antibiotic resistance to transformants. After
transformation, the two microorganisms can be spread onto antibiotic plates in
parallel and incubated at an appropriate temperature. The time to the
appearance of antibiotic resistant colonies of a specified diameter can be
15 determined. The rapid growing microorganisms of the present invention will
form antibiotic resistant colonies of a specified size more rapidly than the
microorganisms presently used for cloning applications. The plates are
incubated at the same temperature and the time to colonies of a specified size
is determined. In the examples below, a colony size of 1 mm diameter was
used; however, any size may be selected and used. A microorganism that
attains the specified size at a faster rate than the reference organism is
considered to be a rapid growing organism.
The present invention also comprises a method of cloning employing
the rapid growing microorganisms of the present invention. A population of
recombinant vectors containing a desired insert may be constructed using
techniques known in the art. For example, DNA of interest may be digested
with one or more restriction enzymes to generate a fragment. The fragment
may be purified on an agarose gel. A vector is prepared by digestion with the
appropriate restriction enzymes. The vector may be further treated with other
enzymes such as alkaline phosphatase or the Klenow fragment of DNA
polymerise, and may be gel purified. The DNA fragment is ligated into the
vector using an appropriate ligase enzyme to generate a population of
recombinant vectors.
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Other methods to produce a population of recombinant vectors may be
used. For example, a population of recombinant vectors may be produced by
recombinational cloning. An insert donor molecule is prepared comprising a
DNA of interest flanked by a first and a second recombination site, wherein
the first and the second recombination site do not recombine with each other.
The insert donor molecule is contacted with a vector donor molecule
comprising a third and a fourth recombination site, wherein the third and the
fourth recombination sites do not recombine with each other. The insert
donor/vector donor mixture is further contacted with one or more site specific
recombination proteins capable of catalyzing recombination between the first
and the third recombination sites and/or the second and the fourth
recombination sites thereby allowing recombination to occur and generating a
population of recombinant vectors.
Once constructed, the population of recombinant vectors is introduced
into competent, rapid growing microorganisms using any one of the many
techniques for the introduction of vector into a microorganism known to those
skilled in the art. The transformed microorganisms are grown and
recombinant microorganisms, i.e. those containing a vector, are selected. In
one embodiment, the genotype of the microorganism is suitable for screening
by alpha complementation and the selection step may include the use of a
blue/white screen on solid plates containing a chromogenic substrate for (3-
galactosidase, such as X-gal. The vectors are isolated from the recombinant
microorganism and analyzed for the presence of the DNA of interest.
The present invention also comprises a method of producing a desired
protein or peptide utilizing the rapid growing microorganisms of the present
invention. The method comprises constructing a recombinant vector
containing a gene encoding the desired protein, transforming the vector into a
competent, rapid growing microorganism and culturing the transformed
microorganism under conditions that cause the transformed microorganism to
produce the desired protein. The recombinant vector may be constructed
using the methodology described above. In one embodiment, the recombinant
vector will include an inducible promoter to control transcription from the
gene coding for the desired protein. In other preferred embodiments, the
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genome of the microorganism will contain a gene for the T7 RNA polymerase
under the control of an inducible promoter. In other preferred embodiments,
the promoter controlling the expression of the T7 RNA polymerase will be
inducible by the addition of salt to the growth media. In preferred
embodiments, the rapid growing microorganism is of the genus Escherichia.
In other preferred embodiments, the rapid growing microorganism is an E.
coli. In other preferred embodiments, the rapid growing microorganism is an
E. coli strain W. In another preferred embodiment, the rapid growing
microorganism does not contain endogenous plasmid. In other preferred
embodiments, the genotype of the microorganism has been altered to
inactivate one or more genes coding for a protease and/or a ribonuclease. In
one such preferred embodiment, the rapid growing microorganism does not
contain a functional lon protease and/or a functional ompT protease. In other
preferred embodiments, the rapid growing microorganism of the present
invention does not have a functional rnaE gene and/or a functional rnaI gene.
In other preferred embodiments the microorganism does not contain functional
lon protease and/or a functional ompT protease and does not contain a
functional ribonuclease encoded by the rnaE gene and/or the rnaI gene.
The present invention also includes kits comprising a carrier or
receptacle being compartmentalized to receive and hold therein at least one
container, wherein the container contains rapid growing microorganisms. The
kit optionally further comprises vectors suitable for cloning. In a preferred
embodiment, the kits may contain a vector suitable for recombinational
cloning. Optionally, the kits of the present invention may contain enzymes
useful for cloning. In a preferred embodiment, the kits may contain one or
more recombination proteins. In a preferred embodiment, the rapid growing
microorganisms may be competent. In some preferred embodiments, the rapid
growing microorganisms may be chemically competent. In other preferred
embodiments, the rapid growing microorganisms may be electrocompetent.
The present invention includes compositions comprising rapid growing
microorganisms. In a preferred embodiment, the rapid growing microorganism
may be a competent microorganism. In some preferred embodiments, the
rapid growing microorganisms may be chemically competent. In other
preferred embodiments, the rapid growing microorganisms may be
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electrocompetent. The compositions of the present invention may optionally
comprise at least one component selected from buffers or buffering salts, one
or more DNA fragments, one or more vectors, one or more recombinant
vectors, one or more recombination proteins and one or more ligases. In a
preferred embodiment, the compositions of the present invention may
comprise a rapid growing microorganism in a glycerol solution. In other
preferred embodiments, compositions of the present invention may comprise
rapid growing microorganisms in a buffer. In preferred embodiments, the
microorganisms of the present invention may be in a competence buffer. In
other preferred embodiments, the compositions of the present invention may
comprise a lyophilized rapid growing microorganism.
