U.S. patent application number 09/848185 was filed with the patent office on 2002-09-12 for high throughput screening for novel enzymes.
This patent application is currently assigned to Diversa Corporation. Invention is credited to Keller, Martin, Short, Jay M..
Application Number | 20020127560 09/848185 |
Document ID | / |
Family ID | 25367333 |
Filed Date | 2002-09-12 |
United States Patent
Application |
20020127560 |
Kind Code |
A1 |
Short, Jay M. ; et
al. |
September 12, 2002 |
High throughput screening for novel enzymes
Abstract
Disclosed is a process for identifying clones having a specified
activity of interest, which process comprises (i) generating one or
more expression libraries derived from nuclei acid directly
isolated from the environment; and (ii) screening said libraries
utilizing a fluorescence activated cell sorter to identify said
clones. More particularly, this is a process for identifying clones
having a specified activity of interest by (i) generating one or
more expression libraries derived from nucleic acid directly or
indirectly isolated from the environment; (ii) exposing said
libraries to a particular substrate or substrates of interest; and
(iii) screening said exposed libraries utilizing a fluorescence
activated cell sorter to identify clones which react with the
substrate or substrates. Also provided is a process for identifying
clones having a specified activity of interest by (i) generating
one or more expression libraries derived from nucleic acid directly
or indirectly isolated from the environment; and (ii) screening
said exposed libraries utilizing an assay requiring
co-encapsulation, a binding event or the covalent modification of a
target, and a fluorescence activated cell sorter to identify
positive clones.
Inventors: |
Short, Jay M.; (Rancho Santa
Fe, CA) ; Keller, Martin; (San Diego, CA) |
Correspondence
Address: |
Lisa A. Haile, Ph.D.
Gray Cary Ware & Freidenrich LLP
Suite 1600
4365 Executive Drive
San Diego
CA
92121-2189
US
|
Assignee: |
Diversa Corporation
|
Family ID: |
25367333 |
Appl. No.: |
09/848185 |
Filed: |
May 3, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09848185 |
May 3, 2001 |
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09636778 |
Aug 11, 2000 |
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09636778 |
Aug 11, 2000 |
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09098206 |
Jun 16, 1998 |
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6174673 |
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09098206 |
Jun 16, 1998 |
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08876276 |
Jun 16, 1997 |
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Current U.S.
Class: |
435/6.14 ;
435/471; 435/7.32 |
Current CPC
Class: |
C12N 15/1055 20130101;
C12Q 1/6811 20130101; C12N 15/1037 20130101; C40B 40/02
20130101 |
Class at
Publication: |
435/6 ; 435/7.32;
435/471 |
International
Class: |
C12Q 001/68; G01N
033/554; G01N 033/569; C12N 015/74 |
Claims
What is claimed is:
1. A method for enriching for target DNA sequences containing at
least a partial coding region for at least one specified activity
in a DNA sample comprising: a) co-encapsulating in a
micro-environment a mixture of target DNA obtained from more than
one organism with a mixture of DNA probes comprising a detectable
marker and at least a portion of a DNA sequence encoding at least
one enzyme having a specified enzyme activity; b) incubating the
co-encapsulated mixture under such conditions and for such time as
to allow hybridization of complementary sequences; and c) screening
for the specified activity.
2. The method of claim 1, further comprising transforming host
cells with recovered target DNA to produce an expression library of
a plurality of clones.
3. The method of claim 1, wherein the organisms are
microorganisms.
4. The method of claim 3, wherein the microorganisms are uncultured
microorganisms.
5. The method of claim 1, further comprising screening the
expression library for the specified enzyme activity.
6. The method of claim 1, wherein the target DNA obtained from the
DNA population is selected by: a) converting double stranded DNA
into single stranded DNA; b) recovering from the converted single
stranded DNA, single stranded target DNA which hybridizes to probe
DNA; c) converting recovered single stranded target DNA to double
stranded DNA; and c) transforming a host cell with the double
stranded DNA of c).
7. A method of FACS screening for an agent that modulates the
activity of a target cell component, wherein the target cell
component and a selectable marker are expressed by a eukaryotic
cell, the method comprising co-encapsulating the agent in a
microenvironment with the recombinant cell expressing the target
cell component and detectable marker and detecting the effect of
the agent on the activity of the cell component.
8. The method of claim 1, wherein said target DNA is gene cluster
DNA.
9. The method of claim 4, wherein the uncultured microorganisms are
derived from an environmental sample.
10. The method of claim 4, wherein the uncultured microorganisms
comprise a mixture of terrestrial microorganisms or marine
microorganisms or airborne microorganisms, or a mixture of
terrestrial microorganisms, marine microorganisms and airborne
microorganisms.
11. The method of claim 2, wherein the clones comprise a construct
selected from the group consisting of phage, plasmids, phagemids,
cosmids, fosmids, viral vectors, and artificial chromosomes.
12. The method of claim 1, wherein the target DNA comprises one or
more operons, or portions thereof, of the DNA population.
13. The method of claim 12, wherein the operon or portions thereof
encodes a complete or partial metabolic pathway.
14. The method of claim 4, wherein the uncultured microorganisms
comprise extremophiles.
15. The method of claim 14, wherein the extremophiles are selected
from the group consisting of thermophiles, hyperthermophiles,
psychrophiles, barophiles, and psychrotrophs.
16. The method of claim 6, wherein the host cell is selected from
the group consisting of a bacterium, fungus, plant cell, insect
cell and animal cell.
17. The method of claim 1, wherein the target DNA encodes a
protein.
18. The method of claim 17, wherein the protein is an enzyme.
19. The method of claim 18, wherein the enzyme is selected from the
group consisting of oxidoreductases, transferases, hydrolases,
lyases, isomerases, and ligases.
20. The method of claim 1, wherein the micro-environment is a
liposome, gel microdrop, bead, agarose, cell, ghost red blood cell
or ghost macrophage.
21. The method of claim 20, wherein the liposomes are prepared from
one or more phospholipids, glycolipids, steroids, alkyl phosphates
or fatty acid esters.
22. The method of claim 21, wherein the phospholipids are selected
from the group consisting of lecithin, sphingomyelin and
dipalmitoyl.
23. The method of claim 20, wherein the steroids are selected from
the group consisting of cholesterol, cholestanol and
lanosterol.
24. The method of claim 1, wherein the detectable marker is a
fluorescent dye, a visible dye, a bioluminescent material, a
chemiluminescent material, a radioactive material, or an enzymatic
substrate.
25. The method of claim 24, wherein the bioluminescent material is
green fluorescent protein (GFP) or red fluorescent protein
(RFP).
26. The method of claim 25, wherein detection of the fluorescent
dye or a visible dye is carried out by fluorometric or
spectrophotometric measurement.
Description
[0001] This application is a divisional of U.S. patent application
Ser. No. 09/636,778, filed Aug. 11, 2000, which is a continuation
of U.S. patent application Ser. No. 09/098,206, filed Jun. 16,
1998, continuation-in-part of U.S. application Ser. No. 08/876,276,
filed Jun. 16, 1997, now abandoned.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the
identification of new bioactive molecules and particularly to
methods for recovering such molecules by co-encapsulation and
fluorescence activated cell sorting (FACS).
BACKGROUND OF THE INVENTION
[0003] There is a critical need in the chemical industry for
efficient catalysts for the practical synthesis of optically pure
materials; enzymes can provide the optimal solution. All classes of
molecules and compounds that are utilized in both established and
emerging chemical, pharmaceutical, textile, food and feed,
detergent markets must meet stringent economical and environmental
standards. The synthesis of polymers, pharmaceuticals, natural
products and agrochemicals is often hampered by expensive processes
which produce harmful byproducts and which suffer from low
enantioselectivity (Faber, 1995; Tonkovich and Gerber, U.S. Dept of
Energy study, 1995). Enzymes have a number of remarkable advantages
which can overcome these problems in catalysis: they act on single
functional groups, they distinguish between similar functional
groups on a single molecule, and they distinguish between
enantiomers. Moreover, they are biodegradable and function at very
low mole fractions in reaction mixtures. Because of their chemo-,
regio- and stereospecificity, enzymes present a unique opportunity
to optimally achieve desired selective transformations. These are
often extremely difficult to duplicate chemically, especially in
single-step reactions. The elimination of the need for protection
groups, selectivity, the ability to carry out multi-step
transformations in a single reaction vessel, along with the
concomitant reduction in environmental burden, has led to the
increased demand for enzymes in chemical and pharmaceutical
industries (Faber, 1995). Enzyme-based processes have been
gradually replacing many conventional chemical-based methods
(Wrotnowski, 1997). A current limitation to more widespread
industrial use is primarily due to the relatively small number of
commercially available enzymes. Only .about.300 enzymes (excluding
DNA modifying enzymes) are at present commercially available from
the >3000 non DNA-modifying enzyme activities thus far
described.
[0004] The use of enzymes for technological applications also may
require performance under demanding industrial conditions. This
includes activities in environments or on substrates for which the
currently known arsenal of enzymes was not evolutionarily selected.
Enzymes have evolved by selective pressure to perform very specific
biological functions within the milieu of a living organism, under
conditions of mild temperature, pH and salt concentration. For the
most part, the non-DNA modifying enzyme activities thus far
described (Enzyme Nomenclature, 1992) have been isolated from
mesophilic organisms, which represent a very small fraction of the
available phylogenetic diversity (Amann et al., 1995). The dynamic
field of biocatalysis takes on a new dimension with the help of
enzymes isolated from microorganisms that thrive in extreme
environments. Such enzymes must function at temperatures above
100.degree. C. in terrestrial hot springs and deep sea thermal
vents, at temperatures below 0.degree. C. in arctic waters, in the
saturated salt environment of the Dead Sea, at pH values around 0
in coal deposits and geothermal sulfur-rich springs, or at pH
values greater than 11 in sewage sludge (Adams and Kelly, 1995).
Enzymes obtained from these extremophilic organisms open a new
field in biocatalysis.
[0005] For example, several esterases and lipases cloned and
expressed from extremophilic organisms are remarkably robust,
showing high activity throughout a wide range of temperatures and
pHs. The fingerprints of five of these esterases show a diverse
substrate spectrum, in addition to differences in the optimum
reaction temperature. As seen in FIG. 1, esterase #5 recognizes
only short chain substrates while #2 only acts on long chain
substrates in addition to a huge difference in the optimal reaction
temperature. These results suggest that more diverse enzymes
fulfilling the need for new biocatalysts can be found by screening
biodiversity. Substrates upon which enzymes act are herein defined
as bioactive substrates.
[0006] Furthermore, virtually all of the enzymes known so far have
come from cultured organisms, mostly bacteria and more recently
archaea (Enzyme Nomenclature, 1992). Traditional enzyme discovery
programs rely solely on cultured microorganisms for their screening
programs and are thus only accessing a small fraction of natural
diversity. Several recent studies have estimated that only a small
percentage, conservatively less than 1%, of organisms present in
the natural environment have been cultured (see Table I, Amann et
al., 1995, Barns et. al 1994, Torvsik, 1990). For example, Norman
Pace's laboratory recently reported intensive untapped diversity in
water and sediment samples from the "Obsidian Pool" in Yellowstone
National Park, a spring which has been studied since the early
1960's by microbiologists (Bams, 1994). Amplification and cloning
of 16S rRNA encoding sequences revealed mostly unique sequences
with little or no representation of the organisms which had
previously been cultured from this pool. This suggests substantial
diversity of archaea with so far unknown morphological,
physiological and biochemical features which may be useful in
industrial processes. David Ward's laboratory in Bozmen, Mont. has
performed similar studies on the cyanobacterial mat of Octopus
Spring in Yellowstone Park and came to the same conclusion, namely,
tremendous uncultured diversity exists (Bateson et al., 1989).
Giovannoni et al. (1990) reported similar results using
bacterioplankton collected in the Sargasso Sea while Torsvik et al.
(1990) have shown by DNA reassociation kinetics that there is
considerable diversity in soil samples. Hence, this vast majority
of microorganisms represents an untapped resource for the discovery
of novel biocatalysts. In order to access this potential catalytic
diversity, recombinant screening approaches are required.
[0007] The discovery of novel bioactive molecules other than
enzymes is also afforded by the present invention. For instance,
antibiotics, antivirals, antitumor agents and regulatory proteins
can be discovered utilizing the present invention.
[0008] 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.
[0009] Some gene families consist of one or more 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 of
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.
[0010] 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. 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.
As indicated, 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.
[0011] 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 huge 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 encoded enzymes, complicating genetic
manipulation and in vitro studies of these genes/proteins. The
method(s) of the present invention facilitate the rapid discovery
of these gene clusters in gene expression libraries.
[0012] Of particular interest are cellular "switches" known as
receptors which interact with a variety of biomolecules, such as
hormones, growth factors, and neurotransmitters, to mediate the
transduction of an "external" cellular signaling event into an
"internal" cellular signal. External signaling events include the
binding of a ligand to the receptor, and internal events include
the modulation of a pathway in the cytoplasm or nucleus involved in
the growth, metabolism or apoptosis of the cell. Internal events
also include the inhibition or activation of transcription of
certain nucleic acid sequences, resulting in the increase or
decrease in the production or presence of certain molecules (such
as nucleic acid, proteins, and/or other molecules affected by this
increase or decrease in transcription). Drugs to cure disease or
alleviate its symptoms can activate or block any of these events to
achieve a desired pharmaceutical effect.
[0013] Transduction can be accomplished by a transducing protein in
the cell membrane which is activated upon an allosteric change the
receptor may undergo upon binding to a specific biomolecule. The
"active" transducing protein activates production of so-called
"second messenger" molecules within the cell, which then activate
certain regulatory proteins within the cell that regulate gene
expression or alter some metabolic process. Variations on the theme
of this "cascade" of events occur. For example, a receptor may act
as its own transducing protein, or a transducing protein may act
directly on an intracellular target without mediation by a second
messenger.
