U.S. patent application number 10/121145 was filed with the patent office on 2002-10-24 for method for screening enzyme activity.
This patent application is currently assigned to Diversa Corporation. Invention is credited to Short, Jay M..
Application Number | 20020155489 10/121145 |
Document ID | / |
Family ID | 46276531 |
Filed Date | 2002-10-24 |
United States Patent
Application |
20020155489 |
Kind Code |
A1 |
Short, Jay M. |
October 24, 2002 |
Method for screening enzyme activity
Abstract
Disclosed is a process for identifying clones having a specified
enzyme activity by screening for the specified enzyme activity in a
library of clones prepared by (i) selectively isolating target
nucleic acid from nucleic acid derived from at least one
microorganism, by use of at least one polynucleotide probe
comprising at least a portion of a nucleic acid sequence encoding
an enzyme having the specified enzyme activity; and (ii)
transforming a host with isolated target nucleic acid to produce a
library of clones which are screened for the specified enzyme
activity.
Inventors: |
Short, Jay M.; (Rancho Santa
Fe, CA) |
Correspondence
Address: |
GARY CARY WARE & FRIENDENRICH LLP
4365 EXECUTIVE DRIVE
SUITE 1600
SAN DIEGO
CA
92121-2189
US
|
Assignee: |
Diversa Corporation
|
Family ID: |
46276531 |
Appl. No.: |
10/121145 |
Filed: |
April 9, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10121145 |
Apr 9, 2002 |
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09421970 |
Oct 20, 1999 |
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6368798 |
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09421970 |
Oct 20, 1999 |
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08944795 |
Oct 6, 1997 |
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6030779 |
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08944795 |
Oct 6, 1997 |
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08692002 |
Aug 2, 1996 |
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6054267 |
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60008317 |
Dec 7, 1995 |
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Current U.S.
Class: |
435/6.18 ;
435/6.1; 536/23.7 |
Current CPC
Class: |
C12N 15/102 20130101;
C12N 9/00 20130101; C12N 9/1029 20130101; C12N 15/1034 20130101;
C12N 9/16 20130101 |
Class at
Publication: |
435/6 ;
536/23.7 |
International
Class: |
C12Q 001/68; C07H
021/04 |
Claims
What is claimed is:
1. A method for making a library containing a plurality of clones,
each clone containing a cDNA or genomic DNA fragment obtained from
a population of uncultivated microorganisms.
2. A method for identifying a desired bioactivity or biomolecule
comprising: (a) producing one or more expression libraries derived
from nucleic acid directly isolated from the environment; (b)
combining a cell-free extract from the expression library or
libraries with crude or partially purified extracts, or pure
proteins from a metabolically rich cell line to form an extract
mixture free from the library and cell line cells; and (c)
screening said mixture to identify an activity or molecule produced
by the extract mixture.
3. A method for identifying a desired bioactivity or biomolecule
comprising: (a) producing one or more expression libraries
containing clones having nucleic acid inserts derived from nucleic
acid directly isolated from the environment; (b) transferring said
clones into a metabolically rich cell line; and (c) screening said
cell line to identify clones having a bioactivity or biomolecule of
interest.
Description
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 08/692,002 filed Aug. 2, 1996, which is a
continuation-in-part of provisional application No. 60/008,317
filed Dec. 7, 1995.
FIELD OF THE INVENTION
[0002] The present invention relates to the production and
screening of expression libraries for enzyme activity and, more
particularly, to obtaining selected polynucleotides from nucleic
acid of a microorganism and to screening of an expression library
for enzyme activity which is produced from selected
polynucleotides.
BACKGROUND OF THE INVENTION
[0003] There is a critical need in the chemical industry for
efficient catalysts for the practical synthesis of optically pure
materials; enzymes can provide the optimal solution. All classes of
molecules and compounds that are utilized in both established and
emerging chemical, pharmaceutical, textile, food and feed,
detergent markets must meet stringent economical and environmental
standards. The synthesis of polymers, pharmaceuticals, natural
products and agrochemicals is often hampered by expensive processes
which produce harmful byproducts and which suffer from low
enantioselectivity. Enzymes have a number of remarkable advantages
which can overcome these problems in catalysis: they act on single
functional groups, they distinguish between similar functional
groups on a single molecule, and they distinguish between
enantiomers. Moreover, they are biodegradable and function at very
low mole fractions in reaction mixtures. Because of their chemo-,
regio- and stereospecificity, enzymes present a unique opportunity
to optimally achieve desired selective transformations. These are
often extremely difficult to duplicate chemically, especially in
single-step reactions. The elimination of the need for protection
groups, selectivity, the ability to carry out multi-step
transformations in a single reaction vessel, along with the
concomitant reduction in environmental burden, has led to the
increased demand for enzymes in chemical and pharmaceutical
industries. Enzyme-based processes have been gradually replacing
many conventional chemical-based methods. A current limitation to
more widespread industrial use is primarily due to the relatively
small number of commercially available enzymes. Only .about.300
enzymes (excluding DNA modifying enzymes) are at present
commercially available from the >3000 non DNA-modifying enzyme
activities thus far described.
[0004] The use of enzymes for technological applications also may
require performance under demanding industrial conditions. This
includes activities in environments or on substrates for which the
currently known arsenal of enzymes was not evolutionarily selected.
Enzymes have evolved by selective pressure to perform very specific
biological functions within the milieu of a living organism, under
conditions of mild temperature, pH and salt concentration. For the
most part, the non-DNA modifying enzyme activities thus far
described have been isolated from mesophilic organisms, which
represent a very small fraction of the available phylogenetic
diversity. The dynamic field of biocatalysis takes on a new
dimension with the help of enzymes isolated from microorganisms
that thrive in extreme environments. Such enzymes must function at
temperatures above 100.degree. C. in terrestrial hot springs and
deep sea thermal vents, at temperatures below 0.degree. C. in
arctic waters, in the saturated salt environment of the Dead Sea,
at pH values around 0 in coal deposits and geothermal sulfur-rich
springs, or at pH values greater than 11 in sewage sludge. Enzymes
obtained from these extremophilic organisms open a new field in
biocatalysis.
[0005] In addition to the need for new enzymes for industrial use,
there has been a dramatic increase in the need for bioactive
compounds with novel activities. This demand has arisen largely
from changes in worldwide demographics coupled with the clear and
increasing trend in the number of pathogenic organisms that are
resistant to currently available antibiotics. For example, while
there has been a surge in demand for antibacterial drugs in
emerging nations with young populations, countries with aging
populations, such as the U.S., require a growing repertoire of
drugs against cancer, diabetes, arthritis and other debilitating
conditions. The death rate from infectious diseases has increased
58% between 1980 and 1992 and it has been estimated that the
emergence of antibiotic resistant microbes has added in excess of
$30 billion annually to the cost of health care in the U.S. alone.
(Adams et al., Chemical and Engineering News, 1995; Amann et al.,
Microbiological Reviews, 59, 1995). As a response to this trend
pharmaceutical companies have significantly increased their
screening of microbial diversity for compounds with unique
activities or specificities.
[0006] There are several common sources of lead compounds (drug
candidates), including natural product collections, synthetic
chemical collections, and synthetic combinatorial chemical
libraries, such as nucleotides, peptides, or other polymeric
molecules. Each of these sources has advantages and disadvantages.
The success of programs to screen these candidates depends largely
on the number of compounds entering the programs, and
pharmaceutical companies have to date screened hundred of thousands
of synthetic and natural compounds in search of lead compounds.
Unfortunately, the ratio of novel to previously-discovered
compounds has diminished with time. The discovery rate of novel
lead compounds has not kept pace with demand despite the best
efforts of pharmaceutical companies. There exists a strong need for
accessing new sources of potential drug candidates.
[0007] The majority of bioactive compounds currently in use are
derived from soil microorganisms. Many microbes inhabiting soils
and other complex ecological communities produce a variety of
compounds that increase their ability to survive and proliferate.
