U.S. patent application number 09/401861 was filed with the patent office on 2002-06-06 for combinatorial enzyme development.
Invention is credited to SHORT, JAY M..
Application Number | 20020068340 09/401861 |
Document ID | / |
Family ID | 41527780 |
Filed Date | 2002-06-06 |
United States Patent
Application |
20020068340 |
Kind Code |
A1 |
SHORT, JAY M. |
June 6, 2002 |
COMBINATORIAL ENZYME DEVELOPMENT
Abstract
Disclosed is a process for obtaining an enzyme having a
specified enzyme activity derived from a heterogeneous DNA
population by screening, for the specified enzyme activity, a
library of clones containing DNA from the heterogeneous DNA
population which have been exposed to directed mutagenesis towards
production of the specified enzyme activity. Also disclosed is a
process for obtaining an enzyme having a specified enzyme activity
by screening, for the specified enzyme activity, a library of
clones containing DNA from a pool of DNA populations which have
been exposed to directed mutagenesis in an attempt to produce in
the library of clones DNA encoding an enzyme having one or more
desired characteristics which can be the same or different from the
specified enzyme activity.
Inventors: |
SHORT, JAY M.; (Encinitas,
CA) |
Correspondence
Address: |
LISA A. HAILE, Ph.D.
GRAY CARY WARE & FREIDENRICH LLP
4365 EXECUTIVE DRIVE
SUITE 1600
SAN DIEGO
CA
92121
US
|
Family ID: |
41527780 |
Appl. No.: |
09/401861 |
Filed: |
September 22, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09401861 |
Sep 22, 1999 |
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09375605 |
Aug 17, 1999 |
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09375605 |
Aug 17, 1999 |
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08651568 |
May 22, 1996 |
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60008316 |
Dec 7, 1995 |
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Current U.S.
Class: |
435/183 ;
435/6.14; 435/69.1; 536/23.1; 536/23.2 |
Current CPC
Class: |
C12N 15/1034 20130101;
C12Y 301/03001 20130101; C12N 9/00 20130101; C12Y 302/01021
20130101; C12N 9/2445 20130101; C12N 9/16 20130101; C12N 15/102
20130101 |
Class at
Publication: |
435/183 ; 435/6;
435/69.1; 536/23.1; 536/23.2 |
International
Class: |
C12Q 001/68 |
Claims
What is claimed is:
1. A process for obtaining an enzyme having a specified enzyme
activity derived from a heterogeneous DNA population, which process
comprises: screening, for the specified enzyme activity, a library
of clones containing DNA from the heterogeneous DNA population
which have been exposed to directed mutagenesis towards production
of the specified enzyme activity.
2. The process of claim 1 which further comprises, prior to said
directed mutagenesis, selectively recovering from the heterogeneous
DNA population DNA which comprises DNA sequences coding for a
common characteristic, which can be the same or different from the
specified enzyme activity.
3. The process of claim 2 wherein recovering the DNA preparation
comprises contacting the DNA population with a specific binding
partner for at least a portion of the coding sequences.
4. The process of claim 3 wherein the specific binding partner is a
solid phase bound hybridization probe.
5. The process of claim 2 wherein the common characteristic is a
class of enzyme activity.
6. The process of claim 5 which comprises recovering DNA from
clones containing DNA from the heterogeneous DNA population which
exhibit the class of enzyme activity.
7. The process of claim 1 wherein the directed mutagenesis is
site-specific directed mutagenesis.
8. The process of claim 1 which further comprises prescreening said
library of clones for an activity, which can be the same or
different from the specified enzyme activity, prior to exposing
them to directed mutagenesis.
9 The process of claim 8 which comprises prescreening said clones
for the expression of a protein of interest.
10. A process for obtaining an enzyme having a specified enzyme
activity, which process comprises: screening, for the specified
enzyme activity, a library of clones containing DNA from a pool of
DNA populations which have been exposed to directed mutagenesis in
an attempt to produce in the library of clones DNA encoding an
enzyme having one or more desired characteristics which can be the
same or different from the specified enzyme activity.
11. The process of claim 10 which further comprises, prior to said
directed mutagenesis, selectively recovering from the heterogeneous
DNA population DNA which comprises DNA sequences coding for at
least one common enzyme characteristic, which can be the same or
different from the specified enzyme activity.
12. The process of claim 11 wherein the at least one common enzyme
characteristic is at least one common enzyme activity.
13. The process of claim 12 wherein recovering the DNA preparation
comprises contacting the DNA population with a specific binding
partner for at least a portion of the coding sequences.
14. The process of claim 13 wherein the specific binding partner is
a solid phase bound hybridization probe.
15. The process of claim 14 which comprises recovering DNA from
clones containing DNA from the heterogeneous DNA population which
exhibit the class of enzyme activity.
16. The process of claim 10 wherein the directed mutagenesis is
site-specific directed mutagenesis.