[0014] Signal transduction is a fundamental area of inquiry in
biology. For instance, ligand/receptor interactions and the
receptor/effector coupling mediated by Guanine nucleotide-binding
proteins (G-proteins) are of interest in the study of disease. A
large number of G protein-linked receptors funnel extracellular
signals as diverse as hormones, growth factors, neurotransmitters,
primary sensory stimuli, and other signals through a set of G
proteins to a small number of second-messenger systems. The G
proteins act as molecular switches with an "on" and "off" state
governed by a GTPase cycle. Mutations in G proteins may result in
either constitutive activation or loss of expression mutations.
[0015] Many receptors convey messages through heterotrimeric G
proteins, of which at least 17 distinct forms have been isolated.
Additionally, there are several different G protein-dependent
effectors. The signals transduced through the heterotrimeric G
proteins in mammalian cells influence intracellular events through
the action of effector molecules.
[0016] Given the variety of functions subserved by G
protein-coupled signal transduction, it is not surprising that
abnormalities in G protein-coupled pathways can lead to diseases
with manifestations as dissimilar as blindness, hormone resistance,
precocious puberty and neoplasia. G-protein-coupled receptors are
extremely important to drug research efforts. It is estimated that
up to 60% of today's prescription drugs work by somehow interacting
with G protein-coupled receptors. However, these drugs were
developed using classical medicinal chemistry and without a
knowledge of the molecular mechanism of action. A more efficient
drug discovery program could be deployed by targeting individual
receptors and making use of information on gene sequence and
biological function to develop effective therapeutics. The present
invention allows one to, for example, study molecules which affect
the interaction of G proteins with receptors, or of ligands with
receptors.
[0017] Several groups have reported cells which express mammalian G
proteins or subunits thereof, along with mammalian receptors which
interact with these molecules. For example, WO92/05244 (Apr. 2,
1992) describes a transformed yeast cell which is incapable of
producing a yeast G protein a subunit, but which has been
engineered to produce both a mammalian G protein .alpha. subunit
and a mammalian receptor which interacts with the subunit. The
authors found that a modified version of a specific mammalian
receptor integrated into the membrane of the cell, as shown by
studies of the ability of isolated membranes to interact properly
with various known agonists and antagonists of the receptor. Ligand
binding resulted in G protein-mediated signal transduction.
[0018] Another group has described the functional expression of a
mammalian adenylyl cyclase in yeast, and the use of the engineered
yeast cells in identifying potential inhibitors or activators of
the mammalian adenylyl cyclase (WO 95/30012). Adenylyl cyclase is
among the best studied of the effector molecules which function in
mammalian cells in response to activated G proteins. "Activators"
of adenylyl cyclase cause the enzyme to become more active,
elevating the cAMP signal of the yeast cell to a detectable degree.
"Inhibitors" cause the cyclase to become less active, reducing the
cAMP signal to a detectable degree. The method describes the use of
the engineered yeast cells to screen for drugs which activate or
inhibit adenylyl cyclase by their action on G protein-coupled
receptors.
[0019] When attempting to identify genes encoding bioactivities of
interest from complex environmental expression libraries, the rate
limiting steps in discovery occur at the both DNA cloning level and
at the screening level. Screening of complex environmental
libraries which contain, for example, 100's of different organisms
requires the analysis of several million clones to cover this
genomic diversity. An extremely high-throughput screening method
has been developed to handle the enormous numbers of clones present
in these libraries.
[0020] In traditional flow cytometry, it is common to analyze very
large numbers of eukaryotic cells in a short period of time. Newly
developed flow cytometers can analyze and sort up to 20,000 cells
per second. In a typical flow cytometer, individual particles pass
through an illumination zone and appropriate detectors, gated
electronically, measure the magnitude of a pulse representing the
extent of light scattered. The magnitude of these pulses are sorted
electronically into "bins" or "channels", permitting the display of
histograms of the number of cells possessing a certain quantitative
property versus the channel number (Davey and Kell, 1996). It was
recognized early on that the data accruing from flow cytometric
measurements could be analyzed (electronically) rapidly enough that
electronic cell-sorting procedures could be used to sort cells with
desired properties into separate "buckets", a procedure usually
known as fluorescence-activated cell sorting (Davey and Kell,
1996).
[0021] Fluorescence-activated cell sorting has been primarily used
in studies of human and animal cell lines and the control of cell
culture processes. Fluorophore labeling of cells and measurement of
the fluorescence can give quantitative data about specific target
molecules or subcellular components and their distribution in the
cell population. Flow cytometry can quantitate virtually any
cell-associated property or cell organelle for which there is a
fluorescent probe (or natural fluorescence). The parameters which
can be measured have previously been of particular interest in
animal cell culture.
[0022] Flow cytometry has also been used in cloning and selection
of variants from existing cell clones. This selection, however, has
required stains that diffuse through cells passively, rapidly and
irreversibly, with no toxic effects or other influences on
metabolic or physiological processes. Since, typically, flow
sorting has been used to study animal cell culture performance,
physiological state of cells, and the cell cycle, one goal of cell
sorting has been to keep the cells viable during and after
sorting.
[0023] There currently are no reports in the literature of
screening and discovery of recombinant enzymes in E. coli
expression libraries by fluorescence activated cell sorting of
single cells. Furthermore there are no reports of recovering DNA
encoding bioactivities screened by expression screening in E. coli
using a FACS machine. The present invention provides these methods
to allow the extremely rapid screening of viable or non-viable
cells to recover desirable activities and the nucleic acid encoding
those activities.
[0024] A limited number of papers describing various applications
of flow cytometry in the field of microbiology and sorting of
fluorescence activated microorganisms have, however, been published
(Davey and Kell, 1996). Fluorescence and other forms of staining
have been employed for microbial discrimination and identification,
and in the analysis of the interaction of drugs and antibiotics
with microbial cells. Flow cytometry has been used in aquatic
biology, where autofluorescence of photosynthetic pigments are used
in the identification of algae or DNA stains are used to quantify
and count marine populations (Davey and Kell, 1996). Thus, Diaper
and Edwards used flow cytometry to detect viable bacteria after
staining with a range of fluorogenic esters including fluorescein
diacetate (FDA) derivatives and CemChrome B, a proprietary stain
sold commercially for the detection of viable bacteria in
suspension (Diaper and Edwards, 1994). Labeled antibodies and
oligonucleotide probes have also been used for these purposes.
[0025] Papers have also been published describing the application
of flow cytometry to the detection of native and recombinant
enzymatic activities in eukaryotes. Betz et al. studied native
(non-recombinant) lipase production by the eukaryote, Rhizopus
arrhizus with flow cytometry. They found that spore suspensions of
the mold were heterogeneous as judged by light-scattering data
obtained with excitation at 633 nm, and they sorted clones of the
subpopulations into the wells of microtiter plates. After
germination and growth, lipase production was automatically assayed
(turbidimetrically) in the microtiter plates, and a representative
set of the most active were reisolated, cultured, and assayed
conventionally (Betz et al., 1984).
[0026] Scrienc et al. have reported a flow cytometric method for
detecting cloned -galactosidase activity in the eukaryotic
organism, S. cerevisiae. The ability of flow cytometry to make
measurements on single cells means that individual cells with high
levels of expression (e.g., due to gene amplification or higher
plasmid copy number) could be detected. In the method reported, a
non-fluorescent compound .beta.-naphthol-.beta.-galact-
opyranoside) is cleaved by .beta.-galactosidase and the liberated
naphthol is trapped to form an insoluble fluorescent product. The
insolubility of the fluorescent product is of great importance here
to prevent its diffusion from the cell. Such diffusion would not
only lead to an underestimation of .beta.-galactosidase activity in
highly active cells but could also lead to an overestimation of
enzyme activity in inactive cells or those with low activity, as
they may take up the leaked fluorescent compound, thus reducing the
apparent heterogeneity of the population.
[0027] One group has described the use of a FACS machine in an
assay detecting fusion proteins expressed from a specialized
transducing bacteriophage in the prokaryote Bacillus subtilis
(Chung, et. al., J. of Bacteriology, Apr. 1994, p. 1977-1984;
Chung, et. al., Biotechnology and Bioengineering, Vol. 47, pp.
234-242 (1995)). This group monitored the expression of a lacZ gene
(encodes b-galactosidase) fused to the sporulation loci in subtilis
(spo). The technique used to monitor b-galactosidase expression
from spo-lacZ fusions in single cells involved taking samples from
a sporulating culture, staining them with a commercially available
fluorogenic substrate for b-galactosidase called C8-FDG, and
quantitatively analyzing fluorescence in single cells by flow
cytometry. In this study, the flow cytometer was used as a detector
to screen for the presence of the spo gene during the development
of the cells. The device was not used to screen and recover
positive cells from a gene expression library or nucleic acid for
the purpose of discovery.
[0028] Another group has utilized flow cytometry to distinguish
between the developmental stages of the delta-proteobacteria
Myxococcus xanthus (F. Russo-Marie, et. al., PNAS, Vol. 90,
pp.8194-8198, September 1993). As in the previously described
study, this study employed the capabilities of the FACS machine to
detect and distinguish genotypically identical cells in different
development regulatory states. The screening of an enzymatic
activity was used in this study as an indirect measure of
developmental changes.
[0029] The lacZ gene from E. coli is often used as a reporter gene
in studies of gene expression regulation, such as those to
determine promoter efficiency, the effects of trans-acting factors,
and the effects of other regulatory elements in bacterial, yeast,
and animal cells. Using a chromogenic substrate, such as ONPG
(o-nitrophenyl-(-D-galactopyranosid- e), one can measure expression
of .beta.-galactosidase in cell cultures; but it is not possible to
monitor expression in individual cells and to analyze the
heterogeneity of expression in cell populations. The use of
fluorogenic substrates, however, makes it possible to determine
.beta.-galactosidase activity in a large number of individual cells
by means of flow cytometry. This type of determination can be more
informative with regard to the physiology of the cells, since gene
expression can be correlated with the stage in the mitotic cycle or
the viability under certain conditions. In 1994, Plovins et al.,
reported the use of fluorescein-Di-.beta.-D-galactopyranoside (FDG)
and C.sub.12-FDG as substrates for .beta.-galactosidase detection
in animal, bacterial, and yeast cells. This study compared the two
molecules as substrates for .beta.-galactosidase, and concluded
that FDG is a better substrate for .beta.-galactosidase detection
by flow cytometry in bacterial cells. The screening performed in
this study was for the comparison of the two substrates. The
detection capabilities of a FACS machine were employed to perform
the study on viable bacterial cells.
[0030] Cells with chromogenic or fluorogenic substrates yield
colored and fluorescent products, respectively. Previously, it had
been thought that the flow cytometry-fluorescence activated cell
sorter approaches could be of benefit only for the analysis of
cells that contain intracellularly, or are normally physically
associated with, the enzymatic activity of small molecule of
interest. On this basis, one could only use fluorogenic reagents
which could penetrate the cell and which are thus potentially
cytotoxic. To avoid clumping of heterogeneous cells, it is
desirable in flow cytometry to analyze only individual cells, and
this could limit the sensitivity and therefore the concentration of
target molecules that can be sensed. Weaver and his colleagues at
MIT and others have developed the use of gel microdroplets
containing (physically) single cells which can take up nutrients,
secret products, and grow to form colonies. The diffusional
properties of gel microdroplets may be made such that sufficient
extracellular product remains associated with each individual gel
microdroplet, so as to permit flow cytometric analysis and cell
sorting on the basis of concentration of secreted molecule within
each microdroplet. Beads have also been used to isolate mutants
growing at different rates, and to analyze antibody secretion by
hybridoma cells and the nutrient sensitivity of hybridoma cells.
The gel microdroplet method has also been applied to the rapid
analysis of mycobacterial growth and its inhibition by
antibiotics.
[0031] The gel microdroplet technology has had significance in
amplifying the signals available in flow cytometric analysis, and
in permitting the screening of microbial strains in strain
improvement programs for biotechnology. Wittrup et al.,
(Biotechnolo.Bioeng. (1993) 42:351-356) developed a
microencapsulation selection method which allows the rapid and
quantitative screening of >106 yeast cells for enhanced
secretion of Aspergillus awamori glucoamylase. The method provides
a 400-fold single-pass enrichment for high-secretion mutants.
[0032] Gel microdroplet or other related technologies can be used
in the present invention to localize as well as amplify signals in
the high throughput screening of recombinant libraries. Cell
viability during the screening is not an issue or concern since
nucleic acid can be recovered from the microdroplet.
[0033] Different types of encapsulation strategies and compounds or
polymers can be used with the present invention. For instance, high
temperature agaroses can be employed for making microdroplets
stable at high temperatures, allowing stable encapsulation of cells
subsequent to heat kill steps utilized to remove all background
activities when screening for thermostable bioactivities.
[0034] There are several hurdles which must be overcome when
attempting to detect and sort E. coli expressing recombinant
enzymes, and recover encoding nucleic acids. FACS systems have
typically been based on eukaryotic separations and have not been
refined to accurately sort single E. coli cells; the low forward
and sideward scatter of small particles like E. coli, reduces the
ability of accurate sorting; enzyme substrates typically used in
automated screening approaches, such as umbelifferyl based
substrates, diffuse out of E. Coli at rates which interfere with
quantitation. Further, recovery of very small amounts of DNA from
sorted organisms can be problematic. The present invention
addresses and overcomes these hurdles and offers a novel screening
approach.
SUMMARY OF THE INVENTION
[0035] The present invention adapts traditional eukaryotic flow
cytometry cell sorting systems to high throughput screening for
expression clones in prokaryotes. In the present invention,
expression libraries derived from DNA, primarily DNA directly
isolated from the environment, are screened very rapidly for
bioactivities of interest utilizing fluorescense activated cell
sorting. These libraries can contain greater than 10.sup.8 members
and can represent single organisms or can represent the genomes of
over 100 different microorganisms, species or subspecies.
[0036] Accordingly, in one aspect, the present invention provides a
process for identifying clones having a specified activity of
interest, which process comprises (i) generating one or more
expression libraries derived from nucleic acid directly isolated
from the environment; and (ii) screening said libraries utilizing a
high throughput cell analyzer, preferably a fluorescence activated
cell sorter, to identify said clones.