These compounds are generally thought to be nonessential for growth
of the organism and are synthesized with the aid of genes involved
in intermediary metabolism hence their name--"secondary
metabolites". Secondary metabolites that influence the growth or
survival of other organisms are known as "bioactive" compounds and
serve as key components of the chemical defense arsenal of both
micro- and macroorganisms. Humans have exploited these compounds
for use as antibiotics, antiinfectives and other bioactive
compounds with activity against a broad range of prokaryotic and
eukaryotic pathogens. Approximately 6,000 bioactive compounds of
microbial origin have been characterized, with more than 60%
produced by the gram positive soil bacteria of the genus
Streptomyces. (Barnes et al., Proc. Nat. Acad. Sci. U.S.A., 91,
1994). Of these, at least 70 are currently used for biomedical and
agricultural applications. The largest class of bioactive
compounds, the polyketides, include a broad range of antibiotics,
immunosuppressants and anticancer agents which together account for
sales of over $5 billion per year.
[0008] Despite the seemingly large number of available bioactive
compounds, it is clear that one of the greatest challenges facing
modern biomedical science is the proliferation of antibiotic
resistant pathogens. Because of their short generation time and
ability to readily exchange genetic information, pathogenic
microbes have rapidly evolved and disseminated resistance
mechanisms against virtually all classes of antibiotic compounds.
For example, there are virulent strains of the human pathogens
Staphylococcus and Streptococcus that can now be treated with but a
single antibiotic, vancomycin, and resistance to this compound will
require only the transfer of a single gene, vana, from resistant
Enterococcus species for this to occur. (Bateson et al., System.
Appl. Microbiol, 12, 1989). When this crucial need for novel
antibacterial compounds is superimposed on the growing demand for
enzyme inhibitors, immunosuppressants and anti-cancer agents it
becomes readily apparent why pharmaceutical companies have stepped
up their screening of microbial diversity for bioactive compounds
with novel properties.
SUMMARY OF THE INVENTION
[0009] The present invention provides a novel approach for
obtaining enzymes for further use, for example, for a wide variety
of industrial applications, for medical applications, for packaging
into kits for use as research reagents and for other applications.
In accordance with the present invention, recombinant enzymes are
generated from microorganisms and are classified by various enzyme
characteristics.
[0010] More particularly, one aspect of the present invention
provides a process for identifying clones having a specified enzyme
activity, which process comprises screening for said specified
enzyme activity in a library of clones prepared by:
[0011] (i) selectively isolating target RNA or genomic DNA or
fragments thereof, from nucleic acid derived from at least one
microorganism, by use of at least one probe polynucleotide
comprising at least a portion of a polynucleotide sequence encoding
an enzyme having the specified enzyme activity; and
[0012] (ii) transforming a host with isolated target cDNA, genomic
DNA or fragments thereof, to produce a library of clones which are
screened, preferably for the specified enzyme activity, using an
activity library screening or nucleic acid library screening
protocol.
[0013] In a preferred embodiment of this aspect, nucleic acid
obtained from at least one microorganism is selected by recovering
from the nucleic acid, polynucleotides which specifically bind,
such as by hybridization, to a probe polynucleotide sequence. The
nucleic acid obtained from the microorganism or microorganisms can
be genomic DNA, RNA or genomic gene library DNA. One could even use
nucleic acid prepared for vector ligation, for instance. The probe
may be directly or indirectly bound to a solid phase by which it is
separated from the nucleic acid which is not hybridized or
otherwise specifically bound to the probe. The process can also
include releasing nucleic acid from said probe after recovering
said hybridized or otherwise bound nucleic acid and amplifying the
nucleic acid so released.
[0014] The invention also provides for screening of the expression
libraries for gene cluster protein product(s) and, more
particularly, to obtaining selected gene clusters from nucleic acid
of a prokaryote or eukaryote and to screening of an expression
library for a desired activity of a protein of related
activity(ies) of a family of proteins which results from expression
of the selected gene cluster nucleic acid of interest.
[0015] More particularly, one embodiment of this aspect provides a
process for identifying clones having a specified protein(s)
activity, which process comprises screening for said specified
enzyme activity in the library of clones prepared by (i)
selectively isolating target gene cluster nucleic acid, from
nucleic acid derived from at least one organism, by use of at least
one probe polynucleotide comprising at least a portion of a
polynucleotide sequence complementary to a nucleic acid sequence
encoding the protein(s) having the specified activity of interest;
and (ii) transforming a host with isolated target gene cluster
nucleic acid to produce a library of such clones which are screened
for the specified activity of interest. For example, if one is
using DNA in a lambda vector one could package the DNA and infect
cells via this route.
[0016] In a particular embodiment of this aspect, gene cluster
nucleic acid obtained from the genomic nucleic acid of the
organism(s) is selected by recovering from the nucleic acid,
nucleic acid which specifically binds, such as by hybridization, to
a probe polynucleotide sequence. The polynucleotide probe may be
directly or indirectly bound to a solid phase by which it is
separated from the nucleic acid which is not hybridized or
otherwise specifically bound to the probe. This embodiment of this
aspect of the process of the invention can also include releasing
bound nucleic acid from said probe after recovering said hybridized
or otherwise bound nucleic acid and amplifying the nucleic acid so
released.
[0017] These and other aspects of the present invention will be
apparent to those skilled in the art from the teachings herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1A shows a photograph of an agarose gel containing
standards and samples a-f described in Example 2. Samples c-f
represent DNA recovered from a genomic DNA library using two
specific DNA probes and amplified using gene specific primers, as
described in Example 2.
[0019] FIG. 1B shows a photograph of an agarose gel containing
standards and samples a-f described in Example 2. Samples c-f
represent DNA recovered from a genomic DNA library using two
specific DNA probes and amplified using vector specific primers, as
described in Example 2.
[0020] FIG. 2 shows a photograph of four colony hybridization
plates. Plates A and B showed positive clones i.e., colonies which
contained DNA prepared in accordance with the present invention,
also contained probe sequence. Plates C and D were controls and
showed no positive clones.
DETAILED DESCRIPTION OF THE INVENTION
[0021] Novel systems to clone and screen for enzymatic activities
and bioactivities of interest in vitro are desirable. The method(s)
of the present invention allow the cloning and discovery of novel
bioactive molecules in vitro, and in particular novel bioactive
molecules derived from uncultivated samples Large size gene
clusters, genes and gene fragments can be cloned and screened using
the method(s) of the present invention. Unlike previous strategies,
the method(s) of the present invention allow one to clone utilizing
well known genetic systems, and to screen in vitro with crude
(impure) preparations.
[0022] The present invention allows one to screen for and identify
genes encoding enzymatic activities and bioactivities of interest
from complex environmental gene expression libraries. The
microorganisms from which the libraries may be prepared include
prokaryotic microorganisms, such as Eubacteria and Archaebacteria,
and lower eukaryotic microorganisms such as fungi, some algae and
protozoa. The microorganisms may be cultured microorganisms or
uncultured microorganisms obtained from environmental samples and
such microorganisms may be extremophiles, such as thermophiles,
hyperthermophiles, psychrophiles and psychrotrophs.
[0023] As previously indicated, the library may be produced from
environmental samples in which case nucleic acid may be recovered
without culturing of an organism or the nucleic acid may be
recovered from a cultured organism.
[0024] Sources of microorganism nucleic acid as a starting material
library from which target nucleic acid is obtained are particularly
contemplated to include environmental samples, such as microbial
samples obtained from Arctic and Antarctic ice, water or permafrost
sources, materials of volcanic origin, materials from soil or plant
sources in tropical areas, etc. Thus, for example, nucleic acid may
be recovered from either a culturable or non-culturable organism
and employed to produce an appropriate recombinant expression
library for subsequent determination of enzyme activity.