Description
[0001] This application is a continuation-in-part of copending U.S.
provisional application No. 60/008,316 which was filed on Dec. 7,
1995.
[0002] This invention relates to the field of protein engineering.
More particularly, the invention relates to the directed
mutagenesis of DNA and screening of clones containing the
mutagenized DNA for resultant specified protein, particularly
enzyme, activity(ies) of interest.
[0003] In one aspect the invention provides a process for obtaining
an enzyme having a specified enzyme activity derived from a
heterogeneous DNA population, which process comprises: screening,
for the specified enzyme activity, a library of clones containing
DNA from the heterogeneous DNA population which have been exposed
to directed mutagenesis towards production of the specified enzyme
activity.
[0004] Another aspect of the invention provides a process for
obtaining an enzyme having a specified enzyme activity, which
process comprises: screening, for the specified enzyme activity, a
library of clones containing DNA from a pool of DNA populations
which have been exposed to directed mutagenesis in an attempt to
produce in the library of clones DNA encoding an enzyme having one
or more desired characteristics, which can be the same or different
from the specified enzyme activity. In a preferred embodiment, the
DNA pool which is subjected to directed mutagenesis is a pool of
DNA which has been selected to encode enzymes having at least one
enzyme characteristic, in particular at least one common enzyme
activity.
[0005] Also provided is a process for obtaining a protein having a
specified activity derived from a heterogeneous population of gene
clusters by screening, for the specified protein activity, a
library of clones containing gene clusters from the heterogeneous
gene cluster population which have been exposed to directed
mutagenesis towards production of specified protein activities of
interest.
[0006] Also provided is a process of obtaining a gene cluster
protein product having a specified activity, by screening, for the
specified protein activity, a library of clones containing gene
clusters from a pool of gene cluster populations which have been
exposed to direct mutagenesis to produce in the library of clones
gene clusters encoding proteins having one or more desired
characteristics, which can be the same or different from the
specified protein activity. Preferably, the pool of gene clusters
which is subjected to directed mutagenesis is one which has been
selected to encode proteins having enzymatic activity in the
synthesis of at least one therapeutic, prophylactic or
physiological regulatory activity.
[0007] The process of either of these aspects can further comprise,
prior to the directed mutagenesis, selectively recovering from the
heterogeneous population of gene clusters, gene clusters which
comprise polycistronic sequences coding for proteins having at
least one common physical, chemical or functional characteristic
which can be the same or different from the activity observed prior
to directed mutagenesis. Preferably, recovering the gene cluster
preparation comprises contacting the gene cluster population with a
specific binding partner, such as a solid phase-bound hybridization
probe, for at least a portion of the gene cluster of interest. The
common characteristic of the resultant protein(s) can be classes of
the types of activity specified above, i.e., such as a series of
enzymes related as parts of a common synthesis pathway or proteins
capable of hormonal, signal transduction or inhibition of metabolic
pathways or their functions in pathogens and the like. The gene
cluster DNA is recovered from clones containing such gene cluster
DNA from the heterogeneous gene cluster population which exhibit
the activity of interest. Preferably, the directed mutagenesis is
site-specific directed mutagenesis. This process can further
include a step of pre-screening the library of clones for an
activity, which can be the same or different from the specified
activity of interest, prior to exposing them to directed
mutagenesis. This activity can result, for example, from the
expression of a protein or related family of proteins of
interest.
[0008] The process of any of these aspects can further comprise,
prior to said directed mutagenesis, selectively recovering from the
heterogeneous DNA population DNA which comprises DNA sequences
coding for enzymes having at least one common characteristic, which
can be the same or different from the specified enzyme activity.
Preferably, recovering the DNA preparation comprises contacting the
DNA population with a specific binding partner, such as a solid
phase bound hybridization probe, for at least a portion of the
coding sequences. The common characteristic can be, for example, a
class of enzyme activity, such as hydrolase activity. DNA is
recovered from clones containing DNA from the heterogeneous DNA
population which exhibit the class of enzyme activity. Preferably,
the directed mutagenesis is site-specific directed mutagenesis. The
process of this aspect can further include a step of prescreening
the library of clones for an activity, which can be the same or
different from the specified enzyme activity, prior to exposing
them to directed mutagenesis. This activity can result, for
example, from the expression of a protein of interest.
[0009] The heterogeneous DNA population from which the DNA library
is derived is a complex mixture of DNA, such as is obtained, for
example, from an environmental sample. Such samples can contain
unculturable or uncultured multiple or single organisms. These
environmental samples can be obtained from, for example, Arctic and
Antarctic ice, water or permafrost sources, materials of volcanic
origin, materials from soil or plant sources in tropical areas,
etc. A variety of known techniques can be applied to enrich the
environmental sample for organisms of interest, including
differential culturing, sedimentation gradient, affinity matrices,
capillary electrophoresis, optical tweezers and fluorescence
activated cell sorting. The samples can also be cultures of a
single organism.