It will be readily apparent to one of ordinary skill in the relevant arts
that other suitable modifications and adaptations to the methods and
applications described herein are obvious and may be made without departing
from the scope of the invention or any embodiment thereof. Having now
described the present invention in detail, the same will be more clearly
understood by reference to the following examples, which are included
herewith for purposes of illustration only and are not intended to be limiting
of
the invention.
EXAMPLE 1
Strain Construction
All strains (listed in table 1) were constructed via bacteriophage P1
mediated transduction (Jeffrey Miller, Experiments in Molecular Genetics,
Cold Spring Harbor Laboratories, 1972, specifically incorporated herein by
reference). E. coli strains containing TnlO insertions suitable for use with
the
P1 transduction technique can be obtained from the University of Wisconsin.
The parental strain for this work was an E. coli W strain designated
ATCC9637 obtained from the American Type Culture Collection (Manassas,
VA). The isolate received was resistant to bacteriophage P1. ATCC9637
was, therefore, converted to a P1 sensitive phenotype by infection with
bacteriophage PlCmts. PlCmts is a bacteriophage Pl derivative which
contains a temperature sensitive repressor and contains the chloramphenicol
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resistance gene. The bacteriophage forms P1 lysogens at 30°C but
replicates
lytically at higher temperatures (>37°C). E. coli W ATCC9637 was mixed
with bacteriophage PlCmts and chloramphenicol resistant colonies (which are
PICm lysogens) were selected on LB chloramphenicol plates at 30°C.
The
chloramphenicol resistant strain was cured of the P1 lysogen by selection for
surviving colonies at 42°C. The surviving colonies are now
chloramphenicol
sensitive. The Pl sensitive derivative of ATCC9637 (BRL3234) was then
used for all subsequent work.
EXAMPLE 2
Construction of E. coli W endA-
Competent cells of BRL3234 were prepared by a modification of the
method of Hanahan (Doug Hanahan, J. Mol. Biol. 166,557, 1983) as described
in United States patent no. 4,981,797 which is specifically incorporated
herein
by reference. The competent cells were transformed with pCM301 plasmid
DNA (Tucker, et al., 1984, Cell 38(1):191-201.), a plasmid which is
temperature sensitive for replication. Transformants were selected on
ampicillin plates at 30°C. The introduction of the pCM301 plasmid into
BRL3234 aided in the identification of endA-derivatives as described below.
Bacteriophage Plvir was grown on an E. coli strain, DB2, which
contains an endA- mutation linked to the nupG::TnlO transposon. The P1
lysate grown on DB3.2 was used to infect BRL3234/pCM301 with selection
for tetracycline resistance. The tetr colonies were then screened for the
linked
endA- mutation by determining the ability of the transductants to degrade the
pCM301 DNA after preparation of miniprep DNA. Those transductants
which degraded the plasmid DNA were endA+ and those which did not
degrade pCM301 plasmid DNA were endA-. The tetracycline resistant, endA
derivative of BRL3234/pCM301 was designated BRL3573. A derivative of
BRL3573 lacking pCM301 was selected by streaking BRL3573 on an LB
plate at 42°C and screening colonies for ampicillin sensitivity.
The ampicillin sensitive derivative of BRL3573 was designated
BRL3574. The nupG::TnlO transposon was cured from BRL3574 using LB
plates containing fusaric acid (Stanley Maloy and William Nunn. J. Bacteriol.
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145:1110, 1981). One tetracycline sensitive derivative of BRL3574 was
designated BRL3580. BRL3580 is E. coli W endA- .
EXAMPLE 3
5 Construction of BRL3582 a recA- E. coli W.
A PlCm lysate was grown on BRL3229. BRL3229 contains a TnlO
transposon linked to a deletion mutation in recA. The Pl lysate was used to
transduce BRL3580 and tetracycline resistant transductants were selected at
30°C on LB plates containing 20 ~,g/mL tetracycline. The transductants
were
10 re-purified once on LB tetracycline plates and were then screened for
sensitivity or resistance to nitrofurantoin on LB plates containing 4 ~g/mL
nitrofurantoin. RecA+ strains are resistant to nitrofurantoin whereas recA-
strains are sensitive to nitrofurantoin (S Jenkins and P. Bennett J.,
Bacteriol.
125:1214, 1976). One tetracycline resistant, nitrofurantoin sensitive
derivative
15 of BRL3580 was designated BRL3582.
EXAMPLE 4
Isolation of E. coli W Derivatives Lacking Native Plasmids
20 ATCC9637 and all strains derived from ATCC9637 up to and
including BRL3580 contain 2 plasmids. The smaller plasmid is approximately
5.5 kb and the larger plasmid is >50 kb. The 5.5 kb plasmid was prepared
from ATCC9637 by Lofstrand Labs (Gaithersburg, MD). A restriction map of
this plasmid is provided in Figure 1.
The restriction map provided cloning sites which could be used to
introduce a gene conferring resistance to ampicillin. The ampicillin
resistance
gene was isolated from plasmid pTrcN2, a pProEX-1 derivative (Life
Technologies, Rockville MD). The source of the ampicillin resistance gene is
not critical. The following protocol will work with pProEX-1 and may be
modified by those skilled in the art depending on the plasmid used as a source
of the ampicillin resistance gene. 1 ~g of plasmid pTrcN2 was digested with
BspHl (New England Biolabs) and the ends filled in with Klenow (Life
Technologies, Inc). The 1008 by DNA fragment containing the ampicillin
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resistance gene was purified by agarose gel electrophoresis. The 5.5 kb
plasmid was digested with SmaI (New England Biolabs) and then treated with
TsAP, a temperature sensitive alkaline phosphatase (Life Technologies, Inc.).