[0025] Bacteria and many eukaryotes have a coordinated mechanism
for regulating genes whose products are involved in related
processes. The genes are clustered, in structures referred to as
"gene clusters," on a single chromosome and are transcribed
together under the control of a single regulatory sequence,
including a single promoter which initiates transcription of the
entire cluster. The gene cluster, the promoter, and additional
sequences that function in regulation altogether are referred to as
an "operon" and can include up to 20 or more genes, usually from 2
to 6 genes. Thus, a gene cluster is a group of adjacent genes that
are either identical or related, usually as to their function.
[0026] Some gene families consist of identical members. Clustering
is a prerequisite for maintaining identity between genes, although
clustered genes are not necessarily identical. Gene clusters range
from extremes where a duplication is generated to adjacent related
genes to cases where hundreds of identical genes lie in a tandem
array. Sometimes no significance is discernable in a repetition of
a particular gene. A principal example of this is the expressed
duplicate insulin genes in some species, whereas a single insulin
gene is adequate in other mammalian species.
[0027] It is important to further research gene clusters and the
extent to which the full length of the cluster is necessary for the
expression of the proteins resulting therefrom. Further, gene
clusters undergo continual reorganization and, thus, the ability to
create heterogeneous libraries of gene clusters from, for example,
bacterial or other prokaryote sources is valuable in determining
sources of novel proteins, particularly including enzymes such as,
for example, the polyketide synthases that are responsible for the
synthesis of polyketides having a vast array of useful activities.
Other types of proteins that are the product(s) of gene clusters
are also contemplated, including, for example, antibiotics,
antivirals, antitumor agents and regulatory proteins, such as
insulin.
[0028] Polyketides are molecules which are an extremely rich source
of bioactivities, including antibiotics (such as tetracyclines and
erythromycin), anti-cancer agents (daunomycin), immunosuppressants
(FK506 and rapamycin), and veterinary products (monensin). Many
polyketides (produced by polyketide synthases) are valuable as
therapeutic agents. Polyketide synthases are multifunctional
enzymes that catalyze the biosynthesis of a hugh variety of carbon
chains differing in length and patterns of functionality and
cyclization. Polyketide synthase genes fall into gene clusters and
at least one type (designated type I) of polyketide synthases have
large size genes and enzymes, complicating genetic manipulation and
in vitro studies of these genes/proteins.
[0029] The ability to select and combine desired components from a
library of polyketides and postpolyketide biosynthesis genes for
generation of novel polyketides for study is appealing. The
method(s) of the present invention make it possible to and
facilitate the cloning of novel polyketide synthases, since one can
generate gene banks with clones containing large inserts
(especially when using the f-factor based vectors), which
facilitates cloning of gene clusters.
[0030] Preferably, the gene cluster nucleic acid is ligated into a
vector, particularly wherein a vector further comprises expression
regulatory sequences which can control and regulate the production
of a detectable protein or protein-related array activity from the
ligated gene clusters. Use of vectors which have an exceptionally
large capacity for exogenous nucleic acid introduction are
particularly appropriate for use with such gene clusters and are
described by way of example herein to include the f-factor (or
fertility factor) of E. coli. This f-factor of E. coli is a plasmid
which affect high-frequency transfer of itself during conjugation
and is ideal to achieve and stably propagate large nucleic acid
fragments, such as gene clusters from mixed microbial samples.
[0031] The term "isolated" means that material is removed from its
original environment (e.g., the natural environment if it is
naturally occurring). For example, a naturally-occurring
polynucleotide or polypeptide present in a living animal is not
isolated, but the same polynucleotide or polypeptide separated from
some or all of the coexisting materials in the natural system, is
isolated.
[0032] The nucleic acid isolated or derived from these
microorganisms can preferably be inserted into a vector or a
plasmid prior to probing for selected polynucleotides. Such vectors
or plasmids are preferably those containing expression regulatory
sequences, including promoters, enhancers and the like. Such
polynucleotides can be part of a vector and/or a composition and
still be isolated, in that such vector or composition is not part
of its natural environment. Particularly preferred phage or plasmid
and methods for introduction and packaging into them are described
in detail in the protocol set forth herein.
[0033] The following outlines a general procedure for producing
libraries from both culturable and non-culturable organisms, which
libraries can be probed to select therefrom nucleic acid sequences
which hybridize to specified probe polynucleotides:
[0034] ENVIRONMENTAL SAMPLE
[0035] Obtain Biomass
[0036] nucleic acid Isolation (various methods for DNA and RNA
isolation)
[0037] For Example:
[0038] Shear DNA (25 gauge needle)
[0039] Blunt DNA (Mung Bean Nuclease)
[0040] Methylate DNA (EcoR I Methylase)
[0041] Ligate to EcoR I linkers (GGAATTCC)
[0042] Cut back linkers (EcoR I Restriction Endonuclease)
[0043] Size Fractionate (Sucrose Gradient)
[0044] Ligate to lambda vector
[0045] Package (in vitro lambda packaging extract)
[0046] Plate on E. coli host and amplify
[0047] The probe polynucleotide used for selectively isolating the
target nucleic acid of interest from the nucleic acid derived from
at least one microorganism can be a full-length coding region
sequence or a partial coding region sequence of nucleic acid for an
enzyme of known activity. The original nucleic acid library can be
preferably probed using mixtures of probes comprising at least a
portion of nucleic acid sequences encoding enzymes having the
specified enzyme activity. These probes or probe libraries are
preferably single-stranded and the microbial nucleic acid which is
probed has preferably been converted into single-stranded form. The
probes that are particularly suitable are those derived from
nucleic acid encoding enzymes having an activity similar or
identical to the specified enzyme activity which is to be
screened.
[0048] The probe polynucleotide should be at least about 10 bases
and preferably at least 15 bases. In one embodiment, the entire
coding region may be employed as a probe. Conditions for the
hybridization in which target nucleic acid is selectively isolated
by the use of at least one polynucleotide probe will be designed to
provide a hybridization stringency of at least about 50% sequence
identity, more particularly a stringency providing for a sequence
identity of at least about 70%.
[0049] Hybridization techniques for probing a microbial nucleic
acid library to isolate target nucleic acid of potential interest
are well known in the art and any of those which are described in
the literature are suitable for use herein to probe nucleic acid
for separation from the remainder of the nucleic acid derived from
the microorganisms. Solution phase hybridizations followed by
binding of the probe to a solid phase is preferable.
[0050] Preferably the probe polynucleotide is "labeled" with one
partner of a specific binding pair (i.e. a ligand) and the other
partner of the pair is bound to a solid matrix to provide ease of
separation of target from its source. The ligand and specific
binding partner can be selected from, in either orientation, the
following: (1) an antigen or hapten and an antibody or specific
binding fragment thereof; (2) biotin or iminobiotin and avidin or
streptavidin; (3) a sugar and a lectin specific therefor; (4) an
enzyme and an inhibitor therefor; (5) an apoenzyme and cofactor;
(6) complementary homopolymeric oligonucleotides; and (7) a hormone
and a receptor therefor. The solid phase is preferably selected
from: (1) a glass or polymeric surface; (2) a packed column of
polymeric beads; and (3) magnetic or paramagnetic particles.
[0051] Further, it is optional but desirable to perform an
amplification of the target nucleic acid that has been isolated. In
this embodiment the target nucleic acid is separated from the probe
polynucleotide after isolation. It is then amplified before being
used to transform hosts. The double stranded nucleic acid selected
to include as at least a portion thereof a predetermined nucleic
acid sequence can be rendered single stranded, subjected to
amplification and reannealed to provide amplified numbers of
selected double stranded nucleic acid. Numerous amplification
methodologies are now well known in the art.
[0052] The selected nucleic acid is then used for preparing a
library for screening by transforming a suitable organism. Hosts,
particularly those specifically identified herein as preferred, are
transformed by artificial introduction of the vectors containing
the target nucleic acid by inoculation under conditions conducive
for such transformation. One could transform with double stranded
circular or linear nucleic acid or there may also be instances
where one would transform with single stranded circular or linear
nucleic acid.
