U.S. patent application number 10/072499 was filed with the patent office on 2002-12-12 for method for screening for enzyme activity.
This patent application is currently assigned to Diversa Corporation. Invention is credited to Short, Jay M..
Application Number | 20020187489 10/072499 |
Document ID | / |
Family ID | 27358580 |
Filed Date | 2002-12-12 |
United States Patent
Application |
20020187489 |
Kind Code |
A1 |
Short, Jay M. |
December 12, 2002 |
Method for screening for 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 DNA
from DNA derived from at least one microorganism, by use of at
least one probe DNA comprising at least a portion of a DNA sequence
encoding an enzyme having the specified enzyme activity; and (ii)
transforming a host with isolated target DNA 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: |
27358580 |
Appl. No.: |
10/072499 |
Filed: |
February 5, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10072499 |
Feb 5, 2002 |
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09557276 |
Apr 24, 2000 |
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6344328 |
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09557276 |
Apr 24, 2000 |
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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: |
435/6.16 ;
536/25.4 |
Current CPC
Class: |
C12N 9/16 20130101; C12Q
2521/543 20130101; C12Q 2565/531 20130101; C12N 9/00 20130101; C12N
15/1034 20130101; C12N 15/102 20130101; C12Q 1/6813 20130101; C12N
9/1029 20130101; C12Q 1/6813 20130101 |
Class at
Publication: |
435/6 ;
536/25.4 |
International
Class: |
C12Q 001/68; C07H
021/04 |
Claims
What is claimed is:
1. A method for enriching for DNA sequences containing at least a
partial coding region for at least one specified protein activity
in a DNA sample comprising selecting and recovering a mixture of
target DNA from a mixture of organisms, by use of a mixture of DNA
probes comprising at least a portion of a DNA sequence encoding at
least one protein having a specified protein activity.
Description
[0001] This application is a continuation-in-part of provisional
application no. 60/008,317 which was filed on Dec. 7, 1995.
[0002] The present invention relates to the production and
screening of expression libraries for enzyme activity and, more
particularly, to obtaining selected DNA from DNA of a microorganism
and to screening of an expression library for enzyme activity which
is produced from selected DNA.
[0003] Industry has recognized the need for new enzymes for a wide
variety of industrial applications. As a result, a variety of
microorganisms have been screened to ascertain whether such
microorganisms have a desired enzyme activity. If such
microorganisms do have the desired enzyme activity, the enzyme is
then recovered from them.
[0004] 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, etc. In accordance with the
present invention, recombinant enzymes are generated from
microorganisms and are classified by various enzyme
characteristics.
[0005] More particularly, one aspect of the present invention
provides a process for identifying clones having a specified enzyme
activity, which process comprises:
[0006] screening for said specified enzyme activity in a library of
clones prepared by
[0007] (i) selectively isolating target DNA, from DNA derived from
at least one microorganism, by use of at least one probe DNA
comprising at least a portion of a DNA sequence encoding an enzyme
having the specified enzyme activity; and
[0008] (ii) transforming a host with isolated target DNA 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.
[0009] In a preferred embodiment of this aspect, DNA obtained from
at least one microorganism is selected by recovering from the DNA,
DNA which spcifically binds, such as by hybridization, to a probe
DNA sequence. The DNA obtained from the microorganism or
microorganisms can be genomic DNA or genomic gene library DNA. One
could even use DNA 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 DNA which is not hybridized or otherwise
specifically bound to the probe. The process can also include
releasing DNA from said probe after recovering said hybridized or
otherwise bound DNA and amplifying the DNA so released.
[0010] 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 DNA 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 DNA of interest.
[0011] 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 DNA, from DNA 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 DNA sequence encoding the protein(s)
having the specified activity of interest; and (ii) transforming a
host with isolated target gene cluster DNA 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.
[0012] In a particular embodiment of this aspect, gene cluster DNA
obtained from the genomic DNA of the organism(s) is selected by
recovering from the DNA, DNA which specifically binds, such as by
hybridization, to a probe DNA sequence. The polynucleotide probe
may be directly or indirectly bound to a solid phase by which it is
separated from the DNA 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 DNA
from said probe after recovering said hybridized or otherwise bound
DNA and amplifying the DNA so released.