The DNAs were mixed, treated with T4 DNA ligase (Life Technologies, Inc)
and transformed into competent ME DHlOB cells (Life Technologies, Inc).
Ampicillin resistant colonies were selected on LB plates containing 100
pg/mL ampicillin. Several ampicillin resistant colonies were grown in
overnight culture and plasmid DNA was prepared and analyzed by
electrophoresis on an agarose gel. All ampicillin resistant clones were found
to have a plasmid with a molecular weight of 6.5 kb. The DH10B cells
containing the plasmid (designated Wamp) were designated BRL3709.
The Wamp plasmid was transformed into competent cells of BRL3580
(E. coli W endA-) with selection for ampicillin resistance. BRL3580, as well
as 5 ampicillin resistant transformants, were grown at 37°C in LB broth
containing 100 ~g/mL ampicillin and the plasmid DNA was isolated and
analyzed by agarose gel electrophoresis. The plasmid DNA from BRL3580
had a molecular weight of 5.5 kb whereas the ampicillin resistant
transformants had plasmid DNA with a molecular weight of 6.5 kb indicating
that the ampicillin resistance gene ~1 kb had been introduced into the 5.5 kb
plasmid to give a 6.5 kb plasmid. Further, the 6.5 kb plasmid containing the
ampicillin resistance gene had displaced the 5.5 kb plasmid. This is the
expected result since both plasmids contained the same origin of replication.
The E. coli W derivatives containing the 6.5 kb Wamp plasmid were
designated BRL3711. Both BRL3580 and BRL3711 also contained the higher
molecular weight (>50 kb) plasmid.
EXAMPLE 5
Curing BRL3711 of the 6.5 kb Wamp Plasmid
BRL3711 was cured of the Wamp plasmid by growth in LB broth
containing SDS. SDS is well known in the literature as a compound which is
used to cure plasmids from E. coli strains (A. Bharathi and H. Polasa, FEMS
Microbiol. Lett, 84:37, 1991, Susana Rosos, Aldo Calzolari, Jose La Torre,
Nora Ghittoni, and Cesar Vasquez, J. Bacteriol 155:402, 1983). BRL3711
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was grown in LB broth containing 10% SDS at 30°C. After the culture
reached the stationary phase, the culture was diluted 1:1000 into fresh LB +
10% SDS for a second cycle. After the second cycle, the survivors were
plated on LB plates 30°C and colonies were screened for sensitivity to
ampicillin. One isolate, designated BRL3718, was found to be sensitive to
ampicillin indicating that the 6.5 kb plasmid had been cured. Miniprep DNA
derived from BRL3711 as well as BRL3718 confirmed that BRL3711 had
both the smaller and larger plasmids but that BRL3718 had only the larger
plasmid.
EXAMPLE 6
Preparation of a Der7vative of the Large Plasmid Containing an Antibiotic
Resistance Gene.
To isolate E. coli W derivatives lacking the larger plasmid, antibiotic
resistance genes were introduced into the larger plasmid using the Genome
Primer System from New England BioLabs. The larger plasmid was isolated
from BRL3718 using the standard alkaline-SDS lysis procedure (J. Sambrook,
E.F. Fritsch, and T. Maniatis. 1989 Molecular Cloning: A Laboratory Manual
2°d Ed. Cold Spring Harbor Laboratory Press. Cold Spring Harbor NY.).
The
Genome Priming System was used according to instructions provided by the
manufacturer.
Approximately 80 ng of target plasmid DNA was mixed with 20 ng of
donor plasmid DNA in a 20 p,L reaction. One donor plasmid, pGSPI, donates
the gene confernng resistance to kanamycin (Km). The second donor
plasmid, pGSP2, donates the gene conferring resistance to chloramphenicol
(Cm). The final reactions were diluted 1:10 in water and electroporated into
EMax DH10B cells (Life Technologies, Inc.). 20 pI. of cells were mixed with
1 p,L of the diluted reaction and the cell-DNA combination was electroporated
at 420 V, 4000 ohms, 2.4 kV, 16000 kV/cm. 10 ~I. were expressed in 1 mL
SOC for 1 hour 37°C. 100 pL of the expression mix were plated on LB
plates
containing either 10 ~g/mL kanamycin for the pGPS 1 reaction or LB plates
containing 12.5 p,g/mL chloramphenicol for the pGPS2 reaction. 8
transformants from each reaction were analyzed. Plasmid DNA from all 16
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colonies had a high molecular weight plasmid which ran on an agarose gel in
approximately the same position as the plasmid DNA isolated from BRL3718.
In addition, several of the plasmid DNAs were again electroporated into EMax
DH10B cells and were shown to confer resistance to either kanamycin or
chloramphenicol on the DH10B cells. It was concluded that the genes
conferring resistance to either kanamycin or chloramphenicol had been
introduced into the large molecular weight plasmid from BRL3718. DH10B
cells containing the high molecular weight plasmid which confers resistance to
kanamycin have been designated BRL3726. DH10B cells containing the high
molecular weight plasmid which confers resistance to chloramphenicol have
been designated BRL3727.
EXAMPLE 7
Construction of Deletion Plasmids.