[0053] The resultant libraries of transformed clones are then
screened for clones which display activity for the enzyme of
interest in a phenotypic assay for enzyme activity.
[0054] Having prepared a multiplicity of clones from nucleic acid
selectively isolated from an organism, such clones are screened for
a specific enzyme activity and to identify the clones having the
specified enzyme characteristics.
[0055] The screening for enzyme activity may be effected on
individual expression clones or may be initially effected on a
mixture of expression clones to ascertain whether or not the
mixture has one or more specified enzyme activities. If the mixture
has a specified enzyme activity, then the individual clones may be
rescreened for such enzyme activity or for a more specific
activity. Thus, for example, if a clone mixture has hydrolase
activity, then the individual clones may be recovered and screened
to determine which of such clones has hydrolase activity.
[0056] As described with respect to one of the above aspects, the
invention provides a process for enzyme activity screening of
clones containing selected nucleic acid derived from a
microorganism which process comprises:
[0057] screening a library for specified enzyme activity, said
library including a plurality of clones, said clones having been
prepared by recovering from nucleic acid of a microorganism
selected nucleic acid, which nucleic acid is selected by
hybridization to at least one nucleic acid sequence which is all or
a portion of a nucleic acid sequence encoding an enzyme having the
specified activity; and
[0058] transforming a host with the selected nucleic acid to
produce clones which are screened for the specified enzyme
activity.
[0059] In one embodiment, a nucleic acid library derived from a
microorganism is subjected to a selection procedure to select
therefrom nucleic acid which hybridizes to one or more probe
nucleic acid sequences which is all or a portion of a nucleic acid
sequence encoding an enzyme having the specified enzyme activity
by:
[0060] (a) rendering -the double-stranded nucleic acid population
into a single-stranded nucleic acid population;
[0061] (b) contacting the single-stranded nucleic acid population
of (a) with the nucleic acid probe bound to a ligand under
conditions permissive of hybridization so as to produce a
double-stranded complex of probe and members of the nucleic acid
population which hybridize thereto;
[0062] (c) contacting the double-stranded complex of (b) with a
solid phase specific binding partner for said ligand so as to
produce a solid phase complex;
[0063] (d) separating the solid phase complex from the
single-stranded nucleic acid population of (b);
[0064] (e) releasing from the probe the members of the population
which had bound to the solid phase bound probe;
[0065] (f) forming double-stranded nucleic acid from the members of
the population of (e);
[0066] (g) introducing the double-stranded nucleic acid of (f) into
a suitable host to form a library containing a plurality of clones
containing the selected nucleic acid; and
[0067] (h) screening the library for the specified enzyme
activity.
[0068] In another embodiment, a nucleic acid library derived from a
microorganism is subjected to a selection procedure to select
therefrom double-stranded nucleic acid which hybridizes to one or
more probe polynucleotide sequences which is all or a portion of a
nucleic acid sequence encoding an enzyme having the specified
enzyme activity by:
[0069] (a) contacting the double-stranded nucleic acid population
with the polynucleotide probe bound to a ligand under conditions
permissive of hybridization so as to produce a complex of probe and
members of the nucleic acid population which hybridize thereto;
[0070] (b) contacting the complex of (a) with a solid phase
specific binding partner for said ligand so as to produce a solid
phase complex;
[0071] (c) separating the solid phase complex from the unbound
nucleic acid population of (b);
[0072] (d) releasing from the probe the members of the population
which had bound to the solid phase bound probe;
[0073] (e) introducing the double-stranded nucleic acid of (d) into
a suitable host to form a library containing a plurality of clones
containing the selected nucleic acid; and
[0074] (f) screening the library for the specified enzyme
activity.
[0075] In another aspect, the process includes a preselection to
recover nucleic acid including signal or secretion sequences. In
this manner it is possible to select from the nucleic acid
population by hybridization as hereinabove described only nucleic
acid which includes a signal or secretion sequence. The following
paragraphs describe the protocol for this embodiment of the
invention, the nature and function of secretion signal sequences in
general and a specific exemplary application of such sequences to
an assay or selection process.
[0076] Another particularly preferred embodiment of this aspect
further comprises, after (a) but before (a) above, the steps
of:
[0077] (i). contacting the double-stranded nucleic acid population
of (a) with a ligand-bound oligonucleotide probe that is
complementary to a secretion signal sequence unique to a given
class of proteins under conditions permissive of hybridization to
form a double-stranded complex;
[0078] (ii). contacting the complex of (a i) with a solid phase
specific binding partner for said ligand so as to produce a solid
phase complex;
[0079] (iii) separating the solid phase complex from the unbound
nucleic acid population;
[0080] (iv) releasing the members of the population which had bound
to said solid phase bound probe; and
[0081] (v) separating the solid phase bound probe from the members
of the population which had bound thereto.
[0082] The nucleic acid which has been selected and isolated to
include a signal sequence is then subjected to the selection
procedure hereinabove described to select and isolate therefrom
nucleic acid which binds to one or more probe nucleic acid
sequences derived from nucleic acid encoding an enzyme(s) having
the specified enzyme activity.
[0083] The pathways by which proteins are sorted and transported to
their proper cellular location are often referred to as protein
targeting pathways. One of the most important elements in all of
these targeting systems is a short amino acid sequence at the amino
terminus of a newly synthesized polypeptide called the signal
sequence. This signal sequence directs a protein to its appropriate
location in the cell and is removed during transport or when the
protein reaches its final destination. Most lysosomal, membrane, or
secreted proteins have an amino-terminal signal sequence that marks
them for translocation into the lumen of the endoplasmic reticulum.
More than 100 signal sequences for proteins in this group have been
determined. The sequences vary in length from 13 to 36 amino acid
residues.
[0084] A phoA expression vector, termed pMG, which, like TaphoA, is
useful in identifying genes encoding membrane-spanning sequences or
signal peptides. Giladi et al., J. Bacteriol., 175(13):41294136,
1993. This cloning system has been modified to facilitate the
distinction of outer membrane and periplasmic alkaline phosphatase
(AP) fusion proteins from inner membrane AP fusion proteins by
transforming pMG recombinants into E. coli KS330, the strain
utilized in the "blue halo" assay first described by Strauch and
Beckwith, Proc. Nat. Acad. Sci. USA, 85:15761580, 1988. The
pMG/KS330r.sup.- cloning and screening approach can identify genes
encoding proteins with clevable signal peptides and therefore can
serve as a first step in the identification of genes encoding
polypeptides of interest.
[0085] The nucleic acid derived from a microorganism(s) is
preferably inserted into an appropriate vector (generally a vector
containing suitable regulatory sequences for effecting expression)
prior to subjecting such nucleic acid to a selection procedure to
select and isolate therefrom nucleic acid which hybridizes to
nucleic acid derived from nucleic acid encoding an enzyme(s) having
the specified enzyme activity.
[0086] As representative examples of expression vectors which may
be used there may be mentioned viral particles, baculovirus, phage,
plasmids, phagemids, cosmids, phosmids, bacterial artificial
chromosomes, viral nucleic acid (e.g. vaccinia, adenovirus, foul
pox virus, pseudorabies and derivatives of SV40), P1-based
artificial chromosomes, yeast plasmids, yeast artificial
chromosomes, and any other vectors specific for specific hosts of
interest (such as bacillus, aspergillus, yeast, etc.) Thus, for
example, the DNA may be included in any one of a variety of
expression vectors for expressing a polypeptide. Such vectors
include chromosomal, nonchromosomal and synthetic DNA sequences.
Large numbers of suitable vectors are known to those of skill in
the art, and are commercially available. The following vectors are
provided by way of example; Bacterial: pQE70, pQE60, pQE-9
(Qiagen), psiXl74, pBluescript SK, pBluescript KS, pNH8A, pNH16a,
pNH18A, pNH46A (Stratagene); pTRC99a, pKK223-3, pKK233-3, pDR540,
pRIT5 (Pharmacia); Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG
(Stratagene), pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any
other plasmid or vector may be used as long as they are replicable
and viable in the host.