[0013] These and other aspects of the present invention will be
apparent to those skilled in the art from the teachings herein.
[0014] FIGS. 1A and 1B. FIG. 1A is 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. FIG. 1B is also 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.
[0015] FIG. 2 is 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.
[0016] 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,
psychrotrophs, etc.
[0017] As indicated above, the library may be produced from
environmental samples in which case DNA may be recovered without
culturing of an organism or the DNA may be recovered from a
cultured organism.
[0018] Sources of microorganism DNA as a starting material library
from which target DNA 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, DNA 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.
[0019] 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.
[0020] 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.
[0021] 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.
[0022] 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.
[0023] 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.
[0024] Preferably, the gene cluster DNA 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 DNA 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 DNA fragments, such as gene
clusters from mixed microbial samples.
[0025] 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.
[0026] The DNA isolated or derived from these microorganisms can
preferably be inserted into a vector or a plasmid prior to probing
for selected DNA. 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.
[0027] The following outlines a general procedure for producing
libraries from both culturable and non-culturable organisms, which
libraries can be probed to select therefrom DNA sequences which
hybridize to specified probe DNA:
ENVIRONMENTAL SAMPLE
[0028] Obtain Biomass
[0029] DNA Isolation (various methods)
[0030] Shear DNA (25 gauge needle)
[0031] Blunt DNA (Mung Bean Nuclease)
[0032] Methylate DNA (EcoR I Methylase)
[0033] Ligate to EcoR I linkers (GGAATTCC)
[0034] Cut back linkers (EcoR I Restriction Endonuclease)
[0035] Size Fractionate (Sucrose Gradient)
[0036] Ligate to lambda vector
[0037] Package (in vitro lambda packaging extract)
[0038] Plate on E. coli host and amplify
[0039] The probe DNA used for selectively isolating the target DNA
of interest from the DNA derived from at least one microorganism
can be a full-length coding region sequence or a partial coding
region sequence of DNA for an enzyme of known activity. The
original DNA library can be preferably probed using mixtures of
probes comprising at least a portion of DNA sequences encoding
enzymes having the specified enzyme activity. These probes or probe
libraries are preferably single-stranded and the microbial DNA
which is probed has preferably been converted into single-stranded
form. The probes that are particularly suitable are those derived
from DNA encoding enzymes having an activity similar or identical
to the specified enzyme activity which is to be screened.
[0040] The probe DNA 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 DNA is selectively isolated by the use of at least
one DNA 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%.
[0041] Hybridization techniques for probing a microbial DNA library
to isolate target DNA 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 DNA for separation from the
remainder of the DNA derived from the microorganisms. Solution
phase hybridizations followed by binding of the probe to a solid
phase is preferable.
[0042] Preferably the probe DNA 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.
[0043] Further, it is optional but desirable to perform an
amplification of the target DNA that has been isolated. In this
embodiment the target DNA is separated from the probe DNA after
isolation. It is then amplified before being used to transform
hosts. The double stranded DNA selected to include as at least a
portion thereof a predetermined DNA sequence can be rendered single
stranded, subjected to amplification and reannealed to provide
amplified numbers of selected double stranded DNA. Numerous
amplification methodologies are now well known in the art.
[0044] The selected DNA 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 DNA
by inoculation under conditions conducive for such transformation.
One could transform with double stranded circular or linear DNA or
there may also be instances where one would transform with single
stranded circular or linear DNA.
[0045] 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.
[0046] Having prepared a multiplicity of clones from DNA
selectively isolated from an organism, such clones are screened for
a specific enzyme activity and to identify the clones having the
specified enzyme characteristics.
[0047] 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.
[0048] As described with respect to one of the above aspects, the
invention provides a process for enzyme activity screening of
clones containing selected DNA derived from a microorganism which
process comprises:
[0049] screening a library for specified enzyme activity, said
library including a plurality of clones, said clones having been
prepared by recovering from DNA of a microorganism selected DNA,
which DNA is selected by hybridization to at least one DNA sequence
which is all or a portion of a DNA sequence encoding an enzyme
having the specified activity; and
[0050] transforming a host with the selected DNA to produce clones
which are screened for the specified enzyme activity.