[0010] 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 are uncultured
microorganisms obtained from environmental samples and such
microorganisms may be extremophiles, such as thermophiles,
hyperthermophiles, psychrophiles, psychrotrophs, etc.
[0011] 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.
[0012] 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.
[0013] 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.
[0014] 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.
[0015] 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.
[0016] 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.
[0017] The DNA can then be isolated by available techniques that
are described in the literature. The IsoQuick.RTM. nucleic acid
extraction kit (MicroProbe Corporation) is suitable for this
purpose.
[0018] The term "derived" or "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.
[0019] The DNA isolated or derived from these microorganisms can
preferably be inserted into a vector prior to probing for selected
DNA. Such vectors 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.
[0020] The following outlines a general procedure for producing
gene libraries from both culturable and non-culturable
organisms.
[0021] Obtain Biomass
[0022] DNA Isolation
[0023] Shear DNA (25 gauge needle)
[0024] Blunt DNA (Mung Bean Nuclease)
[0025] Methylate (EcoR I Methylase)
[0026] Ligate to EcoR I linkers (GGAATTCC)
[0027] Cut back linkers (EcoR I Restriction Endonuclease)
[0028] Size Fractionate (Sucrose Gradient)
[0029] Ligate to lambda vector (Lambda ZAP II and gt11)
[0030] Package (in vitro lambda packaging extract)
[0031] Plate on E. coli host and amplify
[0032] Clones having an enzyme activity of interest are identified
by screening. This screening can be done either by hybridization,
to identify the presence of DNA coding for the enzyme of interest
or by detection of the enzymatic activity of interest.
[0033] The probe DNA used for selectively recovering DNA of
interest from the DNA derived from the at least one uncultured
microorganism can be a full-length coding region sequence or a
partial coding region sequence of DNA for an enzyme of known
activity, a phylogenetic marker or other identified DNA sequence.
The original DNA library can be preferably probed using mixtures of
probes comprising at least a portion of the DNA sequence encoding
the specified 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.
[0034] 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 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 75%.
[0035] Hybridization techniques for probing a microbial DNA library
to isolate 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, particularly those which use a solid phase-bound,
directly or indirectly bound, probe DNA for ease in separation from
the remainder of the DNA derived from the microorganisms.
[0036] 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.
[0037] The library of clones prepared as described above can be
screened directly for enzymatic activity without the need for
culture expansion, amplification or other supplementary procedures.
However, in one preferred embodiment, it is considered desirable to
amplify the DNA recovered from the individual clones such as by
PCR.
[0038] Further, it is optional but desirable to perform an
amplification of the target DNA that has been isolated. In this
embodiment the selectively isolated 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.
[0039] 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.
[0040] 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.
[0041] Having prepared a multiplicity of clones from DNA
selectively isolated from an organism, organism, such clones are
screened for a specific enzyme activity and to identify the clones
having the specified enzyme characteristics.
[0042] 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.
[0043] 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.
[0044] 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(Stratagene); pTRC99a, pKK223-3,
pDR540, pRIT2T (Pharmacia); Eukaryotic: pWLNEO, pXT1, pSG5
(Stratagene) pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia). Any other
plasmid or vector may be used as long as they are replicable and
viable in the host.
[0045] Another type of vector for use in the present invention
contains an f-factor origin of 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 a
"fosmids," or bacterial artificial chromosome (BAC) vectors. These
are derived from the E. coli f-factor which is able to stably
integrate large segments of genomic 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."
[0046] 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.
[0047] 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 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.
[0048] 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.
[0049] 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.
[0050] 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 transformation,
calcium phosphate transfection, DEAE-Dextran mediated transfection,
or electroporation (Davis, L., Dibner, M., Battey, I., Basic
Methods in Molecular Biology, (1986)).
[0051] 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 Sf2 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.
[0052] 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.
[0053] 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.
[0054] 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.
[0055] 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.
[0056] Clones found to have the enzymatic activity for which the
screen was performed are sequenced and then subjected to directed
mutagenesis to develop new enzymes with desired activities or to
develop modified enzymes with particularly desired properties that
are absent or less pronounced in the wild-type enzyme, such as
stability to heat or organic solvents. Any of the known techniques
for directed mutagenesis are applicable to the invention. For
example, particularly preferred mutagenesis techniques for use in
accordance with the invention include those discussed below.
[0057] The term "error-prone PCR" refers to a process for
performing PCR under conditions where the copying fidelity of the
DNA polymerase is low, such that a high rate of point mutations is
obtained along the entire length of the PCR product. Leung, D. W.,
et al., Technique, 1:11-15 (1989) and Caldwell, R. C. & Joyce
G. F., PCR Methods Applic., 2:28-33 (1992).
[0058] The term "oligonucleotide directed mutagenesis" refers to a
process which allows for the generation of site-specific mutations
in any cloned DNA segment of interest. Reidhaar-Olson, J. F. &
Sauer, R. T., et al., Science, 241:53-57 (1988).