[0037] More particularly, the invention provides a process for
identifying clones having a specified activity of interest by (i)
generating one or more expression libraries made to contain nucleic
acid directly or indirectly isolated from the environment; (ii)
exposing said libraries to a particular substrate or substrates of
interest; and (iii) screening said exposed libraries utilizing a
high throughput cell analyzer, preferably a fluorescence activated
cell sorter, to identify clones which react with the substrate or
substrates.
[0038] In another aspect, the invention also provides a process for
identifying clones having a specified activity of interest by (i)
generating one or more expression libraries derived from nucleic
acid directly or indirectly isolated from the environment; and (ii)
screening said exposed libraries utilizing an assay requiring a
binding event or the covalent modification of a target, and a high
throughput cell analyzer, preferably a fluorescence activated cell
sorter, to identify positive clones.
[0039] The invention further provides a method of screening for an
agent that modulates the activity of a target protein or other cell
component (e.g., nucleic acid), wherein the target and a selectable
marker are expressed by a recombinant cell, by co-encapsulating the
agent in a micro-environment with the recombinant cell expressing
the target and detectable marker and detecting the effect of the
agent on the activity of the target cell component.
[0040] In another embodiment, the invention provides a method for
enriching for target DNA sequences containing at least a partial
coding region for at least one specified activity in a DNA sample
by co-encapsulating a mixture of target DNA obtained from a mixture
of organisms with a mixture of DNA probes including a detectable
marker and at least a portion of a DNA sequence encoding at least
one enzyme having a specified enzyme activity and a detectable
marker; incubating the co-encapsulated mixture under such
conditions and for such time as to allow hybridization of
complementary sequences and screening for the target DNA.
Optionally the method further comprises transforming host cells
with recovered target DNA to produce an expression library of a
plurality of clones.
[0041] The invention further provides a method of screening for an
agent that modulates the interaction of a first test protein linked
to a DNA binding moiety and a second test protein linked to a
transcriptional activation moiety by co-encapsulating the agent
with the first test protein and second test protein in a suitable
microenvironment and determining the ability of the agent to
modulate the interaction of the first test protein linked to a DNA
binding moiety with the second test protein covalently linked to a
transcriptional activation moiety, wherein the agent enhances or
inhibits the expression of a detectable protein. Preferably,
screening is by FACS analysis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0042] FIG. 1 illustrates the substrate spectrum fingerprints and
optimum reaction temperatures of five of novel esterases showing
the diversity in these enzymes. EST# indicates the different
enzyme; the temperatures indicate the optimal growth temperatures
for the organisms from which the esterases were isolated; "E"
indicates the relative activity of each esterase enzyme on each of
the given substrates indicated (Hepanoate being the reference).
[0043] FIG. 2 illustrates the cloning of DNA fragments prepared by
random cleavage of target DNA to generate a representative library
as described in Example 1.
[0044] FIG. 3 shows a statistical analysis of the total number of
clones to be tested (e.g. the number of genome equivalents).
Assuming that mechanical shearing and gradient purification results
in normal distribution of DNA fragment sizes with a mean of 4.5 kbp
and variance of 1 kbp, the fraction represented of all possible 1
kbp sequences in a 1.8 Mbp genome is plotted in FIG. 3 as a
function of increasing genome equivalents.
[0045] FIG. 4 illustrates the protocol used in the cell sorting
method of the invention to screen for recombinant enzymes, in this
case using a (library excised into E. coli. The expression clones
of interest are isolated by sorting. The procedure is described in
detail in Examples 1,3 and 4.
[0046] FIG. 5 shows .beta.-galactosidase clones stained with three
different substrates: fluorescein-di-.beta.-D-galactopyranoside
(FDG), C12-fluorescein-di-.beta.-D-galactopyranoside (C12FDG),
chloromethyl-fluorescein-di-.beta.-D-galactopyranoside (CMFDG). E.
coli expressing .beta.-galactosidase from Sulfulobus sulfotaricus
species was grown overnight. Cells were centrifuged and substrate
was loaded with deionized water. After five (5) minutes cells were
centrifuged and transferred into HEPES buffer and heated to
70.degree. C. for thirty (30) minutes. Cells were spotted onto a
slide and exposed to UV light. This illustrates the results of the
experiments described in Example 3.
[0047] FIG. 6 shows a microtiter plate where E. coli cells sorted
in accordance with the invention are dispensed, one cell per well
and grown up as clones which are then stained with
fluorescein-di-.beta.-D-galactop- yranoside (FDG) (10 mM). This
illustrates the results of the experiments described in Example
5.
[0048] FIG. 7 shows the principle type of fluorescence enzyme assay
of deacylation.
[0049] FIG. 8 shows staining of .beta.-galactosidase clones from
the hyperthermophilic archaeon Sulfolobus solfataricus expressed in
E. coli using C.sub.12-FDG as enzyme substrate.
[0050] FIG. 9 shows the synthesis of
5-dodecanoyl-aminofluorescein-di-dode- canoic acid.
[0051] FIG. 10 shows Rhodamine protease substrate. FIG. 11 shows a
compound and process that can be used in the detection of
monooxygenases.
[0052] FIG. 12 is a schematic illustration of combinatorial enzyme
development using directed evolution.
[0053] FIG. 13 is a schematic illustration showing bypassing
barriers to directed evolution.
[0054] FIG. 14 depicts a co-encapsulation assay for a novel
bioactive screen. Cells containing large insert library clones are
coencapsulated with a eukaryotic cell containing a receptor.
Binding of the receptor by a small molecule expressed from the
library ultimately yields expression of a GFP reporter molecule.
Encapsulation can occur in a variety of means, including gel
microdroplets, liposomes, and ghost cells. Cells are screened via
high throughput screening on a fluorescence analyzer.
[0055] FIG. 15 depicts co-encapsulation of test organisms with
pathway clones and sorting based on assays for bioactive expression
of clones, such as affects on growth rates of test organisms. In
this figure, sorting occurs on a FACS machine.
[0056] FIG. 16 depicts micrographs of Streptomyces strains. The
picture on the left represents Streptomyces lividans mycelia, and
the right depicts unicells of another species of Streptomyces which
forms unicells (100X objective phase contrast; taken from an
Olympus microscope).
[0057] FIG. 17 depicts a side scatter versus forward scatter graph
of FACS sorted gel-microdroplets (GMD's) containing a species of
Streptomyces which forms unicells. Empty gel-microdroplets are
distinguished from free cells and debris, also.
[0058] FIG. 18 depicts co-encapsulation of a recombinant host cell
containing a clone expressing a small molecule, or agent (labeled
Bioactive), with another cell harboring a receptor, transducing
protein and other components. Activity of the agent compound on
various components of the cell can be assayed. Encapsulation means
includes gel microdroplets, liposomes, or ghost cells. The agent
can affect ligand/receptor interactions, as depicted, which affect
can be assayed via a variety of methods, including detection of
increase or decrease in presence of second messenger molecules,
detection of transcription or inhibition of transcription of a
target gene in the nucleus of the cell (including reporter molecule
expression), detection of phosphorylation or kinase of molecules
within the cell (all or any of which may be a response to the
enhancement or inhibition of the interaction of the ligand with the
receptor).
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0059] In the present invention, for example, gene libraries
generated from one or more uncultivated microorganisms are screened
for an activity of interest. Expression gene libraries are
generated, clones are either exposed to the substrate or
substrate(s) of interest, hybridized to a probe of interest, or
bound to a detectable ligand and positive clones are identified and
isolated via fluorescence activated cell sorting. Cells can be
viable or non-viable during the process or at the end of the
process, as nucleic acid encoding a positive activity can be
isolated and cloned utilizing techniques well known in the art.
[0060] This invention differs from fluorescense activated cell
sorting, as normally performed, in several aspects. Previously,
FACS machines have been employed in the studies focused on the
analyses of eukaryotic and prokaryotic cell lines and cell culture
processes. FACS has also been utilized to monitor production of
foreign proteins in both eukaryotes and prokaryotes to study, for
example, differential gene expression, etc. The detection and
counting capabilities of the FACS system have been applied in these
examples. However, FACS has never previously been employed in a
discovery process to screen for and recover bioactivities in
prokaryotes. Furthermore, the present invention does not require
cells to survive, as do previously described technologies, since
the desired nucleic acid (recombinant clones) can be obtained from
alive or dead cells. The cells only need to be viable long enough
to produce the compound to be detected, and can thereafter be
either viable or non-viable cells so long as the expressed
biomolecule remains active. The present invention also solves
problems that would have been associated with detection and sorting
of E. Coli expressing recombinant enzymes, and recovering encoding
nucleic acids. Additionally, the present invention includes within
its embodiments any apparatus capable of detecting flourescent
wavelengths associated with biological material, such apparatii are
defined herein as fluorescent analyzers (one example of which is a
FACS).
[0061] The use of a culture-independent approach to directly clone
genes encoding novel enzymes from environmental samples allows one
to access untapped resources of biodiversity. The approach is based
on the construction of "environmental libraries" which represent
the collective genomes of naturally occurring organisms archived in
cloning vectors that can be propagated in suitable prokaryotic
hosts. Because the cloned DNA is initially extracted directly from
environmental samples, the libraries are not limited to the small
fraction of prokaryotes that can be grown in pure culture.
Additionally, a normalization of the environmental DNA present in
these samples could allow more equal representation of the DNA from
all of the species present in the original sample. This can
dramatically increase the efficiency of finding interesting genes
from minor constituents of the sample which may be
under-represented by several orders of magnitude compared to the
dominant species.
[0062] In the evaluation of complex environmental expression
libraries, a rate limiting step previously occurred at the level of
discovery of bioactivities. The present invention allows the rapid
screening of complex environmental expression libraries,
containing, for example, thousands of different organisms. The
analysis of a complex sample of this size requires one to screen
several million clones to cover this genomic biodiversity. The
invention represents an extremely high-throughput screening method
which allows one to assess this enormous number of clones. The
method disclosed allows the screening anywhere from about 30
million to about 200 million clones per hour for a desired
biological activity. This allows the thorough screening of
environmental libraries for clones expressing novel
biomolecules.
[0063] The present invention combines a culture-independent
approach to directly clone genes encoding novel bioactivities from
environmental samples with an extremely high throughput screening
system designed for the rapid discovery of new biomolecules.
[0064] The strategy begins with the construction of gene libraries
which represent the genome(s) of microorganisms archived in cloning
vectors that can be propagated in E. coli or other suitable
prokaryotic hosts. Preferably, "environmental libraries" which
represent the collective genomes of naturally occurring
microorganisms are generated. In this case, because the cloned DNA
is extracted directly from environmental samples, the libraries are
not limited to the small fraction of prokaryotes that can be grown
in pure culture. In addition, "normalization" can be performed on
the environmental nucleic acid as one approach to more equally
represent the DNA from all of the species present in the original
sample. Normalization techniques can dramatically increase the
efficiency of discovery from genomes which may represent minor
constituents of the environmental sample. Normalization is
preferable since at least one study has demonstrated that an
organism of interest can be underrepresented by five orders of
magnitude compared to the dominant species.
[0065] The method of the present invention begins with the
construction of gene libraries which represent the collective
genomes of naturally occurring organisms archived in cloning
vectors that can be propagated in suitable prokaryotic hosts. 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. Libraries 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 is described and
exemplified in detail in co-pending, commonly assigned U.S. Serial
No. 08/657,409, filed Jun. 6, 1996, which is incorporated herein by
reference. Such microorganisms may be extremophiles, such as
hyperthermophiles, psychrophiles, psychrotrophs, halophiles,
alkalophiles, acidophiles, etc.
[0066] 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, genomic 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 or other biological
activity. Prokaryotic expression libraries created from such
starting material which includes DNA from more than one species are
defined herein as multispecific libraries.
[0067] In one embodiment, viable or non-viable cells isolated from
the environment are, prior to the isolation of nucleic acid for
generation of the expression gene library, FACS sorted to separate
prokaryotic cells from the sample based on, for instance, DNA or
AT/GC content of the cells. Various dyes or stains well known in
the art, for example those described in "Practical Flow Cytometry",
1995 Wiley-Liss, Inc., Howard M. Shapiro, M.D., are used to
intercalate or associate with nucleic acid of cells, and cells are
separated on the FACS based on relative DNA content or AT/GC DNA
content in the cells. Other criteria can also be used to separate
prokaryotic cells from the sample, as well. DNA is then isolated
from the cells and used for the generation of expression gene
libraries, which are then screened using the FACS for activities of
interest.
[0068] Alternatively, the nucleic acid is isolated directly from
the environment and is, prior to generation of the gene library,
sorted based on DNA or AT/GC content. DNA isolated directly from
the environment, is used intact, randomly sheared or digested to
general fragmented DNA. The DNA is then bound to an intercalating
agent as described above, and separated on the analyzer based on
relative base content to isolate DNA of interest. Sorted DNA is
then used for the generation of gene libraries, which are then
screened using the analyzer for activities of interest.
[0069] The present invention can further optimize methods for
isolation of activities of interest from a variety of sources,
including consortias of microorganisms, primary enrichments, and
environmental "uncultivated" samples, to make libraries which have
been "normalized" in their representation of the genome populations
in the original samples. and to screen these libraries for enzyme
and other bioactivities. Libraries with equivalent representation
of genomes from microbes that can differ vastly in abundance in
natural populations are generated and screened. This
"normalization" approach reduces the redundancy of clones from
abundant species and increases the representation of clones from
rare species. These normalized libraries allow for greater
screening efficiency resulting in the identification of cells
encoding novel biological catalysts.
[0070] One embodiment for forming a normalized library from an
environmental sample begins with the isolation of nucleic acid from
the sample. This nucleic acid can then be fractionated prior to
normalization to increase the chances of cloning DNA from minor
species from the pool of organisms sampled. DNA can be fractionated
using a density centrifugation technique, such as a cesium-chloride
gradient. When an intercalating agent, such as bis-benzimide is
employed to change the buoyant density of the nucleic acid,
gradients will fractionate the DNA based on relative base content.