[0051] In one embodiment, a DNA library derived from a
microorganism is subjected to a selection procedure to select
therefrom DNA which hybridizes to one or more probe DNA sequences
which is all or a portion of a DNA sequence encoding an enzyme
having the specified enzyme activity by:
[0052] (a) rendering the double-stranded DNA population into a
single-stranded DNA population;
[0053] (b) contacting the single-stranded DNA population of (a)
with the DNA probe bound to a ligand under conditions permissive of
hybridization so as to produce a double-stranded complex of probe
and members of the DNA population which hybridize thereto;
[0054] (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;
[0055] (d) separating the solid phase complex from the
single-stranded DNA population of (b);
[0056] (e) releasing from the probe the members of the population
which had bound to the solid phase bound probe;
[0057] (f) forming double-stranded DNA from the members of the
population of (e);
[0058] (g) introducing the double-stranded DNA of (f) into a
suitable host to form a library containing a plurality of clones
containing the selected DNA; and
[0059] (h) screening the library for the specified enzyme
activity.
[0060] In another embodiment, a DNA library derived from a
microorganism,is subjected to a selection procedure to select
therefrom double-stranded DNA which hybridizes to one or more probe
DNA sequences which is all or a portion of a DNA sequence encoding
an enzyme having the specified enzyme activity by:
[0061] (a) contacting the double-stranded DNA population with the
DNA probe bound to a ligand under conditions permissive of
hybridization so as to produce a complex of probe and members of
the DNA population which hybridize thereto;
[0062] (b) contacting the complex of (a) with a solid phase
specific binding partner for said ligand so as to produce a solid
phase complex;
[0063] (c) separating the solid phase complex from the unbound DNA
population of (b);
[0064] (d) releasing from the probe the members of the population
which had bound to the solid phase bound probe;
[0065] (e) introducing the double-stranded DNA of (d) into a
suitable host to form a library containing a plurality of clones
containing the selected DNA; and
[0066] (f) screening the library for the specified enzyme
activity.
[0067] In another aspect, the process includes a preselection to
recover DNA including signal or secretion sequences. In this manner
it is possible to select from the DNA population by hybridization
as hereinabove described only DNA 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.
[0068] Another particularly preferred embodiment of this aspect
further comprises, after (a) but before (a) above, the steps
of:
[0069] (i). contacting the double-stranded DNA 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;
[0070] (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;
[0071] (iii) separating the solid phase complex from the unbound
DNA population;
[0072] (iv) releasing the members of the population which had bound
to said solid phase bound probe; and
[0073] (v) separating the solid phase bound probe from the members
of the population which had bound thereto.
[0074] The DNA 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 DNA which
binds to one or more probe DNA sequences derived from DNA encoding
an enzyme(s) having the specified enzyme activity.
[0075] 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.
[0076] 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):4129-4136,
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, 85:1576-1580, 1988. The
pMG/KS330r 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.
[0077] The DNA 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 DNA to a selection procedure to select and isolate
therefrom DNA which hybridizes to DNA derived from DNA encoding an
enzyme(s) having the specified enzyme activity.
[0078] 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 DNA (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), psiX174,
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.
[0079] 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."
[0080] The DNA derived from a microorganism(s) may be inserted into
the vector by a variety of procedures. In general, the DNA 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.
[0081] The DNA 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 lacI, 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.
[0082] 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.
[0083] 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.
[0084] The DNA 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 DNA is
preferably already in a vector which includes appropriate control
sequences whereby selected DNA 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)).
[0085] As representative examples of appropriate hosts, there may
be mentioned: bacterial cells, such as E. coli, Streptomyces,
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.
[0086] 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. DNA sequences derived
from the SV40 splice, and polyadenylation sites may be used to
provide the required nontranscribed genetic elements.
[0087] 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.
[0088] 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;
oxidoreductases, 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.
[0089] 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 etc.