Plasmid DNA from the strain BRL3726 (DH10B containing the high
MW plasmid + Km' marker) was prepared. In two separate reactions, 1 ~g of
plasmid DNA was partially digested with 0.5 and 0.1 units of the restriction
enzyme Sau3A I (Life Technologies, Inc.) at 37°C for 15 min. The
reactions
were extracted with phenol/chloroform and precipitated with ethanol. The
DNA from each reaction was ligated using T4 DNA Ligase (Life
Technologies, Inc) and transformed into competent ME DHSa cells (Life
Technologies, Inc). Colonies were selected on LB plates containing 20 ~,g/mL
kanamycin at 37°C. Chemically competent cells were used because they
are
not as efficient in taking up high molecular weight plasmid DNA as
electrocompetent cells.
The plasmid DNA from 10 kanamycin resistant (from the 0.1 U
reaction) colonies was analyzed by agarose gel electrophoresis. The size of
the deletion plasmid DNA ranged from ~4.5-15 kb and the plasmids were
designated deletion 1 - deletion 10. DHSa cells containing these plasmids
were designated BRL3740-1 to BRL3740-10.
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EXAMPLE 8
Curing BRL3718 of the High Molecular Weight Plasmid DNA.
Chemically competent cells of BRL3718 were prepared according to
the method of Hanahan (Hanahan D., 1983 J. Mol Biol 166,557) as modified
according to United States Patent no. 4,981,797. Chemically competent cells
of BRL3718 were transformed with plasmid DNA isolated from BRL3740-1
(deletion 1, ~8 kb) and BRL3740-3 (deletion 3, -10 kb) and kanamycin
resistant colonies were selected on LB plates containing 20 ~g/mL kanamycin
at 37°C. Four colonies from each transformation were streaked for
single-
colony isolates onto LB plates containing 20 ~.g/mL kanamycin at 37°C.
Plasmid DNA was isolated from 4, single-colony isolates and analyzed by
agarose gel electrophoresis.
The high molecular weight plasmid DNA was readily apparent in
miniprep DNA prepared from BRL3718. However, plasmid DNA prepared
from the kanamycin resistant transformants did not indicate the presence of
the high molecular weight plasmid DNA. Rather, plasmid DNAs with
molecular weights characteristic of BRL3740-1 (~8 kb) and BRL3740-3 (~10
kb) were readily visible. It was concluded that the transformation of deletion
1 and deletion 3 plasmid DNA into BRL3718 resulted in replacement of the
high molecular weight plasmid DNA (>50 kb) with deletion 1 and deletion 3
DNA. This is the expected result since the high molecular weight plasmid
DNA, deletion 1 plasmid DNA and deletion 3 plasmid DNA all share the same
origin of replication. The BRL3718 derivatives containing deletion 1 and
deletion 3 plasmid DNA were designated BRL3741 and BRL3742,
respectively.
EXAMPLE 9
Curing BRL3741 and 3742 of the Km' Plasmids.
BRL3741 and BRL3742 were grown overnight in LB broth containing
10% SDS at 30°C. The cultures were diluted 1:1000 into LB broth
containing
10% SDS and incubated again at 30°C. After 2 cycles at 30°C,
dilutions of
these cultures ( 1:104 and 1:106) were applied to LB plates, incubated at
30°C,
and screened for sensitivity to kanamycin. For BRL3741, 15/50 colonies were
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sensitive to kanamycin while 9/50 colonies from BRL3742 were sensitive to
kanamycin. Plasmid DNA from 2 kanamycin sensitive derivatives of
BRL3741 and 2 kanamycin sensitive derivatives of BRL3742 was isolated and
analyzed by agarose gel electrophoresis. No plasmid DNA corresponding to
5 the deletion plasmids was observed on the gel. The BRL3741 derivatives
cured of the deletion 1 plasmid were designated BRL3756. The BRL3742
derivatives cured of the deletion 3 plasmid were designated BRL3757.
EXAMPLE 10
10 Competent Cells of BRL3756 and BRL3757.
Chemically competent cells of BRL3741, BRL3742, BRL3756 and
BRL3757 were prepared according to the method of Hanahan (Hanahan D.,
1983 J. Mol Biol 166,557) as modified according to United States Patent no.
4,981,797. BRL3741 and BRL3742 were streaked on LB plates containing 20
15 p,g/mL kanamycin and the plates were incubated at 28°C for 20 hours.
BRL3756(1), BRL3756(2), BRL3757(1) and BRL3757(2) were streaked on
LB plates and the plates were incubated 28°C for 20 hours. 5-6
colonies of
each strain were picked into 1 mL SOB medium (D, Hanahan J. Mol Biol
166:557 1983). 0.9 mL of the cells were inoculated into 60 mL SOB medium
20 in a 500 mL baffled shake flask. The flasks were placed in an 28°C
incubator
250 rpm. When the OD at 550 nm reached 0.25 - 0.33, the cells were
harvested. 50 mL of cells of each strain were centrifuged (4°C) and the
cells
were re-suspended in 4 mL cold CCMB80 buffer (D. Hanahan, J. Jessee and
F. Bloom Methods in Enzymology 204:63 1991, specifically incorporated
25 herein by reference). The cells were allowed to sit on ice for 20 min. 220
pl.
were placed in NUNC vials and the cells were frozen in a dry ice ethanol bath.
The cells were stored at -70°C.