Nucleic acid from multiple organisms can be separated in this
manner, and this technique can be used to fractionate complex
mixtures of genomes. This can be of particular value when working
with complex environmental samples. Alternatively, the DNA does not
have to be fractionated prior to normalization. Samples are
recovered from the fractionated DNA, and the strands of nucleic
acid are then melted and allowed to selectively reanneal under
fixed conditions (Cot driven hybridization). When a mixture of
nucleic acid fragments is melted and allowed to reanneal under
stringent conditions, the common sequences find their complementary
strands faster than the rare sequences. After an optional
single-stranded nucleic acid isolation step, single-stranded
nucleic acid representing an enrichment of rare sequences is
amplified using techniques well known in the art, such as a
polymerase chain reaction (Barnes, 1994), and used to generate gene
libraries. This procedure leads to the amplification of rare or low
abundance nucleic acid molecules, which are then used to generate a
gene library which can be screened for a desired bioactivity. While
DNA will be recovered, the identification of the organism(s)
originally containing the DNA may be lost. This method offers the
ability to recover DNA from "unclonable" sources.
[0071] Hence, one embodiment for forming a normalized library from
environmental sample(s) is by (a) isolating nucleic acid from the
environmental sample(s); (b) optionally fractionating the nucleic
acid and recovering desired fractions; and (c) optionally
normalizing the representation of the DNA within the population so
as to form a normalized expression library from the DNA of the
environmental sample(s). The "normalization" process is described
and exemplified in detail in co-pending, commonly assigned U.S.
Ser. No. 08/665,565, filed Jun. 18, 1996, which is incorporated
herein by reference.
[0072] The preparation of DNA from the sample is an important step
in the generation of normalized or non-normalized DNA libraries
from environmental samples composed of uncultivated organisms, or
for the generation of libraries from cultivated organisms. DNA can
be isolated from samples using various techniques well known in the
art (Nucleic Acids in the Environment Methods & Applications,
J. T. Trevors, D. D. van Elsas, Springer Laboratory, 1995).
Preferably, DNA obtained will be of large size and free of enzyme
inhibitors or other contaminants. DNA can be isolated directly from
an environmental sample (direct lysis), or cells may be harvested
from the sample prior to DNA recovery (cell separation). Direct
lysis procedures have several advantages over protocols based on
cell separation. The direct lysis technique provides more DNA with
a generally higher representation of the microbial community,
however, it is sometimes smaller in size and more likely to contain
enzyme inhibitors than DNA recovered using the cell separation
technique. Very useful direct lysis techniques have been described
which provide DNA of high molecular weight and high purity (Barns,
1994; Holben, 1994). If inhibitors are present, there are several
protocols which utilize cell isolation which can be employed
(Holben, 1994). Additionally, a fractionation technique, such as
the bis-benzimide separation (cesium chloride isolation) described,
can be used to enhance the purity of the DNA.
[0073] Isolation of total genomic DNA from extreme environmental
samples varies depending on the source and quantity of material.
Uncontaminated, good quality (>20 kbp) DNA is required for the
construction of a representative library. A successful general DNA
isolation protocol is the standard cetyl-trimethyl-ammonium-bromide
(CTAB) precipitation technique. A biomass pellet is lysed and
proteins digested by the nonspecific protease, proteinase K, in the
presence of the detergent SDS. At elevated temperatures and high
salt concentrations, CTAB forms insoluble complexes with denatured
protein, polysaccharides and cell debris. Chloroform extractions
are performed until the white interface containing the CTAB
complexes is reduced substantially. The nucleic acids in the
supernatant are precipitated with isopropanol and resuspended in TE
buffer.
[0074] For cells which are recalcitrant to lysis, a combination of
chemical and mechanical methods with cocktails of various
cell-lysing enzymes may be employed. Isolated nucleic acid may then
further be purified using small cesium gradients.
[0075] Gene libraries can be generated by inserting the DNA
isolated or derived from a sample into a vector or a plasmid. 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
plasmids and methods for introduction and packaging into them are
described herein.
[0076] The following outlines a general procedure for producing
libraries from both culturable and non-culturable organisms: obtain
Biomass DNA Isolation (various methods), shear DNA (for example,
with a 25 gauge needle), blunt DNA, methylate DNA, ligate to
linkers, cut back linkers, size fractionate (for example, use a
Sucrose Gradient), ligate to lambda expression vector, package (in
vitro lambda packaging extract), plate on E. coli host and
amplify.
[0077] As detailed in FIG. 1, cloning DNA fragments prepared by
random cleavage of the target DNA generates a representative
library. DNA dissolved in TE buffer is vigorously passed through a
25 gauge double-hubbed needle until the sheared fragments are in
the desired size range. The DNA ends are "polished" or blunted with
Mung Bean Nuclease, and EcoRI restriction sites in the target DNA
are protected with EcoRI Methylase. EcoRI linkers (GGAATTCC) are
ligated to the blunted/protected DNA using a very high molar ratio
of linkers to target DNA. This lowers the probability of two DNA
molecules ligating together to create a chimeric clone. The linkers
are cut back with EcoRI restriction endonuclease and the DNA is
size fractionated. The removal of sub-optimal DNA fragments and the
small linkers is critical because ligation to the vector will
result in recombinant molecules that are unpackageable, or the
construction of a library containing only linkers as inserts.
Sucrose gradient fractionation is used since it is extremely easy,
rapid and reliable. Although the sucrose gradients do not provide
the resolution of agarose gel isolations, they do produce DNA that
is relatively free of inhibiting contaminants. The prepared target
DNA is ligated to the lambda vector, packaged using in vitro
packaging extracts and grown on the appropriate E. coli.
[0078] As representative examples of expression vectors which may
be used there may be mentioned viral particles, baculovirus, phage,
plasmids, phagemids, cosmids, fosmids, 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: pQE vectors (Qiagen), pBluescript plasmids,
pNH vectors, (ZAP vectors (Stratagene); ptrc99a, pKK223-3, pDR540,
pRIT2T (Pharmacia); Eukaryotic: pXT1, pSG5 (Stratagene), pSVK3,
pBPV, pMSG, pSVLSV40 (Pharmacia). However, any other plasmid or
other vector may be used as long as they are replicable and viable
in the host.
[0079] Another 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 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."
[0080] The DNA sequence in the expression vector is operatively
linked to an appropriate expression control sequence(s) (promoter)
to direct RNA 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.
[0081] 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.
[0082] 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 TRP 1 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), (-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.
[0083] The cloning strategy permits expression via both vector
driven and endogenous promoters; vector promotion may be important
with expression of genes whose endogenous promoter will not
function in E. coli.
[0084] 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.
[0085] 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 is
a prokaryotic cell, such as a bacterial cell. Particularly
preferred host cells are E. coli. 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)). The selection of an appropriate host
is deemed to be within the scope of those skilled in the art from
the teachings herein.
[0086] 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.
[0087] It is also contemplated that expression libraries generated
can be phage display or cell surface display libraries. Numerous
techniques are published in the art for generating such
libraries.
[0088] After the expression libraries have been generated one can
include the additional step of "biopanning" such libraries prior to
screening by cell sorting. The "biopanning" procedure refers to a
process for identifying clones having a specified biological
activity by screening for sequence homology 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
biological having the specified biological activity; and (ii)
optionally transforming a host with isolated target DNA to produce
a library of clones which are screened for the specified biological
activity.
[0089] 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 the DNA sequence encoding
an enzyme 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.
[0090] 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%.
[0091] In nucleic acid hybridization reactions, the conditions used
to achieve a particular level of stringency will vary, depending on
the nature of the nucleic acids being hybridized. For example, the
length, degree of complementarity, nucleotide sequence composition
(e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. DNA)
of the hybridizing regions of the nucleic acids can be considered
in selecting hybridization conditions. An additional consideration
is whether one of the nucleic acids is immobilized, for example, on
a filter.
[0092] An example of progressively higher stringency conditions is
as follows: 2 .times.SSC/0. 1% SDS at about room temperature
(hybridization conditions); 0.2 .times.SSC/0. 1% SDS at about room
temperature (low stringency conditions); 0.2 .times.SSC/0.1% SDS at
about 42.degree. C. (moderate stringency conditions); and 0.1
.times.SSC at about 68.degree. C. (high stringency conditions).
Washing can be carried out using only one of these conditions,
e.g., high stringency conditions, or each of the conditions can be
used, e.g., for 10-15 minutes each, in the order listed above,
repeating any or all of the steps listed. However, as mentioned
above, optimal conditions will vary, depending on the particular
hybridization reaction involved, and can be determined
empirically.
[0093] 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, 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.
[0094] 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.
[0095] 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.
[0096] 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.
[0097] The resultant libraries of transformed clones are then
screened for clones which display activity for the enzyme of
interest.
[0098] 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.
[0099] 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 utilizing a FACS machine for such enzyme activity or for
a more specific activity. Alternatively, encapsulation techniques
such as gel microdroplets, may be employed to localize multiple
clones in one location to be screened on a FACS machine for
positive expressing clones within the group of clones which can
then be broken out into individual clones to be screened again on a
FACS machine to identify positive individual clones. Thus, for
example, if a clone mixture has hydrolase activity, then the
individual clones may be recovered and screened utilizing a FACS
machine to determine which of such clones has hydrolase activity.
As used herein, "small insert library" means a gene library
containing clones with random small size nucleic acid inserts of up
to approximately 5000 base pairs. As used herein, "large insert
library" means a gene library containing clones with random large
size nucleic acid inserts of approximately 5000 up to several
hundred thousand base pairs or greater.
[0100] 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 includes:
[0101] screening a library for specified enzyme activity, said
library including a plurality of clones, said clones having been
prepared by recovering from genomic 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 transforming a host with
the selected DNA to produce clones which are screened for the
specified enzyme activity.
[0102] 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:
[0103] (a) rendering the double-stranded genomic DNA population
into a single-stranded DNA population;
[0104] (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 genomic DNA population which hybridize thereto;
(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;
[0105] (d) separating the solid phase complex from the
single-stranded DNA population of (b);
[0106] (e) releasing from the probe the members of the genomic
population which had bound to the solid phase bound probe;
[0107] (f) forming double-stranded DNA from the members of the
genomic population of (e);
[0108] (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
[0109] (h) screening the library for the specified enzyme
activity.
[0110] 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 genomic 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.
[0111] A particularly preferred embodiment of this aspect further
comprises, after (a) but before (b) above, the steps of:
[0112] (a i). contacting the single-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;
[0113] (a ii). contacting the double-stranded complex of (a i) with
a solid phase specific binding partner for said ligand so as to
produce a solid phase complex;
[0114] (a iii) separating the solid phase complex from the
single-stranded DNA population of (a);
[0115] (a iv) releasing the members of the genomic population which
had bound to said solid phase bound probe; and (a v) separating the
solid phase bound probe from the members of the genomic population
which had bound thereto.
[0116] 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.
[0117] This procedure is described and exemplified in U.S. Ser. No.
08/692,002, filed Aug. 2, 1996, incorporated herein by
reference.
[0118] In-vivo biopanning may be performed utilizing a FACS-based
machine. Complex gene libraries are constructed with vectors which
contain elements which stabilize transcribed RNA. For example, the
inclusion of sequences which result in secondary structures such as
hairpins which are designed to flank the transcribed regions of the
RNA would serve to enhance their stability, thus increasing their
half life within the cell. The probe molecules used in the
biopanning process consist of oligonucleotides labeled with
reporter molecules that only fluoresce upon binding of the probe to
a target molecule. These probes are introduced into the recombinant
cells from the library using one of several transformation methods.
The probe molecules bind to the transcribed target mRNA resulting
in DNA/RNA heteroduplex molecules. Binding of the probe to a target
will yield a fluorescent signal which is detected and sorted by the
FACS machine during the screening process.
[0119] Further, it is possible to combine all the above embodiments
such that a normalization step is performed prior to generation of
the expression library, the expression library is then generated,
the expression library so generated is then biopanned, and the
biopanned expression library is then screened using a high
throughput cell sorting and screening instrument. Thus there are a
variety of options: i.e. (i) one can just generate the library and
then screen it; (ii) normalize the target DNA, generate the
expression library and screen it; (iii) normalize, generate the
library, biopan and screen; or (iv) generate, biopan and screen the
library.
[0120] The library may, for example, be screened for a specified
enzyme activity. For example, the enzyme activity screened for may
be 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.
[0121] 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.
[0122] 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.
[0123] 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:
[0124] Lipase/Esterase
[0125] Enantioselective hydrolysis of esters (lipids)/thioesters,
resolution of racemic mixtures, synthesis of optically active acids
or alcohols from meso-diesters, selective syntheses, regiospecific
hydrolysis of carbohydrate esters, selective hydrolysis of cyclic
secondary alcohols, synthesis of optically active esters, lactones,
acids, alcohols, transesterification of activated/nonactivated
esters, interesterification, optically active lactones from
hydroxyesters, egio- and enantioselective ring opening of
anhydrides, detergents, fat/oil conversion and cheese ripening.
[0126] Protease
[0127] Ester/amide synthesis, peptide synthesis, resolution of
racemic mixtures of amino acid esters, synthesis of non-natural
amino acids and detergents/protein hydrolysis.
[0128] Glycosidase/Glycosyl Transferase
[0129] Sugar/polymer synthesis, cleavage of glycosidic linkages to
form mono, di-and oligosaccharides, synthesis of complex
oligosaccharides, glycoside synthesis using UDP-galactosyl
transferase, transglycosylation of disaccharides, glycosyl
fluorides, aryl galactosides, glycosyl transfer in oligosaccharide
synthesis, diastereoselective cleavage of
.alpha.-glucosylsulfoxides, asymmetric glycosylations, food
processing and paper processing.
[0130] Phosphatase/Kinase
[0131] Synthesis/hydrolysis of phosphate esters, regio- and
enantioselective phosphorylation, introduction of phosphate esters,
synthesize phospholipid precursors, controlled polynucleotide
synthesis, activate biological molecule, selective phosphate bond
formation without protecting groups.
[0132] Mono/Dioxygenase
[0133] Direct oxyfunctionalization of unactivated organic
substrates, hydroxylation of alkane, aromatics, steroids,
epoxidation of alkenes, enantioselective sulphoxidation, regio- and
stereoselective Bayer-Villiger oxidations.