[0090] The clones which are identified as having the specified
enzyme activity may then be sequenced to identify the DNA sequence
encoding an enzyme having the specified activity. Thus, in
accordance with the present invention it is possible to isolate and
identify: (i) DNA 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.
[0091] The present invention may be employed for example, to
identify new enzymes having, for example, the following activities
which may be employed for the following uses:
[0092] 1 Lipase/Esterase
[0093] a. Enantioselective hydrolysis of esters
(lipids)/thioesters
[0094] 1) Resolution of racemic mixtures
[0095] 2) Synthesis of optically active acids or alcohols from
meso-diesters
[0096] b. Selective syntheses
[0097] 1) Regiospecific hydrolysis of carbohydrate esters
[0098] 2) Selective hydrolysis of cyclic secondary alcohols
[0099] c. Synthesis of optically active esters, lactones, acids,
alcohols
[0100] 1) Transesterification of activated/nonactivated esters
[0101] 2) Interesterification
[0102] 3) Optically active lactones from hydroxyesters
[0103] 4) Regio- and enantioselective ring opening of
anhydrides
[0104] d. Detergents
[0105] e. Fat/Oil conversion
[0106] f. Cheese ripening
[0107] 2 Protease
[0108] a. Ester/amide synthesis
[0109] b. Peptide synthesis
[0110] c. Resolution of racemic mixtures of amino acid esters
[0111] d. Synthesis of non-natural amino acids
[0112] e. Detergents/protein hydrolysis
[0113] 3 Glycosidase/Glycosyl Transferase
[0114] a. Sugar/polymer synthesis
[0115] b. Cleavage of glycosidic linkages to form mono, di-and
oligosaccharides
[0116] c. Synthesis of complex oligosaccharides
[0117] d. Glycoside synthesis using UDP-galactosyl transferase
[0118] e. Transglycosylation of disaccharides, glycosyl fluorides,
aryl galactosides
[0119] f. Glycosyl transfer in oligosaccharide synthesis
[0120] g. Diastereoselective cleavage of
.beta.-glucosylsulfoxides
[0121] h. Asymmetric glycosylations
[0122] i. Food processing
[0123] j. Paper processing
[0124] 4 Phosphatase/Kinase
[0125] a. Synthesis/hydrolysis of phosphate esters
[0126] 1) Regio-, enantioselective phosphorylation
[0127] 2) Introduction of phosphate esters
[0128] 3) Synthesize phospholipid precursors
[0129] 4) Controlled polynucleotide synthesis
[0130] b. Activate biological molecule
[0131] C. Selective phosphate bond formation without protecting
groups
[0132] 5 Mono/Dioxygenase
[0133] a. Direct oxyfunctionalization of unactivated organic
substrates
[0134] b. Hydroxylation of alkane, aromatics, steroids
[0135] C. Epoxidation of alkenes
[0136] d. Enantioselective sulphoxidation
[0137] e. Regio- and stereoselective Bayer-Villiger oxidations
[0138] 6 Haloperoxidase
[0139] a. Oxidative addition of halide ion to nucleophilic
sites
[0140] b. Addition of hypohalous acids to olefinic bonds
[0141] C. Ring cleavage of cyclopropanes
[0142] d. Activated aromatic substrates converted to ortho and para
derivatives
[0143] e. 1.3 diketones converted to 2-halo-derivatives
[0144] f. Heteroatom oxidation of sulfur and nitrogen containing
substrates
[0145] g. Oxidation of enol acetates, alkynes and activated
aromatic rings
[0146] 7 Lignin Peroxidase/Diarylpropane Peroxidase
[0147] a. Oxidative cleavage of C-C bonds
[0148] b. Oxidation of benzylic alcohols to aldehydes
[0149] c. Hydroxylation of benzylic carbons
[0150] d. Phenol dimerization
[0151] e. Hydroxylation of double bonds to form diols
[0152] f. Cleavage of lignin aldehydes
[0153] 8 Epoxide Hydrolase
[0154] a. Synthesis of enantiomerically pure bioactive
compounds
[0155] b. Region and enantioselective hydrolysis of epoxide
[0156] c. Aromatic and olefinic epoxidation by monooxygenases to
form epoxides
[0157] d. Resolution of racemic epoxides
[0158] e. Hydrolysis of steroid epoxides
[0159] 9 Nitrile Hydratase/Nitrilase
[0160] a. Hydrolysis of aliphatic nitrites to carboxamides
[0161] b. Hydrolysis of aromatic, heterocyclic, unsaturated
aliphatic nitrites to corresponding acids
[0162] c. Hydrolysis of acrylonitrile
[0163] d. Production of aromatic and carboxamides, carboxylic acids
(nicotinamide, picolinamide, isonicotinamide)
[0164] e. Regioselective hydrolysis of acrylic dinitrile
[0165] f. .alpha.-amino acids from .alpha.-hydroxynitriles
[0166] 10 Transaminase
[0167] a. Transfer of amino groups into oxo-acids
[0168] 11 Amidase/Acylase
[0169] a. Hydrolysis of amides, amidines, and other C--N bonds
[0170] b. Non-natural amino acid resolution and synthesis
EXAMPLE 1
Preparation of a Genomic DNA Library in Lambda ZAPII
[0171] 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:
[0172] 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).