EXAMPLE 11
Evaluation of Time to Ampicillin Resistant Colony
Vials of competent cells (ATCC9637, BRL3718, BRL3741, BRL3742,
BRL3756 and BRL3757) were thawed on ice for 20 min. 100 p,L of cells
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were mixed in a cold Falcon 2059 tube with pUC 19 (5 pL of 10 pg/p,L =
50pg) . The cells were allowed to sit on ice for 15 min. The cells were heat
shocked at 42°C for 45 seconds followed by a 2 minute incubation on
ice. 0.9
mL of room temperature SOC was added to each tube and the tubes were
shaken at 37°C (250 rpm) for 30 minutes. Aliquots of the expression mix
were plated on LB plates containing 100 p,g/mL ampicillin and the plates were
incubated at either 42°C or 37°C. The time to the appearance of
lmm
colonies is shown in table 2. At 37°C, ampicillin resistant colonies of
1 mm
size required between 7.8 and 8.2 hours and there was no significant
difference in time between strains containing both the 5.5 kb plasmid and the
>50 kb plasmid (ATCC9637), strains containing only the >50 kb plasmid
(BRL3718), strains containing the smaller kanamycin resistant plasmid
(BRL3741 and 3742), or strains containing no plasmids (BRL3756 and 3757).
In fact at 42°C colonies of 1 mm size required 7.7 hours for all
strains tested.
It was concluded that the presence or absence of plasmids in E. coli W does
not significantly affect the time to appearance of colonies after
transformation.
EXAMPLE 12
Construction of BRL3734.
Electrocompetent cells of BRL3718 were prepared according to a
modification of the protocol described in Hanahan et. al., Methods in
Enzymology, vol. 204, p. 63 (1991). DNA from BRL3727 isolate 46 was used
to introduce the plasmid into BRL3718. 20 ~,I. of cells were mixed with 1 ~I.
of DNA and the cell-DNA mixture was electroporated at 250 V, 2000 ohms,
1.44 kV, 9.6 kV/cm in the Life Technologies Cell-Porator. 10 ~,I. were
expressed in 1 mL SOC for 60 min 37°C and the expression was plated on
LB
plates containing 12.5 p,g/mL chloramphenicol. After 24 hours the colonies
were re-purified and analyzed. The miniprep DNA contained a plasmid with a
molecular weight approximately the same size as the plasmid found in
BRL3718. The E. coli W strain containing the chloramphenicol resistant high
molecular weight plasmid was designated BRL3734.
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EXAMPLE 13
Curing BRL3734 of the High Molecular Weight Plasmid DNA.
Chemically competent cells of BRL3734 were prepared according to
the method of Hanahan (Hanahan D., 1983 J. Mol Biol 166,557) as modified
according to United States Patent no. 4,981,797. Chemically competent cells
of BRL3734 were transformed with plasmid DNA isolated from BRL3740-1
deletion 1, ~8 kb) and BRL3740-3 ( deletion 3, ~10 kb) and kanamycin
resistant colonies were selected on LB plates containing 20 ~,g/mL kanamycin
at 37°C. Four colonies from each transformation were streaked for
single-
colony isolates onto LB plates containing 20 ~g/mL kanamycin at 37°C.
Plasmid DNA was isolated from 4, single-colony isolates and analyzed by
agarose gel electrophoresis. The high molecular weight plasmid DNA was
readily apparent in miniprep DNA prepared from BRL3734. However,
plasmid DNA prepared from the kanamycin resistant transformants did not
indicate the presence of the high molecular weight plasmid DNA. Rather,
plasmid DNA with molecular weight characteristic of BRL3740-1 (~8 kb) and
BRL3740-3 (~10 kb) were readily visible. Moreover, BRL3734 containing
deletion 1 and deletion 3 plasmids were streaked for single-colony isolates
onto LB containing Km 20 pg/mL and LB containing Cm 12.5 pg/mL plates
to confirm the presence, or absence, of the desired plasmid DNAs. No growth
was observed on the LB + Cm 12.5 pg/mL plates while the formation of
single-colony isolates was observed on Km 20 pg/mL plates. It was
concluded that the transformation of deletion 1 and deletion 3 plasmid DNA
into BRL3734 resulted in replacement of the high molecular weight plasmid
DNA (>50 kb) with deletion 1 and deletion 3 DNA. This is the expected
result since the high molecular weight plasmid DNA, deletion 1 plasmid DNA
and deletion 3 plasmid DNA all share the same origin of replication. The
BRL3734 derivatives containing deletion 1 and deletion 3 plasmid DNA were
designated BRL3745 and BRL3746, respectively.
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EXAMPLE 14
Curing BRL3745 and 3746 of the Km' Plasmids.
BRL3745 and BRL3746 were grown overnight in LB broth containing
10% SDS at 30°C. The cultures were diluted 1:1000 into LB broth
containing
10% SDS and incubated again at 30°C. After 2 cycles at 30°C,
dilutions of
these cultures ( 1:106) were applied to LB plates, incubated at 30°C,
and
screened for sensitivity to kanamycin. For BRL3745, 22/100 colonies were
sensitive to kanamycin while 1/100 colonies from BRL3742 were sensitive to
kanamycin. Plasmid DNA from 3 kanamycin sensitive derivatives of
BRL3745 and the one kanamycin sensitive derivative of 3746 was isolated
and analyzed by agarose gel electrophoresis.
No plasmid DNA corresponding to the deletion 1 and deletion 3
plasmids was observed on the gel after curing. The BRL3745 derivatives
cured of the deletion 1 plasmid were designated BRL3762. The BRL3746
derivative cured of the deletion 3 plasmid were designated BRL3763.
EXAMPLE 15
Competent Cells of BRL3762 and BRL3763.
Chemically competent cells of BRL3745, BRL3746, BRL3762 and
BRL3763 were prepared according to the method of Hanahan (Hanahan D.,
1983 J. Mol Biol 166,557) as modified according to United States Patent no.
4,981,797. BRL3745 and BRL3746 were streaked on LB plates containing 20
~g/mL kanamycin and the plates were incubated at 28°C for 20 hours.