[0134] Haloperoxidase
[0135] Oxidative addition of halide ion to nucleophilic sites,
addition of hypohalous acids to olefinic bonds, ring cleavage of
cyclopropanes, activated aromatic substrates converted to ortho and
para derivatives 1.3 diketones converted to 2-halo-derivatives,
heteroatom oxidation of sulfur and nitrogen containing substrates,
oxidation of enol acetates, alkynes and activated aromatic
rings.
[0136] Lignin Peroxidase/Diarylpropane Peroxidase
[0137] Oxidative cleavage of C-C bonds, oxidation of benzylic
alcohols to aldehydes, hydroxylation of benzylic carbons, phenol
dimerization, hydroxylation of double bonds to form diols, cleavage
of lignin aldehydes.
[0138] Epoxide Hydrolase
[0139] Synthesis of enantiomerically pure bioactive compounds,
regio- and enantioselective hydrolysis of epoxide, aromatic and
olefinic epoxidation by monooxygenases to form epoxides, resolution
of racemic epoxides, hydrolysis of steroid epoxides.
[0140] Nitrile Hydratase/Nitrilase
[0141] Hydrolysis of aliphatic nitriles to carboxamides, hydrolysis
of aromatic, heterocyclic, unsaturated aliphatic nitriles to
corresponding acids, hydrolysis of acrylonitrile, production of
aromatic and carboxamides, carboxylic acids (nicotinamide,
picolinamide, isonicotinamide), regioselective hydrolysis of
acrylic dinitrile, amino acids from hydroxynitriles.
[0142] Transaminase
[0143] Transfer of amino groups into oxo-acids.
[0144] Amidase/Acylase
[0145] Hydrolysis of amides, amidines, and other C-N bonds,
non-natural amino acid resolution and synthesis.
[0146] As indicated, the present invention also offers the ability
to screen for other types of bioactivities. For instance, 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 vectors
which can accept large inserts, such as the f-factor based
vectors), which facilitates cloning of gene clusters.
[0147] Preferably, the gene cluster or pathway 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. As previously indicated, 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. Other examples of vectors include cosmids,
bacterial artificial chromosome vectors, and P1 vectors.
[0148] Lambda vectors can also accommodate relatively large DNA
molecules, have high cloning and packaging efficiencies and are
easy to handle and store compared to plasmid vectors. (-ZAP vectors
(Stratagene Cloning Systems, Inc.) have a convenient subcloning
feature that allows clones in the vector to be excised with helper
phage into the pBluescript phagemid, eliminating the time involved
in subcloning. The cloning site in these vectors lies downstream of
the lac promoter. This feature allows expression of genes whose
endogenous promoter does not function in E. coli.
[0149] The following describes the total number of assays required
to test an entire library:
[0150] The two main factors which govern the total number of clones
that can be pooled and simultaneously screened are (i) the level of
gene expression and (ii) enzyme assay sensitivity. As estimate of
the level of gene expression is that each E. coli cell infected
with lambda will produce 103 copies of the gene product from the
insert. FACS instruments are sufficiently sensitive to detect about
500 to 1000 Fluorescein molecules.
[0151] In order to assess the total number of clones to be tested
(e.g., the number of genome equivalents) a statistical analysis was
performed. Assuming that mechanical shearing and gradient
purification results in a normal distribution of DNA fragment sizes
with a mean of 4.5 kbp and variance of 1 kbp, the fraction
represented of all possible 1 kbp sequences in a 1.8 Mbp genome is
plotted in FIG. 3 as a function of increasing genome
equivalents.
[0152] Based on these results, approximately 2,000 clones (5 genome
equivalents) must be screened to achieve a .about.90% probability
of obtaining a particular gene. This represents the point of
maximal efficiency for library throughput. Assuming that a complex
environmental library contains about 1000 different organisms, at
least 2,000,000 clones have to be screened to achieve a >90%
probability of obtaining a particular gene. This number rises
dramatically assuming that the organisms differ vastly in abundance
in natural populations.
[0153] Substrate can be administered to the cells before or during
the process of the cell sorting analysis. In either case a solution
of the substrate is made up and the cells are contacted therewith.
When done prior to the cell sorting analysis this can be by making
a solution which can be administered to the cells while in culture
plates or other containers. The concentration ranges for substrate
solutions will vary according to the substrate utilized.
Commercially available substrates will generally contain
instructions on concentration ranges to be utilized for, for
instance, cell staining purposes. These ranges may be employed in
the determination of an optimal concentration or concentration
range to be utilized in the present invention. The substrate
solution is maintained in contact with the cells for a period of
time and at an appropriate temperature necessary for the substrate
to permeablize the cell membrane. Again, this will vary with
substrate. Instruments which deliver reagents in stream such as by
poppet valves which seal openings in the flow path until activated
to permit introduction of reagents (e.g. substrate) into the flow
path in which the cells are moving through the analyzer can be
employed for substrate delivery.
[0154] The substrate is one which is able to enter the cell and
maintain its presence within the cell for a period sufficient for
analysis to occur. It has generally been observed that introduction
of the substrate into the cell across the cell membrane occurs
without difficulty. It is also preferable that once the substrate
is in the cell it not "leak" back out before reacting with the
biomolecule being sought to an extent sufficient to product a
detectable response. Retention of the substrate in the cell can be
enhanced by a variety of techniques. In one, the substrate compound
is structurally modified by addition of a hydrophobic tail. In
another certain preferred solvents, such as DMSO or glycerol, can
be administered to coat the exterior of the cell. Also the
substrate can be administered to the cells at reduced temperature
which has been observed to retard leakage of the substrate from the
cell's interior.
[0155] A broad spectrum of substrates can be used which are chosen
based on the type of bioactivity sought. In addition where the
bioactivity being sought is in the same class as that of other
biomolecules for which a number have known substrates, the
bioactivity can be examined using a cocktail of the known
substrates for the related biomolecules which are already known.
For example, substrates are known for approximately 20 commercially
available esterases and the combination of these known substrates
can provide detectable, if not optimal, signal production.
Substrates are also known and available for glycosidases,
proteases, phosphatases, and monoxygenases.
[0156] The substrate interacts with the target biomolecule so as to
produce a detectable response. Such responses can include
chromogenic or fluorogenic responses and the like. The detectable
species can be one which results from cleavage of the substrate or
a secondary molecule which is so affected by the cleavage or other
substrate/biomolecule interaction to undergo a detectable change.
Innumerable examples of detectable assay formats are known from the
diagnostic arts which use immunoassay, chromogenic assay, and
labeled probe methodologies.
[0157] Several enzyme assays described in the literature are built
around the change in fluorescence which results when the phenolic
hydroxyl (or anilino amine) becomes deacylated (or dealkylated) by
the action of the enzyme. FIG. 7 shows the basic principle for this
type of enzyme assay for deacylation. Any emission or activation of
fluorescent wavelengths as a result of any biological process are
defined herein as bioactive fluoresence.
[0158] In comparison to colorimetric assays, fluorescent based
assays are very sensitive, which is a major criteria for single
cell assays. There are two main factors which govern the screening
of a recombinant enzyme in a single cell: i) the level of gene
expression, and ii) enzyme assay sensitivity. To estimate the level
of gene expression one can determine how many copies of the gene
product will be produced by the host cell given the vector. For
instance, one can assume that each E. coli cell infected with
pBluescript phagemid (Stratagene Cloning Systems, Inc.) will
produce .about.10.sup.3 copies of the gene product from the insert.
The FACS instruments are capable of detecting about 500 to 1,000
fluorescein molecules per cell. Assuming that one enzyme turns over
at least one fluorescein based substrate molecule, one cell will
display enough fluorescence to be detected by the optics of a
fluorescence-activated cell sorter (FACS).
[0159] Several methods have been described for using reporter genes
to measure gene expression. These reporter genes encode enzymes not
ordinarily found in the type of cell being studied, and their
unique activity is monitored to determine the degree of
transcription. Nolan et al., developed a technique to analyze
(-galactosidase expression in mammalian cells employing
fluorescein-di-(-D-galactopyranoside (FDG) as a substrate for
(-galactosidase, which releases fluorescein, a product that can be
detected by a fluorescence-activated cell sorter (FACS) upon
hydrolysis (Nolan et al., 1991). A problem with the use of FDG is
that if the assay is performed at room temperature, the
fluorescence leaks out of the positively stained cells. A similar
problem was encountered in other studies of (-galactosidase
measurements in mammalian cells and yeast with FDG as well as other
substrates (Nolan et al, 1988; Wittrup et al., 1988). Performing
the reaction at 0.degree. C. appreciably decreased the extent of
this leakage of fluorescence (Nolan et al., 1988). However this low
temperature is not adaptable for screening for, for instance, high
temperature (-galactosidases. Other fluorogenic substrates have
been developed, such as 5-dodecanoylamino fluorescein
di-(-D-galactopyranoside (C.sub.12-FDG) (Molecular Probes) which
differs from FDG in that it is a lipophilic fluorescein derivative
that can easily cross most cell membranes under physiological
culture conditions. The green fluorescent enzymatic hydrolysis
product is retained for hours to days in the membrane of those
cells that actively express the lacZ reporter gene. In animal cells
C.sub.12-FDG was a much better substrate, giving a signal which was
100 times higher than the one obtained with FDG (Plovins et al.,
1994). However in Gram negative bacteria like E. coli, the outer
membrane functions as a barrier for the lipophilic molecule
C.sub.12-FDG and it only passes through this barrier if the cells
are dead or damaged (Plovins et al). The fact that C.sub.12 retains
FDG substrate inside the cells indicates that the addition of
unpolarized tails may be used for retaining substrate inside the
cells with respect to other enzyme substrates.
[0160] The abovementioned (-galactosidase assays may be employed to
screen single E. coli cells, expressing recombinant
(-D-galactosidase isolated from a hyperthermophilic archaeon such
as Sulfolobus solfataricus, on a fluorescent microscope. Cells are
cultivated overnight, centrifuged and washed in deionized water and
stained with FDG. To increase enzyme activity, cells are heated to
70.degree. C. for 30 minutes and examined with a fluorescence phase
contrast microscope. E. coli cell suspensions of the
(-galactosidase expressing clone stained with C.sub.12-FDG show a
very bright fluorescence inside single cells (FIG. 8).
[0161] The heat treatment of E. coli permeabilizes the cells to
allow the substrate to pass through the membrane. Control strains
containing plasmid DNA without insert and stained with the same
procedure show no fluorescence. Phase contrast microscopy of heated
cells reveals that cells maintain their structural integrity up to
2 hours if heated up to 70.degree. C. The lipophilic tail of the
modified fluorescein-di-(-D-gala- ctopyranoside prevents leakage of
the molecule, even at elevated temperatures. The attachment of a
lipophilic carbon chain changes the solubility of substrates
tremendously. Thus, substrates containing lipophilic carbon chains
can be generated and utilized as screening substrates in the
present invention. For instance, the following activities may be
detected utilized the indicated substrates. Different methods can
be employed for loading substrate inside the cells. Additionally,
DMSO can be used as solvent up to a concentration of 50% in water
to dissolve and load substrates without significantly dropping the
viability of E. coli. Enzyme activity and leakage can be monitored
with fluorescence microscopy.
[0162] Lipases/esterases. An acylated derivative of fluorescein can
be used to detect esterases such as lipases. The fluorophore is
hydrolyzed from the derivative to generate a signal. Acylated
derivatives of fluorescein can be synthesized according to FIG. 9.
Nine molar equivalents of lauric anhydride triethylamine and
N,N-diisopropylethylami- ne are added to a solution of
fluoresceinamine in chloroform. After the reaction is complete, the
product 5-dodecanoyl-aminofluorescein-di-dodeca- noic acid
(C.sub.12-FDC.sub.12) is recrystallized.
[0163] Proteases. Proteases can be assayed in the same way as the
esterases, with an amide being cleaved instead of an ester. There
are now well over 100 different protease substrates available with
an acylated fluorophore at the scissile bond. Rhodamine derivatives
(FIG. 10), have more lipophilic characteristics compared to
fluorescein protrease substrates, therefore they make good
substrates for more general assays.
[0164] Monooxygenases (dealkylases). Compounds such as that
depicted in FIG. 11 can be used to detected monooxygenases.
Hydroxylation of the ethyl group in the compound results in the
release of the resorufin fluorophore. Several unmodified coumarin
derivatives are also commercially available.
[0165] A variety of types of high throughput cell sorting
instruments can be used with the present invention. First there is
the FACS cell sorting instrument which has the advantage of a very
high throughput and individual cell analysis. Other types of
instruments which can be used are robotics instruments and
time-resolved fluorescence instruments, which can actually measure
the fluorescence from a single molecule over an elapsed period of
time. Since they are measuring a single molecule, they can
simultaneously determine its molecular weight, however their
throughput is not as high as the FACS cell sorting instruments.
[0166] When screening with the FACS instrument, the trigger
parameter is set with logarithmic forward side scatter. The
fluorescent signals of positive clones emitted by fluorescein or
other fluorescent substrates is distinguished by means of a
dichroic mirror and acquired in log mode. For example, "active"
clones can be sorted and deposited into microtiter plates. When
sorting clones from libraries constructed from single organisms or
from small microbial consortia, approximately 50 clones can be
sorted into individual microtiter plate wells. When complex
environmental mega-libaries (i.e. libraries containing
.about.10.sup.8 clones which represent >100 organisms) about 500
expressing clones should be collected.
[0167] Plasmid DNA can then be isolated from the sorted clones
using any commercially available automated miniprep machine, such
as that from Autogen. The plasmids are then retransformed into
suitable expression hosts and assayed for activity utilizing
chromogenic agar plate based or automated liquid format assays.
Confirmed expression clones can then undergo RFLP analysis to
determine unique clones prior to sequencing. The inserts which
contain the unique esterase clones can be sequenced, open reading
frames (ORF's) identified and the genes PCR subcloned for
overexpression. Alternatively, expressing clones can be "bulk
sorted" into single tubes and the plasmid inserts recovered as
amplified products, which are then subcloned and transformed into
suitable vector-hosts systems for rescreening.