[0059] The term "assembly PCR" refers to a process which involves
the assembly of a PCR product from a mixture of small DNA
fragments. A large number of different PCR reactions occur in
parallel in the same vial, with the products of one reaction
priming the products of another reaction.
[0060] The term "sexual PCR mutagenesis" refers to forced
homologous recombination between DNA molecules of different but
highly related DNA sequence in vitro, caused by random
fragmentation of the DNA molecule based on sequence homology,
followed by fixation of the crossover by primer extension in a PCR
reaction. Stemmer, W. P., PNAS, USA, 91:10747-10751 (1994).
[0061] The term "in vivo mutagenesis" refers to a process of
generating random mutations in any cloned DNA of interest which
involves the propagation of the DNA in a strain of E. coli that
carries mutations in one or more of the DNA repair pathways. These
"mutator" strains have a higher random mutation rate than that of a
wild-type parent. Propogating the DNA in one of these strains will
eventually generate random mutations within the DNA.
[0062] The term "cassette mutagenesis" refers to any process for
replacing a small region of a double stranded DNA molecule with a
synthetic oligonucleotide "cassette" that differs from the native
sequence. The oligonucleotide often contains completely and/or
partially randomized native sequence.
[0063] The term "recursive ensemble mutagenesis" refers to an
algorithm for protein engineering (protein mutagenesis) developed
to produce diverse populations of phenotypically related mutants
whose members differ in amino acid sequence. This method uses a
feedback mechanism to control successive rounds of combinatorial
cassette mutagenesis. Arkin, A. P. and Youvan, D.C., PNAS, USA,
89:7811-7815 (1992).
[0064] The term "exponential ensemble mutagenesis" refers to a
process for generating combinatorial libraries with a high
percentage of unique and functional mutants, wherein small groups
of residues are randomized in parallel to identify, at each altered
position, amino acids which lead to functional proteins, Delegrave,
S. and Youvan, D.C., Biotechnology Research, 11:1548-1552 (1993);
and random and site-directed mutagenesis, Arnold, F. H., Current
Opinion in Biotechnology, 4:450-455 (1993). All of the references
mentioned above are hereby incorporated by reference in their
entirety.
EXAMPLE 1
Preparation of a Mammalian DNA Library
[0065] The following outlines the procedures used to generate a
gene library from a sample of the exterior surface of a whale bone
found at 1240 meters depth in the Santa Catalina Basin during a
dive expedition.
1 Isolate DNA. IsoQuick Procedure as per manufacturer`s
instructions. Shear DNA 1. Vigorously push and pull DNA through a
25G double-hub needle and 1-cc syringes about 500 times. 2. Check a
small amount (0.5 .mu.g) on a 0.8% agarose gel to make sure the
majority of the DNA is in the desired size range (about 3-6 kb).
Blunt DNA 1. Add: H.sub.2O to a final volume of 405 .mu.l 45 .mu.l
10X Mung Bean Buffer 2.0 .mu.l Mung Bean Nuclease (150 u/.mu.l) 2.
Incubate 37.degree. C., 15 minutes. 3. Phenol/chloroform extract
once. 4. Chloroform extract once. 5. Add 1 ml ice cold ethanol to
precipitate. 6. Place on ice for 10 minutes. 7. Spin in microfuge,
high speed, 30 minutes. 8. Wash with 1 ml 70% ethanol. 9. Spin in
microfuge, high speed, 10 minutes and dry. Methylate DNA 1. Gently
resuspend DNA in 26 .mu.l TE. 2. Add: 4.0 .mu.l 10X EcoR I
Methylase Buffer 0.5 .mu.l SAM (32 mM) 5.0 .mu.l EcoR I Methylase
(40 u/.mu.l) 3. Incubate 37.degree., 1 hour. Insure Blunt Ends 1.
Add to the methylation reaction: 5.0 .mu.l 100 mM MgCl.sub.2 8.0
.mu.l dNTP mix (2.5 mM of each dGTP, dATP, dTTP, dCTP) 4.0 .mu.l
Klenow (5 u/.mu.l) 2. Incubate 12.degree. C., 30 minutes. 3. Add
450 .mu.l 1X STE. 4. Phenol/chloroform extract once. 5. Chloroform
extract once. 6. Add 1 ml ice cold ethanol to precipitate and place
on ice for 10 minutes. 7. Spin in microfuge, high speed, 30
minutes. 8. Wash with 1 ml 70% ethanol. 9. Spin in microfuge, high
speed, 10 minutes and dry. Linker Ligation 1. Gently resuspend DNA
in 7 .mu.l Tris-EDTA (TE). 2. Add: 14 .mu.l Phosphorylated EcoR I
linkers (200 ng/.mu.l) 3.0 .mu.l 10X Ligation Buffer 3.0 .mu.l 10
mM rATP 3.0 .mu.l T4 DNA Ligase (4 Wu/.mu.l) 3. Incubate 4.degree.