BRL3762(1), BRL3762(2), and BRL3763(1) were streaked on LB plates and
the plates were incubated 28°C for 20 hours. 5-6 colonies of each
strain were
picked into 1 mL SOB medium(D, Hanahan J. Mol Biol 166:557 1983). 0.9
mL of the cells were inoculated into 60 mL SOB medium in a 500 mL baffled
shake flask. The flasks were placed in an 28°C incubator 250 rpm. When
the
OD550nm reached 0.25 - 0.33 the cells were harvested. 50 mL of cells of
each strain were centrifuged (4°C) and the cells were re-suspended in 4
mL
cold CCMB80 buffer (D. Hanahan, J. Jessee and F. Bloom Methods in
Enzymology 204:63 1991 ). The cells were allowed to sit on ice for 20 min.
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220 ~L were placed in NUNC vials and the cells were frozen in a dry ice
ethanol bath. The cells were stored at -70°C.
EXAMPLE 16
Evaluation of Time to Ampicillin Resistant Colony_
One vial of competent cells (ATCC9637, BRL3734, BRL3745,
BRL3746, BRL3762 and BRL3763) was thawed on ice for 20 min. 100 ~I. of
cells were mixed in a cold Falcon 2059 tube with pUCl9 (5 ~,L of 10 pg/~L =
50 pg) . The cells were allowed to sit on ice for 15 min. The cells were heat
shocked at 42°C for 45 seconds followed by a 2 minute incubation on
ice. 0.9
mL of room temperature SOC was added to each tube and the tubes were
shaken at 37°C (250 rpm) for 30 minutes. Aliquots of the expression mix
were plated on LB plates containing 100 ~g/mL ampicillin and the plates were
incubated at either 42°C or 37°C. The time to the appearance of
lmm
colonies is shown in table 3. At 37°C, ampicillin resistant colonies of
1 mm
size required 8.0 hours and there was no significant difference in time
between
strains containing both the 5.5 kb plasmid and the >50 kb plasmid
(ATCC9637), strains containing only the >50 kb plasmid (BRL3734), strains
containing the smaller kanamycin resistant plasmid (BRL3745 and 3746), or
strains containing no plasmids (BRL3762 and 3763). At 42°C, colonies of
1
mm size required 7.3 hours for all strains tested. It was concluded that the
presence or absence of plasmids in E. coli W does not significantly affect the
time to appearance of colonies after transformation. In addition, the data in
tables 3 and 4 indicate that incubation of the LB ampicillin plates at
42°C
results in the appearance of ampicillin resistant colonies approximately 0.5
hours faster than on plates incubated at 37°C.
EXAMPLE 17
Comparison of Wildtype E. coli W and E. coli K-12.
Competent cells of Escherichia coli strains ATCC9637 (W), BRL3582
(E. coli W endA- srl::TnlO recA1398), and ATCC33625 (MM294) were
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prepared according to the method of Hanahan (Hanahan D., 1983 J. Mol Biol
166,557) as modified according to United States Patent no. 4,981,797. The
competent cells were prepared using CCMB80 buffer (Hanahan,D., Jessee, J.,
and Bloom, F.R., 1991, Methods in Enzymology 204,63). Max Efficiency
5 DHSa competent cells were obtained from Life Technologies Inc.
The competent cells were thawed on ice for 20 minutes. 100 ~,L of the
cells were transformed with 50 pg of pUCl9 or 50 pg of pBR322 DNA. The
cell-DNA mixture was placed on ice for 30 minutes and then heat shocked at
42°C for 45 seconds. The tubes were then placed on ice for 2 minutes.
0.9
10 mL of SOC (Hanahan 1983) was added to each tube and the tubes were then
shaken at 225 rpm for 1 hour at 37°C. Appropriate dilutions were spread
on
LB plates containing 100 ~,g/mI. ampicillin and the plates were incubated at
37°C. The amount of time in hours to the appearance of 1 mm colonies
was
measured and is shown in Table 4. ATCC9637 yielded colonies in 8 - 8.5
15 hours compared to approximately 10 hours for ATCC33625, another recA+
strain, recA- strains were also compared. BRL3582 yielded colonies in
approximately 10 hours compared to 16 hours for DHSa.
20 EXAMPLE 18
Growth of Transformed Microorganisms at an Elevated Temperature.
Using the protocol described in the preceding example, the effects of
growth an elevated temperature were analyzed. Incubating the transformed
microorganisms on LB ampicillin plates at 42°C resulted in the
appearance of
25 colonies from 0.5 - 1 hour faster compared to plates incubated at
37°C.
Plating the cells on plates made from Circle Grow (Bio101) and containing
ampicillin at 100 p,g/mL resulted in the appearance of colonies from 0.5- 1
hour faster compared to the appearance of colonies on LB plates containing
ampicillin at 100 ~g/mL. Thus, the use of elevated temperatures and/or
30 enriched growth media may facilitate an increased growth rate of the
microorganisms of the present invention.
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31
EXAMPLE 19
Preparation of Derivatives of E. coli W Cured of Plasmids.