[0168] Encapsulation techniques may be employed to localize signal,
even in cases where cells are no longer viable. Gel microdrops
(GMDs) are small (25 to 50 um in diameter) particles made with a
biocompatible matrix. In cases of viable cells, these microdrops
serve as miniaturized petri dishes because cell progeny are
retained next to each other, allowing isolation of cells based on
clonal growth. The basic method has a significant degree of
automation and high throughput; after the colony size signal
boundaries are established, about 106 GMDs per hour can be
automatically processed. Cells are encapsulated together with
substrates and particles containing a positive clones are sorted.
Fluorescent substrate labeled glass beads can also be loaded inside
the GMDs. In cases of non-viable cells, GMDs can be employed to
ensure localization of signal.
[0169] After viable or non-viable cells, each containing a
different expression clone from the gene library are screened on a
FACS machine, and positive clones are recovered, DNA is isolated
from positive clones. The DNA can then be amplified either in vivo
or in vitro by utilizing any of the various amplification
techniques known in the art. In vivo amplification would include
transformation of the clone(s) or subclone(s) of the clones into a
viable host, followed by growth of the host. In vitro amplification
can be performed using techniques such as the polymerase chain
reaction.
[0170] Clones found to have the bioactivity for which the screen
was performed can also be subjected to directed mutagenesis to
develop new bioactivities with desired properties or to develop
modified bioactivities 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 described below.
[0171] 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).
[0172] 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).
[0173] 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.
[0174] The term "sexual PCR mutagenesis" (also known as "DNA
shuffling") 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).
[0175] The term "in vivo mutagenesis" refers to a process of
generating random mutations in any cloned DNA of interest which
involves the propogation 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.
[0176] 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.
[0177] 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).
[0178] 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).
[0179] All of the references mentioned above are hereby
incorporated by reference in their entirety. Each of these
techniques is described in detail in the references mentioned.
[0180] DNA can be mutagenized, or "evolved", utilizing any one or
more of these techniques, and rescreened on the FACS machine to
identify more desirable clones. "Fluorescence screening" as
utilized herein means screening for any activity of interest
utilizing any fluorescent analyzer that detects fluorescence.
Internal control reference genes which either express fluorescing
molecules, such as those encoding green fluorescent protein, or
encode proteins that can turnover fluorescing molecules, such as
beta-galactosidase, can be utilized. These internal controls should
optimally fluoresce at a wavelength which is different from the
wavelength at which the molecule used to detect the evolved
molecule(s) emits. DNA is evolved, recloned in a vector which
co-expresses these proteins or molecules, transformed into an
appropriate host organism, and rescreened utilizing the FACS
machine to identify more desirable clones.
[0181] An important aspect of the invention is that cells are being
analyzed individually. However other embodiments are contemplated
which involve pooling of cells and multiple passage screen. This
provides for a tiered analysis of biological activity from more
general categories of activity, i.e. categories of enzymes, to
specific activities of principle interest such as enzymes of that
category which are specific to particular substrate molecules.
[0182] Members of these libraries can be encapsulated in gel
microdroplets, exposed to substrates of interest, such as
transition state analogs, and screened based on binding via FACS
sorting for activities of interest.
[0183] It is anticipated with the present invention that one could
employ mixtures of substrates to simultaneously detect multiple
activities of interest simultaneously or sequentially. FACS
instruments can detect molecules that fluoresce at different
wavelengths, hence substrates which fluoresce at different
wavelengths and indicate different activities can be employed.
[0184] The fluorescence activated cell sorting screening method of
the present invention allows one to assay several million clones
per hour for a desired bioactivity. This technique provides an
extremely high throughput screening process necessary for the
screening of extreme biodiverse environmental libraries.
[0185] In a preferred embodiment, the present invention provides a
novel method for screening for activities, defined as "agents"
herein, which affect the action of transducing proteins, such as,
for example, G-proteins. In the present invention, cells containing
functional transducing proteins (such as membrane bound
G-proteins), defined herein as "target cells" or "target(s)", are
co-encapsulated with potential agent molecules and screened for
affects agent molecules may have on their actions. Potential agent
molecules are originally derived from a gene library generated from
environmental or other samples, as described herein.
[0186] In particular, agents are molecules encoded by a pathway or
gene cluster, or molecules generated by the expression of said
pathways or clusters. Cells containing nucleic acid expressing the
agent, or cells containing nucleic acid expressing activities which
act within the cell to yield agent molecules can be utilized for
screening. Alternatively, agent molecules can be expressed or
generated prior to screening, and subsequently utilized. Cells
expressing agent molecules, or agent molecules are coencapsulated,
and screened utilizing various methods, such as those described
herein.
[0187] Agent molecules can exist in or be introduced into the
encapsulation particle by various means. Cells expressing genes
encoding proteins which act to generate agent molecules (small
molecules, for example) can be introduced into encapsulation
particles using, for instance, Examples provided herein. Said cells
can be prokaryotic or eukaryotic cells. Prokaryotic cells can be
bacteria, such as E. coli. As previously indicated, genes can
alternatively be expressed outside the encapsulation particle, the
expression product or molecules generated via action of expressed
products (such as small molecules or agent molecules) can be
purified from the host, and said agents may be introduced into the
encapsulation particle with the functional transducing protein(s),
also using the methods described in the Examples below.
[0188] Encapsulation can be in beads, high temperature agaroses,
gel microdroplets, cells, such as ghost red blood cells or
macrophages, liposomes, or any other means of encapsulating and
localizing molecules.
[0189] For example, methods of preparing liposomes have been
described (i.e., U.S. Pat. Nos. 5,653,996, 5393530 and 5,651,981),
as well as the use of liposomes to encapsulate a variety of
molecules U.S. Pat. Nos. 5,595,756, 5,605,703, 5,627,159,
5,652,225, 5,567,433, 4,235,871, 5,227,170). Entrapment of
proteins, viruses, bacteria and DNA in erythrocytes during
endocytosis has been described, as well (Journal of Applied
Biochemistry 4, 418-435 (1982)). Erythrocytes employed as carriers
in vitro or in vivo for substances entrapped during hypo-osmotic
lysis or dielectric breakdown of the membrane have also been
described (reviewed in Ihler, G. M. (1983) J. Pharm. Ther). These
techniques are useful in the present invention to encapsulate
samples for screening.
[0190] "Microenvironment", as used herein, is any molecular
structure which provides an appropriate environment for
facilitating the interactions necessary for the method of the
invention. An environment suitable for facilitating molecular
interactions include, for example, liposomes. Liposomes can be
prepared from a variety of lipids including phospholipids,
glycolipids, steroids, long-chain alkyl esters; e.g., alkyl
phosphates, fatty acid esters; e.g., lecithin, fatty amines and the
like. A mixture of fatty material may be employed such a
combination of neutral steroid, a charge amphiphile and a
phospholipid. Illustrative examples of phospholipids include
lecithin, sphingomyelin and dipalmitoylphos-phatidylcholine.
Representative steroids include cholesterol, cholestanol and
lanosterol. Representative charged amphiphilic compounds generally
contain from 12-30 carbon atoms. Mono- or dialkyl phosphate esters,
or alkyl amines; e.g., dicetyl phosphate, stearyl amine, hexadecyl
amine, dilauryl phosphate, and the like.
[0191] In addition, agents which potentially enhance or inhibit
ligand/receptor interactions may be screened and identified. Thus,
the present invention thus provides a method to screen recombinants
producing drugs which block or enhance interactions of molecules,
such as protein-protein interactions. When screening for compounds
which affect G-protein interactions, host cells expressing
recombinant clones to be screened are co-encapsulated with membrane
bound G-proteins and ligands. Compounds (such as small molecules)
diffuse out of host cells, and enhancement or inhibition of
G-protein interactions can be evaluated via a variety of methods.
Any screening method which allows one to detect an increase or
decrease in activity or presence of an intracellular compound or
molecule, including nucleic acids and proteins, which results from
enhancement or inhibition of ligand/receptor interactions,
transducers, such as G-protein interactions, or cascade events
occurring inside a cell are useful in the present invention.
[0192] For example, the adenylyl cyclase method described above can
be utilized in the present invention. Other assays which detect
effects, or changes, modulated by effectors are useful in the
present invention. The change, or signal, must be detectable
against the background, or basal activity of the effector in the
absence of the potential small molecule or drug. The signal may be
a change in the growth rate of the cells, or other phenotypic
changes, such as a color change or luminescence. Production of
functional gene products may be impacted by the effect, as well.
For example, the production of a functional gene product which is
normally regulated by downstream or direct effects created by the
transducer or effector can be altered and detected. Said functional
genes may include reporter molecules, such as green fluorescent
protein, or red fluorescent protein (Biosci Biotechnol Biochem 1995
Oct; 59(10):1817-1824), or other detectable molecules. These
"functional genes" are used as marker genes. "Marker genes" are
engineered into the host cell where desired. Modifications to their
expression levels causes a phenotypic or other change which is
screenable or selectable. If the change is selectable, a phenotypic
change creates a difference in the growth or survival rate between
cells which express the marker gene and those which do not, or a
detectable modification in expression levels of reporter molecules
within or around cells. If the change is screenable, the phenotype
change creates a difference in some detectable characteristic of
the cells, by which the cells which express the marker may be
distinguished from those which do not. Selection is preferable to
screening.
[0193] Rapid assays which measure direct readouts of
transcriptional activity are useful in the present invention. For
example, placing the bacterial gene encoding lacZ under the control
of the FUS1 promoter, activation of the yeast pheromone response
pathway can be detected in less than an hour by monitoring the
ability of permeabilized yeast to produce color from a chromogenic
substrate. Activation of other response pathways may be assayed via
similar strategies. Genes encoding detectable molecules, or which
create a detectable signal via modification of another molecules,
can be utilized to analyze activation or suppression of a
response.
[0194] The use of fluorescent proteins and/or fluorescent groups
and quenching groups in close proximity to one another to assay the
presence of enzymes or nucleic acid sequences has been reported (WO
97/28261 and WO 95/13399). In the first of these reactions,
fluorescent proteins having the proper emission and excitation
spectra are put in physically close proximity to exhibit
fluorescence energy transfer. Substrates for enzyme activities are
placed between the two proteins, such that cleavage of the
substrate by the presence of the enzymatic activity separates the
proteins enough to change the emission spectra. Another group
utilizes a fluorescent protein and a quencher molecule in close
proximity to exhibit "collisional quenching" properties whereby the
fluorescence of the fluorescent protein is diminished simply via
the proximity of the quenching group. Probe nucleic acid sequences
are engineered between the two groups, and a hybridization event
between the probe sequence and a target in a sample separates the
protein from the quencher enough to yield a fluorescent signal.
Still another group has reported a combination of the above
strategies, engineering a molecule which utilizes an enzyme
substrate flanked by a fluorescent protein on one end and a
quencher on the other (EP 0 428 000). It is recognized that these
types assays can be employed in the method of the present invention
to detect modifications in nucleic acid production (transcriptional
activation or repression) and/or enzyme or other protein production
(translational modifications) which results from inhibition of or
improved association of interacting molecules, such as ligands and
receptors, or which results from actions of bioactive compounds
directly on transcription of particular molecules.
[0195] Fluorescent proteins encoded by genes which can be used to
transform host cells and employed in a screen to identify compounds
of interest are particularly useful in the present invention.
Substrates are localized into the encapsulation means by a variety
of methods, including but not limited to the method described
herein in the Example below. Cells can also be engineered to
contain genes encoding fluorescing molecules. For example,
transcriptionally regulated genes can be linked to reporter
molecule genes to allow expression (or lack of expression) of the
reporter molecule to facilitate detection of the expression of the
transcriptionally regulated gene. For example, if the ultimate
effect of an agonist or antagonist interacting to enhance or
inhibit the binding of a ligand to a receptor, or to enhance or
inhibit the effects of any molecule in a pathway, is
transcriptional activation or repression of a gene of interest the
cell, it is useful to be able to link the activated gene to a
reporter gene to facilitate detection of the expression.
[0196] Cells can be engineered in variety of ways to allow the
assay of the effect of compounds on cellular "events". An "event",
as utilized herein, means any cellular function which is modified
or event which occurs in response to exposure of the cell, or
components of the cell, to molecules expressed by, or ultimately
yielded by the expression of, members of gene libraries derived
from samples and generated according to the methods described
herein. For example, cellular events which can be detected with
commercially available products include changes in transmembrane pH
(i.e., BCECF pH indicator sold by BioRad Laboratories, Inc.,
Hercules, Calif.), cell cycle events, such as cell proliferation,
cytotoxicity and cell death (i.e., propidium iodide,
5-bromo-2'-deoxy-uridine (BrdU), Annexin-V-FLUOS, and TUNEL
(method) sold by Boehringer-Mannheim Research Biochemicals), or
production of proteins, such as enzymes. In many instances, the
cascade of events begun by membrane protein interactions with other
molecules involves modifications, such as phosphorylation or
dephosphorylation, of molecules within the cell. Molecules, such as
fluorescent substrates, which facilitate detection of these events
are useful in the present invention to screen libraries expressing
activities of interest. ELISA or calorimetric assays can also be
adapted to single cell screening to be utilized to screen libraries
according to the present invention.
[0197] Probe nucleic acid sequences designed according to the
method described above can also be utilized in the present
invention to "enrich" a population for desirable clones. "Enrich",
as utilized herein, means reducing the number and/or complexity of
an original population of molecules. For example, probes are
designed to identify specific polyketide sequences, and utilized to
enrich for clones encoding polyketide pathways. Figure X depicts
in-situ hybridization of encapsulated fosmid clones with specific
probes of interest, in this case polyketide synthase gene probes.
Fosmid libraries are generated in E. coli according to the methods
described in the Example herein. Clones are encapsulated and grown
to yield encapsulated clonal populations. Cells are lysed and
neutralized, and exposed to the probe of interest. Hybridization
yields a positive fluorescent signal which can be sorted on a
fluorescent cell sorter. Positives can be further screened via
expression, or activity, screening. Thus, this aspect of the
present invention facilitates the reduction of the complexity of
the original population to enrich for desirable pathway clones.