C., overnight. EcoR1 Cutback 1. Heat kill ligation reaction
68.degree. C., 10 minutes. 2. Add: 237.9 .mu.l H.sub.2O 30 .mu.l
10X EcoR I Buffer 2.1 .mu.l EcoR I Restriction Enzyme (100 u/.mu.l)
3. Incubate 37.degree. C., 1.5 hours. 4. Add 1.5 .mu.l 0.5 M EDTA.
5. Place on ice. Sucrose Gradient (2.2 ml) Size Fractionation 1.
Heat sample to 65.degree. C., 10 minutes. 2. Gently load on 2.2 ml
sucrose gradient. 3. Spin in mini-ultracentrifuge, 45K, 20.degree.
C., 4 hours (no brake). 4. Collect fractions by puncturing the
bottom of the gradient tube with a 20 G needle and allowing the
sucrose to flow through the needle. Collect the first 20 drops in a
Falcon 2059 tube then collect 10 1-drop fractions (labelled 1-10).
Each drop is about 60 .mu.l in volume. 5. Run 5 .mu.l of each
fraction on a 0.8% agarose gel to check the size. 6. Pool fractions
1-4 (about 10-1.5 kb) and, in a separate tube, pool fractions 5-7
(about 5-0.5 kb). 7. Add 1 ml ice cold ethanol to precipitate and
place on ice for 10 minutes. 8. Spin in microfuge, high speed, 30
minutes. 9. Wash with 1 ml 70% ethanol. 10. Spin in microfuge, high
speed, 10 minutes and dry. 11. Resuspend each in 10 .mu.l TE
buffer. Test Ligation to Lambda Arms 1. Plate assay to get an
approximate concentration. Spot 0.5 .mu.l of the sample on agarose
containing ethidium bromide along with standards (DNA samples of
known concentration). View in UV light and estimate concentration
compared to the standards. Fraction 1-4 = >1.0 .mu.g/.mu.l.
Fraction 5-7 = 500 ng/.mu.l. 2. Prepare the following ligation
reactions (5 .mu.l reactions) and incubate 4.degree. C.,
overnight:
[0066]
2 Lambda 10X arms T4 DNA Ligase 10 mM (gt11 and Insert Ligase (4
Sample H.sub.2O Buffer rATP ZAP) DNA Wu/.mu.) Fraction 1-4 0.5
.mu.l 0.5 .mu.l 0.5 .mu.l 1.0 .mu.l 2.0 .mu.l 0.5 .mu.l Fraction
5-7 0.5 .mu.l 0.5 .mu.l 0.5 .mu.l 1.0 .mu.l 2.0 .mu.l 0.5 .mu.l
[0067]
3 Test Package and Plate 1. Package the ligation reactions
following manufacturer`s protocol. Package 2.5 .mu.l per packaging
extract (2 extracts per ligation). 2. Stop packaging reactions with
500 .mu.l SM buffer and pool packaging that came from the same
ligation. 3. Titer 1.0 .mu.l of each on appropriate host
(OD.sub.600 = 1.0) [XLI-Blue MRF for ZAP and Y1088 for gt11] Add
200 .mu.l host (in mM MgSO.sub.4) to Falcon 2059 tubes Inoculate
with 1 .mu.l packaged phage Incubate 37.degree. C., 15 minutes Add
about 3 ml 48.degree. C. top agar [50 ml stock containing 150 .mu.l
IPTG (0.5M) and 300 .mu.l X-GAL (350 mg/ml)] Plate on 100 mm plates
and incubate 37.degree. C, overnight. 4. Efficiency results: gt11:
1.7 .times. 10.sup.4 recombinants with 95% background ZAP II: 4.2
.times. 10.sup.4 recombinants with 66% background Contaminants in
the DNA sample may have inhibited the enzymatic reactions, though
the sucrose gradient and organic extractions may have removed them.
Since the DNA sample was precious, an effort was made to "fix" the
ends for cloning: Re-Blunt DNA 1. Pool all left over DNA that was
not ligated to the lambda arms (Fractions 1-7) and add H.sub.2O to
a final volume of 12 .mu.l. Then add: 143 .mu.l H.sub.2O 20 .mu.l
10X Buffer 2 (from Stratagene`s cDNA Synthesis Kit) 23 .mu.l
Blunting dNTP (from Stratagene`s cDNA Synthesis Kit) .sup. 2.0
.mu.l Pfu (from Stratagene"s cDNA Synthesis Kit) 2. Incubate
72.degree. C., 30 minutes. 3. Phenol/chloroform extract once. 4.
Chloroform extract once. 5. Add 20 .mu.l 3M NaOAc and 400 .mu.l ice
cold ethanol to precipitate. 6. Place at -20.degree. C., overnight.