[0173] 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 10x mung bean buffer (45 .mu.l) and mung bean
nuclease (2.0 .mu.l, 150 U/.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).
[0174] EcoR I sites in the DNA must be protected from future
enzymatic reactions. To accomplish this, the DNA is incubated with
10x EcoR I methylase (5.0 .mu.l, 40 U/.mu.l) for 1 hour at
37.degree. C.
[0175] 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, 1x 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).
[0176] 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), 10x 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.
[0177] 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), 10x 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 0.5 M EDTA and the DNA is placed on ice.
[0178] 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).
[0179] 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.
[0180] 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 10x 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.
[0181] 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: 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-4-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 (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
[0182] 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.
[0183] 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.
[0184] 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-bromo-4-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 2
Hybridization Selection and Production of Expression Library
[0185] 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.
[0186] 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.
PROTOCOL
[0187] 1. Six samples of 4 .mu.g of rescued, single-stranded DNA
from library #17 were prepared in 3X SSC buffer. Final reaction
volumes were 30 .mu.l.
[0188] 2. To these solutions was added one of the following:
[0189] a) nothing
[0190] b) 100 ng of biotinylated probe from an unrelated
sequence
[0191] c,d) 100 ng of biotinylated probe from organism #13 DNA
polymerase gene
[0192] e,f) 100 ng of biotinylated probe from organism #17 DNA
polymerase gene
[0193] 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.
[0194] 3. These mixtures were denatured by heating to 95.degree. C.
for 2 minutes.
[0195] 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.
[0196] 5. 50 .mu.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 3X SSC and resuspended in 60 .mu.l of 3X SSC containing 100
.mu.g of sonicated salmon sperm DNA.
[0197] 6. The DNA/bead mixtures were washed 2 times at room
temperature in 0.1X SSC/0.1% SDS, 2 times at 42.degree. C. in 0.1X
SSC/0.1% SDS for 10 minutes each and 1 additional wash at room
temperature with 3X SSC.
[0198] 7. The bound DNA was eluted by heating the beads to
70.degree. C. for 15 minutes in 50 .mu.l TE.
[0199] 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.
[0200] 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).
[0201] 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.
[0202] 11. The digoxigenin system from Boehringer Mannheim was used
to probe the colonies using the organism #17 probe.
RESULTS
PCR Quantitation
[0203] 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.
[0204] 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
Colony Hybridization
[0205] FIG. 2 is a photograph of four colony hybridization plates
resulting from Plates A and B show positive clones ie., 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.
[0206] 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 3
Construction of a Stable, Large Insert DNA Library of Picoplankton
Genomic DNA
[0207] Cell collection and preparation of DNA. Agarose plugs
containing concentrated picoplankton cells were prepared from
samples collected on an oceanographic cruise from Newport, Oreg. to
Honolulu, Hi. 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 2X
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 (1OmM 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.
[0208] 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.
[0209] 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.
[0210] 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, Nuc. 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.
[0211] 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.
* * * * *