These clones can the be utilized for further downstream screening.
For example, these clones can be expressed to yield backbone
structures (defined herein), which can the be decorated in
metabolically rich hosts, and finally screened for an activity of
interest. Alternatively, clones can be expressed to yield small
molecules directly, which can be screened for an activity of
interest. Further more, multiple probes can be designed and
utilized to allow "multiplex" screening and/or enrichment.
"Multiplex" screening and/or enrichment as used herein means that
one is screening and/or enriching for more than desirable outcome,
simultaneously.
[0198] Detectable molecules may be added as substrates to be
utilized in screening assays, or genes encoding detectable
molecules may be utilized in the method of the present
invention.
[0199] The present invention provides for strategies to utilize
high throughput screening mechanisms described herein to allow for
the enrichment for desirable activities from a population of
molecules. In one aspect of the present invention, cells are
screened for the presence of ubiquitous molecules, such as
thioesterase activities, to allow one to enrich for cells producing
desirable bioactivities, such as those encoded by polyketide
pathways. A variety of screening mechanisms can be employed. For
example, identifying and recovering cells possessing thioesterase
activities allows one to enrich for cells potentially containing
polyketide activities. For example, for aromatic polyketides, the
polyketide synthase consists of a single set of enzyme activities,
housed either in a single polypeptide chain (type 1) or on separate
polypeptides (type II), that act in every cycle. In contrast,
complex polyketides are synthesized on multifunctional PKSs that
contain a distinct active site for every catalyzed step in chain
synthesis. Type I polyketide scaffolds are generated and cleaved
from the acyl carrier protein in a final action by a
thioesterase-cylcase activity (thioester bond cleaved). One group
has even demonstrated that moving the location of the thioester
bond along a polyketide pathway clone dictates where the polyketide
scaffold will be clipped from the carrier protein (Cortes J., et.
al., Science, Vol. 258, 9 June 1995). Hybridization (homology)
screening can be employed to identify cells containing thioesterase
activities. If hybridization screening is utilized, sequences
(partial or complete) of genes encoding known thioesterases can be
utilized as identifying probes. Alternatively, probes containing
probing sequences derived from known thioesterase activity genes,
flanked by fluorescing molecules and/or quenching molecules, such
as those described above, can be utilized. Labeled substrates can
also be utilized in screening assays.
[0200] In another aspect of the present invention, screening using
a fluorescent analyzer which requires single cell detection, such
as a FACS machine, is utilized as a high throughput method to
screen specific types of filamentous bacteria and fungi which form
myceliates, such as Actinomyces or Streptomyces. In particular,
screening is performed on filamentous fungi and bacteria which
have, at one stage of their life cycle, unicells or monocells
(multinucleoid cells fragment to produce monocells). Typically,
spores of myceliate organisms germinate to make substrate mycelia
(during which phase antibiotics are potentially produced), which
then form arial mycelia. Arial mycelia eventually fragment to make
more spores. Any filamentous bacteria or fungi which forms
monocells during one stage of its life cycle can be screened for an
activity of interest. Previously, this was not done because a
branching network of multinucleoid (fungal like) cells forms with
certain species. In a preferred embodiment, the present invention
presents a particular species, Streptomyces venezuelae, for
screening utilizing a fluorescent analyzer which requires single
cell detection. The method of the present invention allows one to
perform high throughput screening of myceliates for production of,
for example, novel small molecules and bioactives. These cell types
can be recombinant or non-recombinant.
[0201] Streptomyces venezuelae, unlike most other Streptomyces
species, has been shown to sporulate in liquid grown culture. In
some media, it also fragments into single cells when the cultures
reach the end of vegetative growth. Because the production of most
secondary metabolites, including bioactive small molecules, occurs
at the end of log growth, it is possible to screen for Streptomyces
venezuelae fragmented cells that are producing bioactives by a
fluorescence analyzer, such as a FACS machine, given the natural
fluorescence of some small molecules.
[0202] In one aspect of the present invention, any Streptomyces or
Actinomyces species that can be manipulated to produce single cells
or fragmented mycelia is screened for a characteristic of interest.
It is preferable to screen cells at the stage in their life cycle
when they are producing small molecules for purposes of the present
invention.
[0203] A fluorescence-based method for the selection of recombinant
plasmids has been reported (BioTechniques 19:760-764, November
1995). Escherichia coli strains containing plasmids for the
overexpression of the gene encoding uroporphyrinogen III
methyltransferase accumulate fluorescent porphyrinoid compounds,
which, when illuminated with ultraviolet light, causes recombinant
cells to fluoresce with a bright red color. Replacement or
disruption of the gene with other DNA fragments results in the loss
of enzymatic activity and nonfluorescent cells.
[0204] Uroporphyrinogen III methyltransferase is an enzyme that
catalyzes the S-adenosyl-l-methionine (SAM)-dependent addition of
two methyl groups to uroporphyrinogen III methyltransferase to
yield dihydrosirohydro-chlorin necessary for the synthesis of
siroheme, factor F430 and vitamin B12. The substrate for this
enzyme, uroporphyrinogen III (derived from y-aminolevulinic acid)
is a ubiquitous compound found not only in these pathways, but also
in the pathways for the synthesis of the other so-called "pigments
of life", heme and chlorophyll. Dihydrosirohydrochlorin is oxidated
in the cell to produce a fluorescent compound sirohydochlorin
(Factor II) or modified again by uroporphyrinogen III
methyltransferase to produce trimethylpyrrocorphin, another
fluorescent compound. These fluorescent compounds fluoresce with a
bright red to red-orange color when illuminated with UV light (300
nm).
[0205] Bacterial uroporphyrinogen III methylases have been purified
from E. coli (1), Pseudomonas (2), Bacillus (3) and
Methanobacterium (4). A Bacillus stearothermophilus
uroporphyrinogen III methylase has been cloned sequenced and
expressed in E. coli (Biosci Biotechnol Biochem 1995 Oct;
59(10):1817-1824).
[0206] In the method of the present invention, the fluorescing
properties of this and other similar compounds can are utilized to
screen for compounds of interest, as described previously, or are
utilized to enrich for the presence of compounds of interest. Host
cells expressing recombinant clones potentially encoding gene
pathways are screened for fluorescing properties. Thus, cells
producing fluorescent proteins or metabolites can be identified.
Pathway clones expressed in E. coli or other host cells, can yield
bioactive compounds or "backbone structures" to bioactive compounds
(which can subsequently be "decorated" in other host cells, for
example, in metabolically rich organisms). The "backbone structure"
is the fundamental structure that defines a particular class of
small molecules. For example, a polyketide backbone will differ
from that of a lactone, a glycoside or a peptide antibiotic. Within
each class, variants are produced by the addition or subtraction of
side groups or by rearrangement of ring structures ("decoration" or
"decorated"). Ring structures present in aromatic bioactive
compounds are known in some instance to yield a fluorescent signal,
which can be utilized to distinguish these cells from the
population. Certain of these structures can also provide absorbance
characteristics which differ from the background absorbance of a
non-recombinant host cell, and thus can allow one to distinguish
these cells from the population, as well. Recombinant cells
potentially producing bioactive compounds or "backbone" structures
can be identified and separated from a population of cells, thus
enriching the population for desirable cells. Thus, the method of
the present invention also facilitates the discovery of novel
aromatic compounds encoded by gene pathways, for example, encoded
by polyketide genes, directly from environmental or other
samples.
[0207] Compounds can also be generated via the modification of host
porphyrin-like molecules by gene products derived from these
samples. Thus, one can screen for recombinant clone gene products
which modify a host porphyrin-like compound to make it
fluoresce.
[0208] In yet another aspect of the present invention, cells
expressing molecules of interest are sorted into 96-well or
384-well plates, specifically for further downstream manipulation
and screening for recombinant clones. In this aspect of the present
invention, the a fluorescence analyzer, such as a FACS machine is
employed not to distinguish members of and evaluate populations or
to screen as previously published, but to screen and recover
positives in a manner that allows further screens to be performed
on samples selected. For example, typical stains used for
enumeration can affect cell viability, therefore these types of
stains were not employed for screening and selecting for further
downstream manipulation of cells, specifically for the purpose, for
example, of recovering nucleic acid which encodes an activity of
interest. In particular, cells containing recombinant clones can be
identified and sorted into multi-well plates for further downstream
manipulation. There are various ways of screening for the presence
of a recombinant clone in a cell. Genes encoding fluorescent
proteins, such as green fluorescent protein (Biotechniques
19(4):650-655, 1995), or the gene encoding uroporphyrinogen III
methyltransferase (Bio Techniques 19:760-764, November 1995) can be
utilized in the method of the present invention as reporters to
allow detection of recombinant clones. Recombinant clones are
sorted for further downstream screening for an activity of
interest. Screening may be for an enzyme, for example, or for a
small molecule, and may be performed using any variety of methods,
including those described or referred to herein.
[0209] In yet another aspect of the present invention, desirable
existing compounds are modified, and evaluated for a more desirable
compound. Existing compounds or compound libraries are exposed to
molecules generated via the expression of small or large insert
libraries generated in accordance with the methods described
herein. Desirable modifications of these existing compounds by
these molecules are detected and better lead compounds are screened
for utilizing a fluorescence analyzer, such as a FACS machine. For
example, E. coli cells expressing clones yielding small molecules
are exposed to one or more existing compounds, which are
subsequently screened for desirable modifications. Alternatively,
cells are co-encapsulated with one or more existing compounds, and
screened simultaneously to identify desirable modifications to the
compound. Examples of modifications include covalent or
non-covalent modifications. Covalent modifications include
incorporation, transfer and cleavage modifications, such as the
addition or transfer of methyl groups or phosphate groups to a
compound, or the cleavage of a peptide or other bond to yield an
active compound. Non-covalent modifications include conformational
changes made to a molecule via addition or disruption of, for
example, hydrogen bonds, ionic bonds, and/or Van der Wals forces.
Modified compounds can be screened by various means, including
those described herein.
[0210] Alternatively, existing compounds are utilized to modify the
molecules generated via the expression of large or small insert
clones, and desirable modifications of the molecules are screened
for via fluorescence screening, utilizing various methods,
including those described herein.
[0211] In another aspect of the present invention, molecules
derived from expressed clones are exposed to organisms to enrich
for potential compounds which cause growth inhibition or death of
cells. For example, cultures of Staphylococcus aureus are
co-encapsulated with compounds generated via expression of clones,
or with cells expressing clones, and allowed to grow for a period
of time by exposure to select media. Co-encapsulated products are
then stained and screened for via fluorescence screening. Stains
which allow detection of live cells can be utilized, allowing
positives, which in this case would have no fluorescence, to be
recovered. Alternatively, forward and side scatter characteristics
are used to enrich for positives. Less or no growth of
Staphylococus or other organisms being evaluated will yield
capsules with less forward and/or side scatter.
[0212] In another aspect of the present invention clones expressing
useful bioactivities are screened in-vivo. In this aspect, host
cells are stimulated to internalize recombinant cells, and used to
screen for bioactivities generated by these recombinant cells which
can cause host cell death or modify an internal molecule or
compound within the host.
[0213] Many bacterial pathogens survive in phagocytes, such as
macrophages, by coordinately regulating the expression of a wide
spectrum of genes. A microbes ability to survive killing by
phagocytes correlates with its ability to cause disease. Hence, the
identification of genes that are preferentially transcribed in the
intracellular environment of the host is central to understanding
of how pathogenic organisms mount successful infection.
[0214] Valdivia and Falkow have reported a selection methodology to
identify genes from pathogenic organisms that are induced upon
association with host cells or tissues. The group noted that
fourteen Salmonella typhimuium genes, under control of at least
four independent regulatory circuits, were identified to be
selectively induced in host macrophages. The methodology is based
on differential fluorescence induction (DFI) for the rapid
identification of bacterial genes induced upon association with
host cells that would work independently of drug susceptibility and
nutritional requirements.
[0215] Differential fluorescence induction is employed in one
aspect of the present invention to screen macrophages harboring
bacterial clones carrying any virulence gene fused to a reporter
molecule and a clone of a putative bioactive pathway. Macrophage
cells are coinfected in the method of the present invention with
clones of pathways potentially encoding useful bioactives, and
plasmids or other vectors encoding virulence factors. Thus, one
aspect of the present invention allows one to screen recombinant
bioactive molecules that inhibit transcriptionally active reporter
gene fusions in macrophage or other phagocyte cells. Bioactive
molecules which inhibit virulence factors in-vivo are identified
via a lack of expression of the reporter molecule, for example red
or green fluorescent proteins. This method allows for the rapid
screening for pathways encoding bioactive compounds specifically
inhibiting a virulence factor or other gene product. Thus the
screen allows one to identify biologically relevant molecules
active in mammalian cells.
[0216] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description, utilize the
present invention to its fullest extent. The following examples are
to be considered illustrative and thus are not limiting of the
remainder of the disclosure in any way whatsoever.
Example 1
DNA Isolation and Library Construction
[0217] The following outlines the procedures used to generate a
gene library from an environmental sample.
[0218] DNA isolation. DNA is isolated using the IsoQuick Procedure
as per manufacturer's instructions (Orca, Research Inc., Bothell,
WA). DNA can be normalized according to Example 2 below. Upon
isolation the DNA is sheared by pushing and pulling the DNA through
a 25G double-hub needle and a 1-cc syringes about 500 times. A
small amount is run on a 0.8% agarose gel to make sure the majority
of the DNA is in the desired size range (about 3-6 kb).
[0219] Blunt-ending DNA. The DNA is blunt-ended by mixing 45 .mu.l
of 10X Mung Bean Buffer, 2.0 .mu.l Mung Bean Nuclease (150 u/.mu.l)
and water to a final volume of 405 .mu.l. The mixture is incubate
at 37.degree. C. for 15 minutes. The mixture is phenol/chloroform
extracted followed by an additional chloroform extraction. One ml
of ice cold ethanol is added to the final extract to precipitate
the DNA. The DNA is precipitated for 10 minutes on ice. The DNA is
removed by centrifugation in a microcentrifuge for 30 minutes. The
pellet is washed with 1 ml of 70% ethanol and repelleted in the
microcentrifuge. Following centrifugation the DNA is dried and
gently resuspended in 26 .mu.l of TE buffer.