7. Spin in microfuge, high speed, 30 minutes. 8. Wash with 1 ml 70%
ethanol. 9. Spin in microfuge, high speed, 10 minutes and dry. (Do
NOT Methylate DNA since it was already methylated in the first
round of processing) Adaptor Ligation 1. Gently resuspend DNA in 8
.mu.l EcoR I adaptors (from Stratagene`s cDNA Synthesis Kit). 2.
Add: 1.0 .mu.l 10X Ligation Buffer 1.0 .mu.l 10 mM rATP 1.0 .mu.l
T4 DNA Ligase (4 Wu/.mu.l ) 3. Incubate 4.degree. C., 2 days. (Do
NOT cutback since using ADAPTORS this time. Instead, need to
phosphorylate) Phosphorylate Adaptors 1. Heat kill ligation
reaction 70.degree. C., 30 minutes. Add: 1.0 .mu.l 10X Ligation
Buffer 2.0 .mu.l 10 mM rATF 6.0 .mu.l H.sub.2O 1.0 .mu.l PNK (from
Stratagene`s cDNA Synthesis Kit). 3. Incubate 37.degree. C., 30
minutes. 4. Add 31 .mu.l H.sub.2O and 5 .mu.l 10X STE. 5. Size
fractionate on a Sephacryl S-500 spin column (pool fractions 1-3).
6. Phenol/chloroform extract once. 7. Chloroform extract once. 8.
Add ice cold ethanol to precipitate. 9. Place on ice, 10 minutes.
10. Spin in microfuge, high speed, 30 minutes. 11. Wash with 1 ml
70% ethanol. 12. Spin in microfuge, high speed, 10 minutes and dry.
13. Resuspend in 10.5 .mu.l TE buffer. Do not plate assay. Instead,
ligate directly to arms as above except use 2.5 .mu.l of DNA and no
water. Package and titer as above. Efficiency results: gt11: 2.5
.times. 10.sup.6 recombinants with 2.5% background ZAP II: 9.6
.times. 10.sup.5 recombinants with 0% background Amplification of
Libraries (5.0 .times. 10.sup.5 recombinants from each library) 1.
Add 3.0 ml host cells (0D.sub.600 = 1.0) to two 50 ml conical tube.
2. Inoculate with 2.5 .times. 10.sup.5 pfu per conical tube. 3.
Incubate 37.degree. C., 20 minutes. 4. Add top agar to each tube to
a final volume of 45 ml. 5. Plate the tube across five 150 mm
plates. 6. Incubate 37.degree. C., 6-8 hours or until plaques are
about pin-head in size. 7. Overlay with 8-10 ml SM Buffer and place
at 4.degree. C. overnight (with gentle rocking if possible).
Harvest Phage 1. Recover phage suspension by pouring the SM buffer
off each plate into a 50-ml conical tube. 2. Add 3 ml chloroform,
shake vigorously and incubate at room temperature, 15 minutes. 3.
Centrifuge at 2K rpm, 10 minutes to remove cell debris. 4. Pour
supernatant into a sterile flask, add 500 .mu.l chloroform. 5.
Store at 4.degree. C. Titer Amplified Library 1. Make serial
dilutions: 10.sup.-5 = 1 .mu.l amplified phage in 1 ml SM Buffer
10.sup.-6 = 1 .mu.l of the 10.sup.-3 dilution in 1 ml SM Buffer 2.
Add 200 .mu.l host (in 10 mM MgSO.sub.4) to two tubes. 3. Inoculate
one with 10 .mu.l 10.sup.-6 dilution (10.sup.-5). 4. Inoculate the
other with 1 .mu.l 10.sup.-6 dilution (10.sup.-6). 5. Incubate
37.degree. C., 15 minutes. 6. Add about 3 ml 48.degree. C. top
agar. [50 ml stock containing 150 .mu.l IPTG (0.5 M) and 375 .mu.l
X-GAL (350 mg/ml)] 7. Plate on 100 mm plates and incubate
37.degree. C., overnight. 8. Results: gt11 1.7 .times. 10.sup.11/ml
ZAP II: 2.0 .times. 10.sup.10/ml
EXAMPLE 2
Enzymatic Activity Assay
[0068] The following is a representative example of a procedure for
screening an expression library, prepared in accordance with
Example 1, for hydrolase activity.
[0069] Plates of the library prepared as described in Example 1 are
used to multiply inoculate a single plate containing 200 .mu.L of
LB Amp/Meth, glycerol in each well. This step is performed using
the High Density Replicating Tool (HDRT) of the Beckman Biomek with
a 1% bleach, water, isopropanol, air-dry sterilization cycle
between each inoculation. The single plate is grown for 2 h at
37.degree. C. and is then used to inoculate two white 96-well
Dynatech microtiter daughter plates containing 250 .mu.L of LB
Amp/Meth, glycerol in each well. The original single plate is
incubated at 37.degree. C. for 18 h, then stored at -80.degree. C.