An isolate of E. coli W that has been cured of plasmid, such as
BRL3762, BRL3763, BRL3756 or BRL3757, is used to construct derivatives
having genotypes desirable for biotechnology applications. Using the P1
transduction technique described above, strains having one or more useful
genetic alterations are prepared. Useful genetic alterations include: a recA-
genotype such as recAllrecAl3 or recA deletions, a lacZ- genotype that
allows alpha complementation such as lacX74 lacZO1VI15 or other lacZ
deletion, a protease deficient genotype such as Olon and/or ompT~, an
endonuclease minus genotype such as endAl, a genotype suitable for M13
phage infection by including the F' episome, a restriction negative,
modification positive genotype such as hsdRl7(rK , mK+), a restriction
negative, modification negative genotype such as hsdS20(rB-, mB-), a
methylase deficient genotype such as mcrA and/or mcrB and/or mrr, a
genotype suitable for taking up large plasmids such as deoR, a genotype
containing suppressor mutations such as supE and/or supF. Other suitable
modifications are known to those skilled in the art and such modifications are
considered to be within the scope of the present invention.
In a preferred embodiment, the rapid growing microorganisms of the
present invention contains a modified lac operon that permits alpha
complementation. In order to support alpha complementation, it was
necessary to introduce a deletion into the N-terminal region of the genomic ~i-
galactosidase gene. First, a lacX74 mutation was introduced into BRL3756
and BRL3757 by P1 transduction with a lysate prepared on BRL3759 which
contains the lacX74 mutation linked to a TnlO insertion. Strains containing
the lacX74 insertion are tetracycline resistant as a result of the TnlO
insertion.
Strains were selected on tetracycline containing plates and the resultant
strains
were designated BRL3760 (derived from BRL3756) and BRL3761 (derived
from BRL3757). The strains were cured of the TnlO insertion by growth in
the presence of fusaric acid and the resultant tetracycline sensitive strains
containing the lacX74 mutation were designated BRL3766 and BRL769.
These strains were made competent using the modified method of Hanahan as
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32
disclosed above and were then transformed with plasmid containing the alpha
fragment of the (3-galactosidase gene. The plasmid containing strains were
transduced using a lysate prepared on and E. coli strains carrying the
~80dlacZOMIS deletion mutation linked to a TnlO insertion in the trp gene.
As a result of the insertion in the trp gene, strains carrying this mutation
require tryptophan in the growth media. Tetracycline resistant strains were
selected and were designated BRL3776 (derived from BRL3756 via BRL3760
and BRL3766) and BRL3778 (derived from BRL3757 via BRL3761 and
BRL3769). These strains are lacX74 ~80dlacZ~MIS trp-::TnlO. To restore
the wild type trp gene, strains BRL3776 and BRL3778 were transduced with a
P1 lysate prepared on E. coli DHSa and selected on minimal media minus
tryptophan. The strains were spontaneously cured of the alpha fragment
containing plasmid and the final alpha complementation strains BRL3781
(from BRL3776) and BRL3784 (from (BRL3778) were isolated. These
strains are lacX74 ~80dlacZOMIS. BRL3781 and BRL3784 were deposited
at the Agricultural Research Service Culture Collection (NRRL, 1815 North
University Street, Peoria, Illinois, 61064) on June 17, 1999. The deposits
were made under the terms of the Budapest Treaty. BRL3781 has been given
accession number NRRL No. B-30143 and BRL3784 has been given
accession NRRL No. B-30144.
Those skilled in the art will appreciate that other modifications to the
genome of the rapid growing microorganisms of the present invention are
possible using the techniques described above. E. coli containing a desired
mutation linked to a TnlO insertion are readily available from sources well
known to those skilled in the art. The desired mutation can be inserted into
the
genome of a rapid growing microorganism using P1 transduction and then the
TnlO can be cured by growth in the presence of fusaric acid.
In preferred embodiments, the rapid growing microorganisms of the
present invention will carry an inducible T7 polymerise gene. In preferred
embodiments, the T7 polymerise gene will be under the control of a salt
inducible promoter as described by Bhandari, et al., J. Bacteriology,
179(13):4403-4406, 1997 which is specifically incorporated herein by
reference. The T7 polymerise gene may be under the control of the salt
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33
inducible promoters of the proU locus. Alternatively, the T7 polymerase gene
may be under the control of other salt inducible promoters. Other suitable
inducible promoters include the lac promoter, the trp promoter, the tac
promoter as well as any other inducible promoter known to those skilled in the
art. The selection of the appropriate promoters and construction of strains
carrying the T7 polymerase under the control of a given promoter are well
within the abilities of those of ordinary skill in the art. Optionally,
embodiments containing an inducible T7 polymerise gene may contain
mutations in one or more protease genes and mutations in one or more
ribonuclease genes. Such mutations may be inserted into the genome using
the methods described above.
EXAMPLE 20
Identification of Rapid Growing Microorganisms
Other microorganisms will be screened to identify rapid growing
strains. Isolates to be screened are plated on an appropriate solid medium and
grown to a defined colony size. The time to reach the defined colony size is
compared to the time taken by an E. coli K or other strains described herein
to
reach the same colony size. The microorganisms to be screened include, but
are not limited to, microorganisms such as those of the genera Escherichia sp.
(particularly E. coli and, more specifically, E. coli strains B, C, W and K)),
Klebsiella sp., Streptomyces sp., Streptocococcus sp., Shigella sp.,
Staphylococcus sp., Erwinia sp., Klebsiella sp., Bacillus sp. (particularly B.
cereus, B. subtilis, and B. megaterium), Serratia sp., Pseudomonas sp.
(particularly P. aeruginosa and P. syringae) and Salmonella sp. (particularly
S. typhi or S. typhimurium). A plasmid confernng an antibiotic resistance is
transformed into the microorganism to screened using the techniques
described above. The transformed microorganisms are then plated on a solid
medium containing antibiotic and then incubated at an appropriate temperature
until colonies of a defined size are observed.