[0087] A particularly preferred type of vector for use in the
present invention contains an f-factor origin replication. The
f-factor (or fertility factor) in E. coli is a plasmid which
effects high frequency transfer of itself during conjugation and
less frequent transfer of the bacterial chromosome itself. A
particularly preferred embodiment is to use cloning vectors,
referred to as "fosmids" or bacterial artificial chromosome (BAC)
vectors. These are derived from E. coli f-factor which is able to
stably integrate large segments of DNA. When integrated with DNA
from a mixed uncultured environmental sample, this makes it
possible to achieve large genomic fragments in the form of a stable
"environmental DNA library."
[0088] The nucleic acid derived from a microorganism(s) may be
inserted into the vector by a variety of procedures. In general,
the nucleic acid sequence is inserted into an appropriate
restriction endonuclease site(s) by procedures known in the art.
Such procedures and others are deemed to be within the scope of
those skilled in the art.
[0089] The nucleic acid sequence in the expression vector is
operatively linked to an appropriate expression control sequence(s)
(promoter) to direct mRNA synthesis. Particular named bacterial
promoters include lac, lacZ, T3, T7, gpt, lambda P.sub.R, P.sub.L
and trp. Eukaryotic promoters include CMV immediate early, HSV
thymidine kinase, early and late SV40, LTRs from retrovirus, and
mouse metallothionein-I. Selection of the appropriate vector and
promoter is well within the level of ordinary skill in the art. The
expression vector also contains a ribosome binding site for
translation initiation and a transcription terminator. The vector
may also include appropriate sequences for amplifying expression.
Promoter regions can be selected from any desired gene using CAT
(chloramphenicol transferase) vectors or other vectors with
selectable markers.
[0090] In addition, the expression vectors preferably contain one
or more selectable marker genes to provide a phenotypic trait for
selection of transformed host cells such as dihydrofolate reductase
or neomycin resistance for eukaryotic cell culture, or such as
tetracycline or ampicillin resistance in E. coli.
[0091] Generally, recombinant expression vectors will include
origins of replication and selectable markers permitting
transformation of the host cell, e.g., the ampicillin resistance
gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived
from a highly-expressed gene to direct transcription of a
downstream structural sequence. Such promoters can be derived from
operons encoding glycolytic enzymes such as 3-phosphoglycerate
kinase (PGK), .alpha.-factor, acid phosphatase, or heat shock
proteins, among others. The heterologous structural sequence is
assembled in appropriate phase with translation initiation and
termination sequences, and preferably, a leader sequence capable of
directing secretion of translated protein into the periplasmic
space or extracellular medium.
[0092] The nucleic acid selected and isolated as hereinabove
described is introduced into a suitable host to prepare a library
which is screened for the desired enzyme activity. The selected
nucleic acid is preferably already in a vector which includes
appropriate control sequences whereby selected nucleic acid which
encodes for an enzyme may be expressed, for detection of the
desired activity. The host cell can be a higher eukaryotic cell,
such as a mammalian cell, or a lower eukaryotic cell, such as a
yeast cell, or the host cell can be a prokaryotic cell, such as a
bacterial cell. Introduction of the construct into the host cell
can be effected by calcium phosphate transfection, DEAE-Dextran
mediated transfection, or electroporation (Davis, L., Dibner, M.,
Battey, I., Basic Methods in Molecular Biology, (1986)).
[0093] As representative examples of appropriate hosts, there may
be mentioned: bacterial cells, such as E. coli, Streptonyces,
Salmonella typhimurium; fungal cells, such as yeast; insect cells
such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO,
COS or Bowes melanoma; adenoviruses; plant cells, etc. The
selection of an appropriate host is deemed to be within the scope
of those skilled in the art from the teachings herein.
[0094] With particular references to various mammalian cell culture
systems that can be employed to express recombinant protein,
examples of mammalian expression systems include the COS-7 lines of
monkey kidney fibroblasts, described by Gluzman, Cell, 23:175
(1981), and other cell lines capable of expressing a compatible
vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.
Mammalian expression vectors will comprise an origin of
replication, a suitable promoter and enhancer, and also any
necessary ribosome binding sites, polyadenylation site, splice
donor and acceptor sites, transcriptional termination sequences,
and 5' flanking nontranscribed sequences. nucleic acid sequences
derived from the SV40 splice, and polyadenylation sites may be used
to provide the required nontranscribed genetic elements.
[0095] Host cells are genetically engineered (transduced or
transformed or transfected) with the vectors. The engineered host
cells can be cultured in conventional nutrient media modified as
appropriate for activating promoters, selecting transformants or
amplifying genes. The culture conditions, such as temperature, pH
and the like, are those previously used with the host cell selected
for expression, and will be apparent to the ordinarily skilled
artisan.
[0096] The library may be screened for a specified enzyme activity
by procedures known in the art. For example, the enzyme activity
may be screened for one or more of the six IUB classes;
oxid6reductases, transferases, hydrolases, lyases, isomerases and
ligases. The recombinant enzymes which are determined to be
positive for one or more of the IUB classes may then be rescreened
for a more specific enzyme activity.
[0097] Alternatively, the library may be screened for a more
specialized enzyme activity. For example, instead of generically
screening for hydrolase activity, the library may be screened for a
more specialized activity, i.e. the type of bond on which the
hydrolase acts. Thus, for example, the library may be screened to
ascertain those hydrolases which act on one or more specified
chemical functionalities, such as: (a) amide (peptide bonds), i.e.
proteases; (b) ester bonds, i.e. esterases and lipases; (c)
acetals, i.e., glycosidases.
[0098] The clones which are identified as having the specified
enzyme activity may then be sequenced to identify the nucleic acid
sequence encoding an enzyme having the specified activity. Thus, in
accordance with the present invention it is possible to isolate and
identify: (i) nucleic acid encoding an enzyme having a specified
enzyme activity, (ii) enzymes having such activity (including the
amino acid sequence thereof) and (iii) produce recombinant enzymes
having such activity.
[0099] Having thus disclosed exemplary embodiments of the present
invention, it should be noted by those skilled in the art that the
disclosures are exemplary only and that various other alternatives,
adaptations and modifications may be made within the scope of the
present invention. Accordingly, the present invention is not
limited to the specific embodiments as illustrated herein.
[0100] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description, utilize the
present invention to its fullest extent. The following examples are
to be considered illustrative and thus are not limiting of the
remainder of the disclosure in any way whatsoever.