The two condensed daughter plates are incubated at 37.degree. C.
also for 18 h. The condensed daughter plates are then heated at
70.degree. C. for 45 min. to kill the cells and inactivate the host
E. coli enzymes. A stock solution of 5 mg/mL morphourea
phenylalanyl-7-amino-4-trifluoromethyl coumarin (MuPheAFC, the
`substrate`) in DMSO is diluted to 600 .mu.M with 50 mM pH 7.5
Hepes buffer containing 0.6 mg/mL of the detergent dodecyl
maltoside. 1
[0070] Fifty .mu.L of the 600 .mu.M MuPheAFC solution is added to
each of the wells of the white condensed plates with one 100 .mu.L
mix cycle using the Biomek to yield a final concentration of
substrate of .about.100 .mu.M. The fluorescence values are recorded
(excitation=400 nm, emission=505 nm) on a plate reading fluorometer
immediately after addition of the substrate (t=0). The plate is
incubated at 70.degree. C. for 100 min, then allowed to cool to
ambient temperature for 15 additional minutes. The fluorescence
values are recorded again (t=100). The values at t=0 are subtracted
from the values at T=100 to determine if an active clone is
present.
[0071] The data will indicate whether one of the clones in a
particular well is hydrolyzing the substrate. In order to determine
the individual clone which carries the activity, the source library
plates are thawed and the individual clones are used to singly
inoculate a new plate containing LB Amp/Meth, glycerol. As above,
the plate is incubated at 37.degree. C. to grow the cells, heated
at 70.degree. C. to inactivate the host enzymes, and 50 .mu.L of
600 .mu.M MuPheAFC is added using the Biomek.
[0072] After addition of the substrate the t=0 fluorescence values
are recorded, the plate is incubated at 70.degree. C., and the
t=100 min. values are recorded as above. These data indicate which
plate the active clone is in.
[0073] The enantioselectivity value, E, for the substrate is
determined according to the equation below: 1 E = ln [ ( 1 - c ( 1
+ ee p ) ] ln [ ( 1 - c ( 1 - ee p ) ]
[0074] where ee.sub.p=the enantiomeric excess (ee) of the
hydrolyzed product and c=the percent conversion of the reaction.
See Wong and Whitesides, Enzymes in Synthetic Organic Chemistry,
1994, Elsevier, Tarrytown, N.Y., pp. 9-12.
[0075] The enantiomeric excess is determined by either chiral high
performance liquid chromatography (HPLC) or chiral capillary
electrophoresis (CE). Assays are performed as follows: two hundred
.mu.L of the appropriate buffer is added to each well of a 96-well
white microtiter plate, followed by 50 .mu.L of partially or
completely purified enzyme solution; 50 .mu.L of substrate is added
and the increase in fluorescence monitored versus time until 50% of
the substrate is consumed or the reaction stops, whichever comes
first.
EXAMPLE 3
Directed Mutagenesis of Positive Enzyme Activity Clones
[0076] Directed mutagenesis was performed on two different enzymes
(alkaline phosphatase and .beta.-glycosidase), using the two
different strategies described here, to generate new enzymes which
exhibit a higher degree of activity than the wild-type enzymes.
Alkaline Phosphatase
[0077] The XL1-Red strain (Stratagene) was transformed with genomic
clone 27s3a plasmid pBluescript) encoding the alkaline phosphatase
gene from the organism OC9a according to the manufacturer's
protocol. A 5 ml culture of LB+0.1 mg/ml ampicillin was inoculated
with 200 .mu.l of the transformation. The culture was allowed to
grow at 37.degree. C. for 30 hours. A miniprep was then performed
on the culture, and screening was performed by transforming 2 .mu.l
of the resulting DNA into XL-1 Blue cells (Stratagene) according to
the manufacturer's protocol and following procedure outlined below
(after "Transform XL1 Blue cells). The mutated OC9a phosphatase
took 10 minutes to develop color and the wild type enzyme took 30
minutes to develop color in the screening assay.
[0078] Standard Alkaline Phosphatase Screening Assay
[0079] Transform XL1 Red strain Inoculate 5 ml LB/amp culture with
200 .mu.l transformation and incubate at 37.degree. C. for 30
hours.fwdarw.Miniprep DNA.fwdarw.Transform XL1 Blue
cells.fwdarw.Plate on LB/amp plates.fwdarw.Lift colonies with
Duralon UV (Stratagene) or HATF (Millipore) membranes.fwdarw.Lyse
in chloroform vapors for 30 seconds.fwdarw.Heat kill for 30 minutes
at 85.degree. C..fwdarw.Develop filter at room temperature in BCIP
buffer.fwdarw.Watch as filter develops and identify and pick
fastest developing colonies ("positives").fwdarw.Re- streak
"positives" onto a BCIP plate
[0080] BCIP Buffer:
[0081] 20 mm CAPS pH 9.0
[0082] 1 mm MgCl.sub.2
[0083] 0.01 mm ZnCl.sub.2
[0084] 0.1 mg/ml BCIP
Beta-Glycosidase
[0085] This protocol was used to mutagenize Thermococcus 9N2
Beta-Glycosidase.