[0220] Methylation of DNA. The DNA is methylated by mixing 4 .mu.l
of 10X EcoR I Methylase Buffer, 0.5 .mu.l SAM (32 mM), 5.0 .mu.l
EcoR I Methylase (40 u/.mu.l) and incubating at 37.degree. C., 1
hour. In order to insure blunt ends, add to the methylation
reaction: 5.0 .mu.l of 100 mM MgCl.sub.2, 8.0 .mu.l of dNTP mix
(2.5 mM of each dGTP, dATP, dTTP, dCTP), 4.0 .mu.l of Klenow (5
u/.mu.l) and incubate at 12.degree. C. for 30 minutes.
[0221] After 30 minutes add 450 .mu.l 1X STE. The mixture is
phenol/chloroform extracted once followed by an additional
chloroform extraction. One ml of ice cold ethanol is added to the
final extract to precipitate the DNA. The DNA is precipitated for
10 minutes on ice. The DNA is removed by centrifugation in a
microcentrifuge for 30 minutes. The pellet is washed with 1 ml of
70% ethanol, repelleted in the microcentrifuge and allowed to dry
for 10 minutes.
[0222] Ligation. The DNA is ligated by gently resuspending the DNA
in 8 .mu.l EcoR I adaptors (from Stratagene's cDNA Synthesis Kit),
1.0 .mu.l of 10X Ligation Buffer, 1.0 .mu.l of 10 mM rATP, 1.0
.mu.l of T4 DNA Ligase (4 Wu/.mu.l ) and incubating at 4.degree. C.
for 2 days. The ligation reaction is terminated by heating for 30
minutes at 70.degree. C.
[0223] Phosphorylation of adaptors. The adaptor ends are
phosphorylated by mixing the ligation reaction with 1.0 .mu.l of
10X Ligation Buffer, 2.0 .mu.l of 10 mM rATP, 6.0 .mu.l of
H.sub.2O, 1.0 .mu.l of polynucleotide kinase (PNK) and incubating
at 37.degree. C. for 30 minutes. After 30 minutes 31 .mu.l H.sub.2O
and 5 ml 10X STE are added to the reaction and the sample is size
fractionate on a Sephacryl S-500 spin column. The pooled fractions
(1-3) are phenol/chloroform extracted once followed by an
additional chloroform extraction. The DNA is precipitated by the
addition of ice cold ethanol on ice for 10 minutes. The precipitate
is pelleted by centrifugation in a microfuge at high speed for 30
minutes. The resulting pellet is washed with 1 ml 70% ethanol,
repelleted by centrifugation and allowed to dry for 10 minutes. The
sample is resuspended in 10.5 .mu.l TE buffer. Do not plate.
Instead, ligate directly to lambda arms as above except use 2.5
.mu.l of DNA and no water.
[0224] Sucrose Gradient (2.2 ml) Size Fractionation. Stop ligation
by heating the sample to 65.degree. C. for 10 minutes. Gently load
sample on 2.2 ml sucrose gradient and centrifuge in
mini-ultracentrifuge at 45K, 20.degree. C. for 4 hours (no brake).
Collect fractions by puncturing the bottom of the gradient tube
with a 20G 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 (labeled 1-10). Each drop is about 60
.mu.l in volume. Run 5 .mu.l of each fraction on a 0.8% agarose gel
to check the size. Pool fractions 1-4 (about 10-1.5 kb) and, in a
separate tube, pool fractions 5-7 (about 5-0.5 kb). Add 1 ml ice
cold ethanol to precipitate and place on ice for 10 minutes. Pellet
the precipitate by centrifugation in a microfuge at high speed for
30 minutes. Wash the pellets by resuspending them in 1 ml 70%
ethanol and repelleting them by centrifugation in a microfuge at
high speed for 10 minutes and dry. Resuspend each pellet in 10
.mu.l of TE buffer.
[0225] Test Ligation to Lambda Arms. Plate assay by spotting 0.5
.mu.l of the sample on agarose containing ethidium bromide along
with standards (DNA samples of known concentration) to get an
approximate concentration. View the samples using 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.
[0226] Prepare the following ligation reactions (5 .mu.l reactions)
and incubate 4.degree. C., overnight:
1 Lambda T4 DN 10X Ligase 10 mM arms Insert Ligase Sample H.sub.2O
Buffer rATP (ZAP) DNA Wu/(1) 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
[0227] Test Package and Plate. Package the ligation reactions
following manufacturer's protocol. Stop packaging reactions with
500 .mu.l SM buffer and pool packaging that came from the same
ligation. Titer 1.0 .mu.l of each pooled reaction on appropriate
host (OD.sub.600=1.0) [XLI-Blue MRF]. Add 200 .mu.l host (in mM
MgSO.sub.4) to Falcon 2059 tubes, inoculate with 1 .mu.l packaged
phage and incubate at 37.degree. C. for 15 minutes. Add about 3 ml
48.degree. C. top agar [50 ml stock containing 150 .mu.l IPTG (0.5
M) and 300 .mu.l X-GAL (350 mg/ml)] and plate on 100 mm plates.
Incubate the plates at 37.degree. C., overnight.
[0228] Amplification of Libraries (5.0.times.10.sup.5 recombinants
from each library). Add 3.0 ml host cells (OD.sub.600=1.0) to two
50 ml conical tube and inoculate with 2.5.times.10.sup.5 pfu of
phage per conical tube. Incubate at 37.degree. C. for 20 minutes.
Add top agar to each tube to a final volume of 45 ml. Plate each
tube across five 150 mm plates. Incubate the plates at 37.degree.
C. for 6-8 hours or until plaques are about pin-head in size.
Overlay the plates with 8-10 ml SM Buffer and place at 4.degree. C.
overnight (with gentle rocking if possible).
[0229] Harvest Phage. Recover phage suspension by pouring the SM
buffer off each plate into a 50-ml conical tube. Add 3 ml of
chloroform, shake vigorously and incubate at room temperature for
15 minutes. Centrifuge the tubes at 2K rpm for 10 minutes to remove
cell debris. Pour supernatant into a sterile flask, add 500 .mu.l
chloroform and store at 4.degree. C.
[0230] Titer Amplified Library. Make serial dilutions of the
harvested phage (for example, 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). Add 200 .mu.l host (in 10 mM MgSO.sub.4) to two tubes.
Inoculate one tube with 10 .mu.l 10.sup.-6 dilution (10.sup.-5).
Inoculate the other tube with 1 .mu.l 10.sup.-6 dilution
(10.sup.-6). Incubate at 37.degree. C. for 15 minutes.
[0231] Add about 3 ml 48.degree. C. top agar [50 ml stock
containing 150 .rho.l IPTG (0.5 M) and 375 .mu.l X-GAL (350 mg/ml)]
to each tube and plate on 100 mm plates. Incubate the plates at
37.degree. C., overnight.
[0232] Excise the ZAP II library to create the pBLUESCRIPT library
according to manufacturers protocols (Stratagene).
EXAMPLE 2
Normalization
[0233] Prior to library generation, purified DNA can be normalized.
DNA is first fractionated according to the following protocol. A
sample composed of genomic DNA is purified on a cesium-chloride
gradient. The cesium chloride (Rf=1.3980) solution is filtered
through a 0.2 .mu.m filter and 15 ml is loaded into a 35 ml
OptiSeal tube (Beckman). The DNA is added and thoroughly mixed. Ten
micrograms of bis-benzimide (Sigma; Hoechst 33258) is added and
mixed thoroughly. The tube is then filled with the filtered cesium
chloride solution and spun in a VTi50 rotor in a Beckman L8-70
Ultracentrifuge at 33,000 rpm for 72 hours. Following
centrifugation, a syringe pump and fractionator (Brandel Model 186)
are used to drive the gradient through an ISCO UA-5 UV absorbance
detector set to 280 nm. Peaks representing the DNA from the
organisms present in an environmental sample are obtained.
Eubacterial sequences can be detected by PCR amplification of DNA
encoding rRNA from a 10-fold dilution of the E. coli peak using the
following primers to amplify:
2 [0221] Forward primer: 5'-AGAGTTTGATCCTGGCTCAG-3' [0222] Reverse
primer: 5'-GGTTACCTTGTTACGACTT-3'
[0234] Recovered DNA is sheared or enzymatically digested to 3-6 kb
fragments. Lone-linker primers are ligated and the DNA is sized
selected. Size-selected DNA is amplified by PCR, if necessary.
[0235] Normalization is then accomplished as follows by
resuspending double-stranded DNA sample in hybridization buffer
(0.12 M NaH.sub.2PO.sub.4, pH 6.8/0.82 M NaC1/1 mM EDTA/0.1% SDS).
The sample is overlaid with mineral oil and denatured by boiling
for 10 minutes. Sample is incubated at 68.degree. C. for 12-36
hours. Double-stranded DNA is separated from single-stranded DNA
according to standard protocols (Sambrook, 1989) on hydroxyapatite
at 60.degree. C. The single-stranded DNA fraction is desalted and
amplified by PCR. The process is repeated for several more rounds
(up to 5 or more).
EXAMPLE 3
Cell Staining Prior to FACS Screening
[0236] Gene libraries, including those generated as described in
Example 1, can be screened for bioactivities of interest on a FACS
machine as indicated herein. A screening process begins with
staining of the cells with a desirable substrate according to the
following example.
[0237] A gene library is made from the hyperthermophilic archaeon
Sulfulobus solfataricus in the .lambda.-ZAPII vector according to
the manufacturers instructions (Stratagene Cloning Systems, Inc.,
La Jolla, Calif.), and excised into the pBLUESCRIPT plasmid
according to the manufacturers instructions (Stratagene). DNA was
isolated using the IsoQuick DNA isolation kit according to the
manufacturers instructions (Orca, Inc., Bothell, Wash.).
[0238] To screen for .beta.-galactosidase activity, cells are
stained as follows. Cells are cultivated overnight at 37.degree. C.
in an orbital shaker at 250 rpm. Cells are centrifuged to collect
about 2.times.10.sup.7 cells (0.1 ml of the culture), resuspended
in 1 ml of deionized water, and stained with
C.sub.2-Fluoroscein-Di-(-D-galactopyran- oside (FDG). Briefly, 0.5
ml of cells are mixed with 50 .mu.l C.sub.12-FDG staining solution
(1 mg C.sub.12-FDG in 1 ml of a mixture of 98% H.sub.2O, 1% DMSO,
1% EtOH) and 50 .mu.l Propidium iodide (PI) staining solution (50
.mu.g/ml of distilled water). The sample is incubated in the dark
at 37.degree. C. with shaking at 150 rpm for 30 minutes. Cells are
then heated to 70.degree. C. for 30 minutes (this step can be
avoided if sample is not derived from a hyperthermophilic
organism).
EXAMPLE 4
Screening of Expression Libraries by FACS and Recovery of Genetic
Information of Sorted Organisms
[0239] The excised .lambda.-ZAP II library is incubated for 2 hours
and induced with IPTG. Cells are centrifuged, washed and stained
with the desired enzyme substrate, for example
C.sub.12-Fluoroscein-Di-(-D-galacto- pyranoside (FDG) as in Example
3. Clones are sorted on a commercially available FACS machine, and
positives are collected. Cells are lysed according to standard
techniques (Current Protocols in Molecular Biology, 1987) and
plasmids are transformed into new host by electroporation using
standard techniques. Transformed cells are plated for secondary
screening. The procedure is illustrated in FIG. 5. Sorted organisms
can be grown and plated for secondary screening.
EXAMPLE 5
Sorting Directly on Microtiter Plates
[0240] Cells can be sorted in a FACS instrument directly on
microtiter plates in accordance with the present invention. Sorting
in this fashion facilitates downstream processing of positive
clones.
[0241] E. coli cells containing .beta.-galactosidase genes are
exposed to a staining solution in accordance with Example 3. These
cells are then left to sit on ice for three minutes. For the cell
sorting procedure they are diluted 1:100 in deionized water or in
Phosphate Buffered Saline solution according to the manufacturers
protocols for cell sorting. The cells are then sorted by the FACS
instrument into microtiter plates, one cell per well. The sorting
criteria is fluorescein fluorescence indicating
.beta.-galactosidase activity or PI for indicating the staining of
dead cells (unlike viable cells, dead cells have no membrane
potential; hence PI remains in the cell with dead cells and is
pumped out with live cells). Results as observed on the microtiter
plate are shown in FIG. 6.
3 TABLE 1 Habitat Cultured (%) Seawater 0.001-0.1 Freshwater 0.25
Mesotrophic lake 0.01-1.0 Unpolluted esturine waters 0.1-3.0
Activated sludge 1.0-15.0 Sediments 0.25 Soil 0.3
EXAMPLE 6
Production of Single Cells or Fragmented Mycelia
[0242] Inoculate 25 ml MYME media (see recipe below) in 250 ml
baffled flask with 100 .mu.l of Streptomyces 10712 spore suspension
and incubated overnight @ 30.degree. C. 250 rpm. After 24 hour
incubation, transfer 10 ml to 50 ml conical polypropylene
centrifuge tube and centrifuge @ 4,000 rpm for 10 minutes @
25.degree. C. Decant supernatant and resuspend pellet in 10 ml 0.05
M TES buffer. Sort cells into MYM agar plates (sort 1 cell per
drop, 5 cells per drop, 10 cells per drop) and incubate plates @
30.degree. C.
[0243] MYME media (Yang, et. al., 1995 J. Bacteriol. 177(21):
6111-6117) contains: 10.3% sucrose, 1% maltose, 0.5% peptone, 0.3%
yeast extract, 0.3% maltose extract, 5 mM MgCl2 and 1% glycine
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[0249] It will be apparent to those skilled in the art that various
modifications and variations can be made to the compounds and
processes of this invention. Thus, it is intended that the present
invention cover such modifications and variations, provided they
come within the scope of the appended claims and their equivalents.
Accordingly, the invention is limited only by the following
claims.
* * * * *