EXAMPLE 1
Examples of Enzymes Which can be Identified by the Invention
[0101] Lipase/Esterase
[0102] a. Enantioselective hydrolysis of esters
(lipids)/thioesters
[0103] 1) Resolution of racemic mixtures
[0104] 2) Synthesis of optically active acids or alcohols from
meso-diesters
[0105] b. Selective syntheses
[0106] 1) Regiospecific hydrolysis of carbohydrate esters
[0107] 2) Selective hydrolysis of cyclic secondary alcohols
[0108] c. Synthesis of optically active esters, lactones, acids,
alcohols
[0109] 1) Transesterification of activated/nonactivated esters
[0110] 2) Interesterification
[0111] 3) Optically active lactones from hydroxyesters
[0112] 4) Regio- and enantioselective ring opening of
anhydrides
[0113] d. Detergents
[0114] e. Fat/Oil conversion
[0115] f. Cheese ripening
[0116] 2 Protease
[0117] a. Ester/amide synthesis
[0118] b. Peptide synthesis
[0119] c. Resolution of racemic mixtures of amino acid esters
[0120] d. Synthesis of non-natural amino acids
[0121] e. Detergents/protein hydrolysis
[0122] 3 Glycosidase/Glycosyl Transferase
[0123] a. Sugar/polymer synthesis
[0124] b. Cleavage of glycosidic linkages to form mono, di-and
oligosaccharides
[0125] c. Synthesis of complex oligosaccharides
[0126] d. Glycoside synthesis using UDP-galactosyl transferase
[0127] e. Transglycosylation of disaccharides, glycosyl fluorides,
aryl galactosides
[0128] f. Glycosyl transfer in oligosaccharide synthesis
[0129] g. Diastereoselective cleavage of
.beta.-glucosylsulfoxides
[0130] h. Asymmetric glycosylations
[0131] i. Food processing
[0132] j. Paper processing
[0133] 4 Phosphatase/Kinase
[0134] a. Synthesis/hydrolysis of phosphate esters
[0135] 1) Regio-, enantioselective phosphorylation
[0136] 2) Introduction of phosphate esters
[0137] 3) Synthesize phospholipid precursors
[0138] 4) Controlled polynucleotide synthesis
[0139] b. Activate biological molecule
[0140] c. Selective phosphate bond formation without protecting
groups
[0141] 5 Mono/Dioxygenase
[0142] a. Direct oxyfunctionalization of unactivated organic
substrates
[0143] b. Hydroxylation of alkane, aromatics, steroids
[0144] c. Epoxidation of alkenes
[0145] d. Enantioselective sulphoxidation
[0146] e. Regio- and stereoselective Bayer-Villiger oxidations
[0147] 6 Haloperoxidase
[0148] a. Oxidative addition of halide ion to nucleophilic
sites
[0149] b. Addition of hypohalous acids to olefinic bonds
[0150] c. Ring cleavage of cyclopropanes
[0151] d. Activated aromatic substrates converted to ortho and para
derivatives
[0152] e. 1.3 diketones converted to 2-halo-derivatives
[0153] f. Heteroatom oxidation of sulfur and nitrogen containing
substrates
[0154] g. Oxidation of enol acetates, alkynes and activated
aromatic rings
[0155] 7 Lignin Peroxidase/Diarylpropane Peroxidase
[0156] a. Oxidative cleavage of C-C bonds
[0157] b. Oxidation of benzylic alcohols to aldehydes
[0158] c. Hydroxylation of benzylic carbons
[0159] d. Phenol dimerization
[0160] e. Hydroxylation of double bonds to form diols
[0161] f. Cleavage of lignin aldehydes
[0162] 8 Epoxide Hydrolase
[0163] a. Synthesis of enantiomerically pure bioactive
compounds
[0164] b. Regio- and enantioselective hydrolysis of epoxide
[0165] c. Aromatic and olefinic epoxidation by monooxygenases to
form epoxides
[0166] d. Resolution of racemic epoxides
[0167] e. Hydrolysis of steroid epoxides
[0168] 9 Nitrile Hydratase/Nitrilase
[0169] a. Hydrolysis of aliphatic nitrites to carboxamides
[0170] b. Hydrolysis of aromatic, heterocyclic, unsaturated
aliphatic nitrites to corresponding acids
[0171] c. Hydrolysis of acrylonitrile
[0172] d. Production of aromatic and carboxamides, carboxylic acids
(nicotinamide, picolinamide, isonicotinamide)
[0173] e. Regioselective hydrolysis of acrylic dinitrile
[0174] f. .alpha.-amino acids from .alpha.-hydroxynitriles
[0175] 10 Transaminase
[0176] a. Transfer of amino groups into oxo-acids
[0177] 11 Amidase/Acylase
[0178] a. Hydrolysis of amides, amidines, and other C--N bonds
[0179] b. Non-natural amino acid resolution and synthesis
EXAMPLE 2
Preparation of a Genomic DNA Library in Lambda ZAPII
[0180] Cloning DNA fragments prepared by random cleavage of the
target DNA generates the most representative library. An aliquot of
DNA (50-100 .mu.g) isolated from biomass is prepared as
follows:
[0181] The DNA is sheared by vigorous passage through a 25 gauge
double-hub needle attached to 1-ml syringes. An aliquot (0.5 .mu.g)
is electrophoresed through a 0.8% agarose gel to confirm that the
majority of the sheared DNA is within the desired size range (3-6
kb).
[0182] The sheared DNA is "polished" or made blunt-ended by
treatment with mung bean nuclease. First, the sheared DNA is
brought up to a volume of 405 .mu.l with Tris/EDTA (TE buffer) and
incubated with lOx mung bean buffer (45 .mu.l) and mung bean
nuclease (2.0 .mu.l, 150 .mu.l) for 15 minutes at 37.degree. C. The
reaction is extracted once with phenol/chloroform and then once
with chloroform alone. The DNA is precipitated by adding ice-cold
ethanol (1 ml) and is placed on ice for 10 minutes. The precipitate
is spun in a microcentrifuge (high speed, 30 minutes), washed with
70% ethanol (1 ml), microcentrifuged again (high speed, 10
minutes), dried and resuspended in TE buffer (26 .mu.l).
[0183] EcoR I sites in the DNA must be protected from future
enzymatic reactions. To accomplish this, the DNA is incubated with
10.times.EcoR I methylase (5.0 .mu.l, 40 U/.mu.l) for 1 hour at
37.degree. C.
[0184] The DNA is further treated to ensure that it is blunt-ended
by incubation with 100 mM MgCl.sub.2 (5.0 .mu.l), dNTP mix (8.0
.mu.l, 2.5 mM of each dGTP, dATP, dCTP, dTTP) and DNA polymerase I
large (Klenow) fragment (4.0 .mu.l, 5 U/.mu.l) for 30 minutes at
12.degree. C. Then, 1.times.sodium chloride/Tris/EDTA (STE buffer)
450 .mu.l) is added and the reaction is extracted once with
phenol/chloroform and then once with chloroform alone. The DNA is
precipitated by adding ice-cold ethanol (1 ml) and is placed on ice
for 10 minutes. The precipitate is spun in a microcentrifuge (high
speed, 30 minutes), washed with 70% ethanol (1 ml),
microcentrifuged again (high speed, 10 minutes), dried and
resuspended in TE buffer (7.0 .mu.l).
[0185] The blunt-ended DNA is made compatible with the vector
cloning site by ligating to EcoR I linkers using a very high molar
ratio of linkers to DNA. This lowers the probability of two DNA
molecules ligating together creating a chimeric clone and increases
the probability of linkers ligating to both ends of the DNA
molecules. The ligation reaction is performed by adding EcoR I
linkers [GGAATTCC] (14 .mu.l, 200 ng/.mu.l), 10.times.ligase buffer
(3.0 .mu.l), 10 mM rATP (3.0 .mu.l) and T4 DNA ligase (3.0 .mu.l, 4
WU/.mu.l) and incubating at 4.degree. C overnight.
[0186] The ligation reaction is terminated by heating to 68.degree.
C. for 10 minutes. The linkers are digested to create EcoR I
overhangs by incubation with water (238 .mu.l), 10.times.EcoR I
buffer (30 .mu.l) and EcoR I restriction endonuclease (2.0 .mu.l,
100 U/.mu.l) for 1.5 hours at 37.degree. C. The digestion reaction
is discontinued by adding 0.5M EDTA and the DNA is placed on
ice.
[0187] The DNA is size fractionated through a sucrose gradient
which is rapid, reliable and relatively free of inhibiting
contaminants. The removal of sub-optimal DNA fragments and the
small linkers is critical because ligation to the vector can result
in recombinant molecules that are too large and unpackageable by in
viro lambda packaging extracts or can result in the construction of
a "linker library." The DNA sample is heated to 65.degree. C. for
10 minutes and loaded on a 10-ml sucrose gradient (40% w/v). The
gradient is spun in an ultracentrifuge at room temperature, 25K for
16 hours. Fractions are collected from the gradient by puncturing
the bottom of the gradient tube with an 18 gauge needle and
collecting the sucrose solution which flows through the needle (10
drops per fraction). A small aliquot (20 .mu.l) of each fraction is
analyzed by 0.8% agarose gel electrophoresis and the fractions
containing DNA in the desired size range (3-6 kb) is precipitated
by adding ice cold ethanol (1 ml). The precipitate is spun in a
microcentrifuge (high speed, 30 minutes), washed with 70% ethanol
(1 ml), microcentrifuged again (high speed, 10 minutes), dried and
resuspended in TE buffer (5-10 .mu.l).