[0086] PCR Reaction
[0087] 2 microliters dNTP's (10 mM Stocks)
[0088] 10 microliters 10.times. PCR Buffer
[0089] 0.5 microliters Vector DNA-31G1A-100 nanograms
[0090] 20 microliters 3' Primer (100 pmol)
[0091] 20 microliters 5' Primer (100 pmol)
[0092] 16 microliters MnCl 4H.sub.2O (1.25 mM Stock)
[0093] 24.5 microliters H.sub.2O
[0094] 1 microliter Taq Polymerase (5.0 Units)
[0095] 100 microliters total
[0096] Reaction Cycle
[0097] 95.degree. C. 15 seconds
[0098] 58.degree. C. 30 seconds
[0099] 72.degree. C. 90 seconds
[0100] 25 cycles (10 minute extension at 72.degree. C.-4.degree. C.
incubation)
[0101] Run 5 microliters on a 1% agarose gel to check the
reaction.
[0102] Purify on a Qiaquick column (Qiagen).
[0103] Resuspend in 50 microliters H.sub.2O.
[0104] Restriction Digest
[0105] 25 microliters purified PCR product
[0106] 10 microliters NEB Buffer #2
[0107] 3 microliters Kpn I (10 U/microliter)
[0108] 3 microliters EcoR1 (20 U/microliter)
[0109] 59 microliters H.sub.2O
[0110] Cut for 2 hours at 37.degree. C.
[0111] Purify on a Qiaquick column (Qiagen).
[0112] Elute with 35 microliters H.sub.2O.
[0113] Ligation
[0114] 10 microliters Digested PCR product
[0115] 5 microliters Vector (cut with EcoRI/KpnI and phosphatased
with shrimp alkaline phosphatase
[0116] 4 microliters 5.times. Ligation Buffer
[0117] 1 microliter T4 DNA Ligase (BRL)
[0118] Ligate overnight.
[0119] Transform into M15pREP4 cells using electroporation.
[0120] Plate 100 or 200 microliters onto LB amp meth kan plates,
grow overnight at 37 degrees celsius.
[0121] Beta-Glycosidase Assay
[0122] Perform glycosidase assay to screen for mutants as follows.
The filter assay uses buffer Z (see recipe below) containing 1
mg/ml of the substrate
5-bromo-4-chloro-3-indolyl-.beta.-o-glucopyranoside (XGLU)
(Diagnostic Chemicals Limited or Sigma).
[0123] Z-Buffer: (referenced in Miller, J. H. (1992) A Short Course
in Bacterial Genetics, p. 445.)
4 per liter: Na.sub.2HPO.sub.4--7H.sub.2O 16.1 g
NaH.sub.2PO.sub.4--H.sub.2O 5.5 g KCl 0.75 g MgSO.sub.4--7H.sub.2O
0.246 g 6-mercaptoethanol 2.7 ml Adjust pH to 7.0
[0124] (1) Perform colony lifts using Millipore HATF membrane
filters.
[0125] (2) Lyse colonies with chloroform vapor in 150 mm glass
petri dishes.
[0126] (3) Transfer filters to 100 mm glass petri dishes containing
a piece of Whatman 3MM filter paper saturated with Z buffer
containing 1 mg/ml XGLU. After transferring filter bearing lysed
colonies to the glass petri dish, maintain dish at room
temperature.
[0127] (4) "Positives" were observed as blue spots on the filter
membranes ("positives" are spots which appear early). Use the
following filter rescue technique to retrieve plasmid from lysed
positive colony. Use pasteur pipette (or glass capillary tube) to
core blue spots on the filter membrane. Place the small filter disk
in an Epp tube containing 20 .mu.l water. Incubate the Epp tube at
75.degree. C. for 5 minutes followed by vortexing to elute plasmid
DNA off filter. Transform this DNA into electrocompetent E. coli
cells. Repeat filter-lift assay on transformation plates to
identify "positives." Return transformation plates to 37.degree. C.
incubator after filter lift to regenerate colonies. Inoculate 3 ml
LBamp liquid with repurified positives and incubate at 37.degree.
C. overnight. Isolate plasmid DNA from these cultures and sequence
plasmid insert.
EXAMPLE 4
Construction of a Stable, Large Insert DNA Library of Picoplankton
Genomic DNA
[0128] Cell Collection and Preparation of DNA.
[0129] 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 (10 mM 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.
[0130] 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.
[0131] 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.
[0132] 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.
[0133] 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.
* * * * *