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34
EXAMPLE 21
Cloning Usin~pid Growing Microorganisms.
The rapid growing microorganisms identified above may be used to
clone DNA fragments. A population of recombinant vectors comprising a
DNA insert having a desired sequence is constructed as described above. The
vector may contain a DNA sequence coding for an antibiotic resistance gene
and/or may contain one or more marker genes. The population of recombinant
vectors is transformed into a rapid growing microorganism rendered
competent by any conventional technique. For example, the microorganism is
rendered competent by chemical means using the technique of Hanahan
discussed above. Alternatively, the microorganism is made competent for
electroporation by removing the growth media and placing the microorganism
in a medium of low ionic strength. Any method of making the microorganism
competent that allows the microorganism to take up exogenously applied
DNA and, in particular, recombinant plasmids, is suitable for the practice of
the instant invention.
Competent microorganisms are contacted with some or all of the
population of recombinant vectors under conditions suitable to cause the
uptake of the recombinant vectors into the competent microorganism.
Suitable conditions may include a heat shock. For example, the mixture of
cells and population of recombinant vectors are heated to 42°C for 45
seconds.
Alternatively, suitable conditions may include subjecting a mixture of
microorganism and recombinant vector to an electric field.
After the recombinant vector is taken up by the microorganism, the
microorganism is grown for a period of time sufficient to allow the expression
of an antibiotic resistance gene. After any such period, the microorganism
containing the recombinant vector is spread on plates containing the
appropriate antibiotic and incubated until colonies are visible. In a
preferred
embodiment, the plates are incubated from about 4 hours to about 16 hours. In
other preferred embodiments, the plates are incubated from about 4 hours to
about 8 hours and in other preferred embodiments, the plates are incubated
from about 4 hours to about six hours. In a preferred embodiment, the
incubation step is performed at a temperature above 37°C at which
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temperature the microorganism containing the recombinant plasmid grows
more rapidly than it grows at 37°C. In another preferred embodiment,
the
incubation step is performed at 42°C.
After colonies become visible, some or all of the colonies will be
5 selected to be grown in liquid culture. The selection process may be by any
conventional means. In a preferred embodiment, the microorganism and
vector will permit alpha complementation and the selection is by blue/white
screening on X-gal plates in the presence of IPTG. In other preferred
embodiments, the selection is by detecting the presence or absence of a marker
10 gene present on the vector. Suitable marker genes include, but are not
limited
to, the gene coding for luciferase, the gene coding for chloramphenicol acetyl
transferase and the gene coding for (3-glucuronidase.
The selected colonies are grown in liquid culture for a period of time
sufficient to produce a quantity of recombinant microorganisms suitable for
15 analysis. The recombinant vector will be isolated from the microorganisms.
In a preferred embodiment, the period of growth in liquid culture will be from
about 2 hours to about 16 hours. In other preferred embodiments, the period
of growth in liquid culture will be from about 2 hours to about 8 hours and in
other preferred embodiments, the period of growth in liquid culture will be
20 from about 2 hours to about 4 hours.
The recombinant vector is isolated by any conventional means. In a
preferred embodiment, the recombinant vector is isolated by an alkaline lysis
"mini-prep" technique. Optionally, the isolation may employ a column
purification step. The isolated vector is analyzed by any conventional
25 technique, for example, by agarose gel electrophoresis of the plasmid with
or
without prior digestion of the plasmid with one or more restriction enzymes.
Other suitable techniques include sequencing of the plasmid. Techniques for
determining the DNA sequence of a plasmid are well known to those skilled in
the art.
30 Having now fully described the present invention in some detail by
way of illustration and example for purposes of clarity of understanding, it
will be obvious to one of ordinary skill in the art that the same can be
performed by modifying or changing the invention within a wide and
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36
equivalent range of conditions, formulations and other parameters without
affecting the scope of the invention or any specific embodiment thereof, and
that such modifications or changes are intended to be encompassed within the
scope of the appended claims.
All publications, patents and patent applications mentioned in this
specification are indicative of the level of skill of those skilled in the art
to
which this invention pertains, and are herein incorporated by reference to the
same extent as if each individual publication, patent or patent application
was
specifically and individually indicated to be incorporated by reference.
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37
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CA 02375482 2001-12-18
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CA 02375482 2001-12-18
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39
TABLE 2: Time in hours to ampicillin resistant colonies after transformation
with pUC 19 DNA.
Time to lmm colony size
STRAIN 42C 37C
ATCC 9637 7.7 8.2
BRL3718 7.7 7.8
BRL3741 7.7 8.2
BRL3742 7.7 7.8
BRL3756 ( 1 ) 7.7 8.2
BRL3756 (2) 7.7 8.2
BRL3757 (1) 7.7 7.8
BRL3757 (2) 7.7 8.2
TABLE 3: Time in hours to ampicillin resistant colonies after transformation
with pUC 19 DNA.
Time to lmm colony size
STRAIN 42C 37C
ATCC 9637 7.3 8.0
BRL3734 7.3 8.0
BRL3745 7.3 8.0
BRL3746 7.3 8.0
BRL3762 ( 1 ) 7.3 8.0
BRL3762(2) 7.3 8.0
BRL3763 7.3 8.0
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Table 4: Time in hours to ampicillin resistant colonies after transformation
with pUC 19 DNA
Time to lmm colony size
5
STRAIN pUCl9 Bp 8322
ATCC 9637 (W) recA+ 8.0 8.5
10 BRL3582(6) W recA- 10.25 ND
MM294 recA+ 10.25 10.25
DHSa recA- 16.0 16.0