[0188] A plate assay is performed on the resuspended DNA to acquire
an approximate concentration by spotting 0.5 .mu.l of the DNA on
0.8% agarose containing ehtidium bromide (5 .mu.g/ml). The DNA is
visually compared to spotted DNA standards of known concentration
by viewing on a UV light box.
[0189] The DNA is ligated to Lambda ZAP II cloning vector arms
which were digested with EcoR I restriction enzyme and
dephosphorylated. The ligation reaction has a final volume of 5.0
.mu.l and contains 10.times.ligase buffer (0.5 .mu.l), 10 mM rATP
(0.5 .mu.l), Lambda ZAP II arms (1.0 .mu.l, 1.0 .mu.g/.mu.l), DNA
(.ltoreq.2.5 .mu.l, 200 ng), T4 DNA ligase (0.5 .mu.l, 4 WU/.mu.l)
and water (as needed to bring the final volume up to 5.0 .mu.l).
The ligation reaction is incubated overnight at 4.degree. C.
[0190] The ligation reaction is packaged using two in vitro lambda
packaging extracts (2.5 .mu.l of ligation per extract) in
accordance with the manufacturer's protocol. The packaging
reactions are stopped with the addition of sodium
chloride/MgSO.sub.4/Tris/gelatin (SM buffer) (500 .mu.l) and pooled
for a total volume of 1 ml per ligation reaction. The packaged
phage are titrated on a suitable host, for example, XL1-Blue MRF'
E. coli cells, as follows:
[0191] Host cells (200 .mu.l, OD.sub.600=1.0 in MgSO.sub.4) are
aliquotted into tubes, inoculated with the packaged phage (1 .mu.l)
and incubated for 15 minutes at 37.degree. C. Top agar (3 ml,
48.degree. C.) containing Isopropyl-.beta.-D-thio-galactopyranoside
(IPTG) (1.5 mM) and
5-bromo-chloro-3-indoyl-.alpha.-D-galactopyranoside (X-gal) (2.5
mglml) is added to each tube, plated onto 100-mm petri dishes
containing bottom agar and incubated at 37.degree. C. overnight.
The number of plaque forming units (pfu) are calculated as follows.
Typical results are 5.0.times.10.sup.5-1.0.times.10.sup.6 pfu/ml
with a 5% background (nonrecombinants).
(# clear pfu).times.(1,000 .mu.l packaged phage)=# recombinant
pfu/ml
[0192] A portion of the library (.gtoreq.2.5.times.10.sup.5 pfu) is
amplified as follows. Host cells (3 ml, OD.sub.600=1.0 in
MgSO.sub.4) are aliquotted into 50-ml conical tubes, inoculated
with packaged phage (.gtoreq.2.5.times.10.sup.5 pfu) and incubated
for 20 minutes at 37.degree. C. Top agar (40 ml, 48.degree. C.) is
added to each tube and plated across five 150-mm petri dishes
containing bottom agar and incubated at 37.degree. C. for 6-8 hours
or until plaques are about pinhead in size.
[0193] The phage particles are harvested by overlaying the plates
with SM buffer (8-10 ml) and maintaining the plates at 4.degree. C.
overnight with gentle rocking. The phage elute into the SM buffer
and are recovered by pouring the SM buffer off of each plate into a
50-ml conical tube. Chloroform (3 ml) is added to each tube which
is then shaken vigorously, incubated at room temperature for 15
minutes and centrifuged (2,000 rpm, 10 minutes) to remove cell
debris. The supernatant (amplified library) is then decanted into a
fresh 50-ml conical tube to which chloroform (500 .mu.l) is added
and stored at 4.degree. C. for later use.
[0194] The amplified library is titered as follows. Serial
dilutions of the amplified library are preapred in SM buffer
(10.sup.-5, 10.sup.-6). Host cells (200 .mu.l, OD.sub.600=1.0 in
MgSO.sub.4) are aliquotted into two tubes, inoculated with the
diluted phage (1 .mu.l from each dilution) and incubated for 15
minutes at 37.degree. C. Top agar (3 ml, 48.degree. C.) containing
Isopropyl-.beta.-D-thio-galactopyranoside (IPTG) (1.5 mM) and
5-bromo4-chloro-3-indoyl-.beta.-D-galactopyranoside (X-gal) (2.5
mg/ml) is added to each tube, plated onto 100-mm petri dishes
containing bottom agar and incubated at 37.degree. C. overnight.
The number of plaque forming units are calculated. Typical results
are 1.0.times.10.sup.10 pfu/ml with a 5% background
(nonrecombinants).
EXAMPLE 3
Hybridization Selection and Production of Expression Library
[0195] Starting with a plasmid library prepared as described in
Example 1, hybridization selection and preparation of the
expression library were performed according to the protocol
described in this example. The library can contain DNA from
isolated microorganisms, enriched cultures or environmental
samples.
[0196] Single-stranded DNA is made in one of two ways: 1) The
plasmid library can be grown and the double-stranded plasmid DNA
isolated. The double-stranded DNA is made single-stranded using F1
gene II protein and Exonuclease III. The gene II protein nicks the
double-stranded plasmids at the F1 origin and the Exo III digests
away the nicked strand leaving a single-stranded circle. This
method is used by Life Technologies in their GeneTrapper.TM. kit;
2) the second method involves the use of a helper phage to "rescue"
one of the strands of the double-stranded plasmids. The plasmid
library is grown in a small overnight culture. A small aliquot of
this is mixed with VCS-M13 helper phage and again grown overnight.
The next morning the phagemids (virus particles containing
single-stranded DNA) are recovered from the media and used in the
following protocol.
[0197] PROTOCOL
[0198] 1. Six samples of 4 .mu.g of rescued, single-stranded DNA
from library #17 were prepared in 3.times.SSC buffer. Final
reaction volumes were 30 .mu.l.
[0199] 2. To these solutions was added one of the following:
[0200] a) nothing
[0201] b) 100 ng of biotinylated probe from an unrelated
sequence
[0202] c,d) 100 ng of biotinylated probe from organism #13 DNA
polymerase gene
[0203] e,f) 100 ng of biotinylated probe from organism #17 DNA
polymerase gene
[0204] Biotinylated probes were prepared by PCR amplification of
fragments of .about.1300 bp in length coding for a portion of the
DNA polymerase gene of these organisms. The amplification products
were made using biotinylated dUTP in the amplification mix. This
modified nucleotide is incorporated throughout the DNA during
synthesis. Unincorporated nucleotides were removed using the QIAGEN
PCR Clean-up kit.
[0205] 3. These mixtures were denatured by heating to 95.degree. C.
for 2 minutes.
[0206] 4. Hybridization was performed for 90 minutes at 70.degree.
C. for samples a, b, d and f. Samples c and e were hybridized at
60.degree. C.
[0207] 5. 50 .parallel.l of washed and blocked MPG beads were added
and mixed to each sample. These mixtures were agitated every 5
minutes for a total of 30 minutes. MPG beads are sent at 1 mg/ml in
buffer containing preservative so 6 sets of 100 .mu.l were washed 2
times in 3.times.SSC and resuspended in 60 .mu.l of 3>SSC
containing 100 .mu.g of sonicated salmon sperm DNA.
[0208] 6. The DNA/bead mixtures were washed 2 times at room
temperature in 0.1.times.SSC/0. 1% SDS, 2 times at 42.degree. C. in
0.1.times.SSC/0.1% SDS for 10 minutes each and 1 additional wash at
room temperature with 3.times.SSC.
[0209] 7. The bound DNA was eluted by heating the beads to
70.degree. C. for 15 minutes in 50 .mu.l TE.
[0210] 8. Dilutions of the eluted DNAs were made and PCR
amplification was performed with either gene specific primers or
vectors specific primers. Dilutions of the library DNA were used as
standards.
[0211] 9. The DNA inserts contained within the DNA were amplified
by PCR using vector specific primers. These inserts were cloned
using the TA Cloning system (Invitrogen).
[0212] 10. Duplicates of 92 white colonies and 4 blue colonies from
samples d and f were grown overnight and colony lifts were prepared
for Southern blotting.
[0213] 11. The digoxigenin system from Boehringer Mannheim was used
to probe the colonies using the organism #17 probe.
[0214] RESULTS
[0215] PCR Quantitation
[0216] FIGS. 1A and 1B. FIG. 1A is a photograph of the
autoradiogram resulting from the Southern hybridization agarose gel
electrophoresis columns of DNA from sample solutions a-f in Example
2, when hybridized with gene specific primers. FIG. 1B is a
photograph of the autoradiogram resulting from the Southern
hybridization agarose gel electrophoresis columns of DNA from
sample solutions a-f in Example 2, when hybridized with vector
specific primers.
[0217] The gene specific DNA amplifications of samples a and b
demonstrate that non-specific binding to the beads is minimal. The
amount of DNA bound under the other conditions results in the
following estimates of enrichment.
1 gene specific equivalent total enrichment c 50 ng 100 pg 500X d
50 ng 30 pg 1667X e 20 ng 50 pg 400X f 20 ng 20 pg 1000X
[0218] Colony Hybridization
[0219] FIG. 2 is a photograph of four colony hybridization plates
resulting from Plates A and B show positive clones i.e., colonies
containing sequences contained in the probe and which contain DNA
from a library prepared in accordance with the invention. Plates C
and D were controls and showed no positive clones.
[0220] Seven of 92 colonies from the panned sample were positive
for sequences contained in the probe. No positive clones were found
in the unpanned sample.
EXAMPLE 4
Construction of a Stable, Large Insert DNA Library of Picoplankton
Genomic DNA
[0221] Cell collection and preparation of DNA. Agarose plugs
containing concentrated picoplankton cells were prepared from
samples collected on an oceanographic cruise from Newport, Oregon
to Honolulu, Hawaii. Seawater (30 liters) was collected in Niskin
bottles, screened through 10 .mu.m Nitex, and concentrated by
hollow fiber filtration (Amicon DC10) through 30,000 MW cutoff
polyfulfone filters. The concentrated bacterioplankton cells were
collected on a 0.22 .mu.m, 47 mm Durapore filter, and resuspended
in 1 ml of 2.times.STE buffer (1M NaCl, 0.1M EDTA, 10 mM Tris, pH
8.0) to a final density of approximately 1.times.10.sup.10 cells
per ml. The cell suspension was mixed with one volume of 1% molten
Seaplaque LMP agarose (FMC) cooled to 40.degree. C., and then
immediately drawn into a 1 ml syringe. The syringe was sealed with
parafilm and placed on ice for 10 min. The cell-containing agarose
plug was extruded into 10 ml of Lysis Buffer (lOmM Tris pH 8.0, 50
mM NaCl, 0.1M EDTA, 1% Sarkosyl, 0.2% sodium deoxycholate, 1 mg/ml
lysozyme) and incubated at 37.degree. C. for one hour. The agarose
plug was then transferred to 40 mls of ESP Buffer (1% Sarkosyl, 1
mg/ml proteinase K, in 0.5M EDTA), and incubated at 55.degree. C.
for 16 hours. The solution was decanted and replaced with fresh ESP
Buffer, and incubated at 55.degree. C. for an additional hour. The
agarose plugs were then placed in 50 mM EDTA and stored at
4.degree. C. shipboard for the duration of the oceanographic
cruise.
[0222] One slice of an agarose plug (72 .mu.l) prepared from a
sample collected off the Oregon coast was dialyzed overnight at
4.degree. C. against 1 mL of buffer A (100 mM NaCl, 10 mM Bis Tris
Propane-HCl, 100 .mu.g/ml acetylated BSA: pH 7.0 @ 25.degree. C.)
in a 2 mL microcentrifuge tube. The solution was replaced with 250
.mu.l of fresh buffer A containing 10 mM MgCl.sub.2 and 1 mM DTT
and incubated on a rocking platform for 1 hr at room temperature.
The solution was then changed to 250 .mu.l of the same buffer
containing 4U of Sau3A1 (NEB), equilibrated to 37.degree. C. in a
water bath, and then incubated on a rocking platform in a
37.degree. C. incubator for 45 min. The plug was transferred to a
1.5 ml microcentrifuge tube and incubated at 68.degree. C. for 30
min to inactivate the enzyme and to melt the agarose. The agarose
was digested and the DNA dephosphorylased using Gelase and
HK-phosphatase (Epicentre), respectively, according to the
manufacturer's recommendations. Protein was removed by gentle
phenol/chloroform extraction and the DNA was ethanol precipitated,
pelleted, and then washed with 70% ethanol. This partially digested
DNA was resuspended in sterile H.sub.2O to a concentration of 2.5
ng/.mu.l for ligation to the pFOS1 vector.
[0223] PCR amplification results from several of the agarose plugs
(data not shown) indicated the presence of significant amounts of
archaeal DNA. Quantitative hybridization experiments using rRNA
extracted from one sample, collected at 200 m of depth off the
Oregon Coast, indicated that planktonic archaea in (this assemblage
comprised approximately 4.7% of the total picoplankton biomass
(this sample corresponds to "PACI"-200 m in Table 1 of DeLong et
al., high abundance of Archaea in Antarctic marine picoplankton,
Nature, 371:695-698, 1994). Results from archaeal-biased rDNA PCR
amplification performed on agarose plug lysates confirmed the
presence of relatively large amounts of archaeal DNA in this
sample. Agarose plugs prepared from this picoplankton sample were
chosen for subsequent fosmid library preparation. Each 1 ml agarose
plug from this site contained approximately 7.5.times.10.sup.5
cells, therefore approximately 5.4.times.10.sup.5 cells were
present in the 72 .mu.l slice used in the preparation of the
partially digested DNA.
[0224] Vector arms were prepared from pFOS1 as described (Kim et
al., Stable propagation of casmid sized human DNA inserts in an F
factor based vector, Nucl. Acids Res., 20:10832-10835, 1992).
Briefly, the plasmid was completely digested with AstII,
dephosphorylated with HK phosphatase, and then digested with BamHI
to generate two arms, each of which contained a cos site in the
proper orientation for cloning and packaging ligated DNA between
35-45 kbp. The partially digested picoplankton DNA was ligated
overnight to the PFOS1 arms in a 15 .mu.l ligation reaction
containing 25 ng each of vector and insert and 1U of T4 DNA ligase
(Boehringer-Mannheim). The ligated DNA in four microliters of this
reaction was in vitro packaged using the Gigapack XL packaging
system (Stratagene), the fosmid particles transfected to E. coli
strain DH10B (BRL), and the cells spread onto LB.sub.cm15 plates.
The resultant fosmid clones were picked into 96-well microliter
dishes containing LB.sub.cm15 supplemented with 7% glycerol.
Recombinant fosmids, each containing ca. 40 kb of picoplankton DNA
insert, yielded a library of 3.552 fosmid clones, containing
approximately 1.4.times.10.sup.8 base pairs of cloned DNA. All of
the clones examined contained inserts ranging from 38 to 42 kbp.
This library was stored frozen at -80.degree. C. for later
analysis.
[0225] Numerous modifications and variations of the present
invention are possible in light of the above teachings; therefore,
within the scope of the claims, the invention may be practiced
other than as particularly described.
* * * * *