PCI‘
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`
`
`Al
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`we 82/ 02562
`
`
`
`
`(51) International Patent Classification 3 :
`(11) International Publication Number:
`,
`-
`C12Q 1/18, 1/04, 1/06
`
`
`(43) International Publication Date:
`5 August 1982 (05.08.82)
`
`
`
`C12M 1/34
`
`Published
`
`(21) International Application Number:
`PCT/U582/00124
`With international search report.
`
`
`Before the expiration of the time limitfor amending the
`claims and to be republished in the event of the receipt
`
`of amendments.
`
`
`
`I (22) International Filing Date:
`
`29 January 1982 (29.0l.82)
`
`(31) Priority Application Number:
`
`229,484
`
`(32) Priority Date:
`
`29 January 1981 (29.01.81)
`
`(33) Priority Country:
`
`US
`
`(71x72) ApplicantEnd Inventor: WEAVER, James, C. [US/
`US]; P. O. Box 429, Sudbury, MA 01776 (US).
`
`(74) Agents: ENGELLENNER, Thomas, J. et al.; Massa-
`chusetts Institute of Technology, 77 Massachusetts
`Avenue, Cambridge, MA 02139 (US).
`
`(81) Designated States: BE (European patent), DE (Euro-
`pean patent), FR (European patent), GB (European
`patent), JP.
`
`
`
`
`
`
`
`
`
`
`
`
`
`(54) Title: PROCESS FOR ISOLATING MIRCOBIOLOGICALLY ACTIVE MATERIAL
`
` (57) Abstract
`
`
`
`
`
`
`Molecules or cells of materials exhibiting a microbiological activity, suppression or interaction of interest are isolat-
`ed from a large population of similar molecules, cells or the like. The activity of the microbiologically active molecules or
`living matter of interest is measured by suspending the population in a liquid medium capable of forming a gel. The result-
`ant suspension is formed into dr0plets which are caused to gel. The gel droplets (GNDs) are treated to effect desirable al-
`teration of the microbiologically active material and the amount of metabolites or reaction products of the desired altera-
`tion within each of the gel droplets. Alternatively, incubation is carried out such that each GMD initially containing one
`cell contains many, a microcolony, which can be tested for desirable properties while retaining sufficient viable cells for
`futher growth and harvesting for further testing and/or production. The gel droplets are then separated based upon the
`measured or sensed characteristic, metabolite or reaction product.
`
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`

`

`WE’I’EQ."' Lti‘t‘
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`FOR 11E PURPOSES 0F HVFORMAHON ONLY
`
`Codes used to identify States party to the PCT on the front pages ofpamphlets publishing international ap-
`plications under the PCT.
`
`United States of America
`
`AT
`AU
`BE
`BR
`
`Austria
`Australia
`Belgium
`Brazil
`Central African Republic
`Congo
`Switzerland
`Cameroon
`Germany. Federal Republic of
`Denmark
`Finland
`France
`Gabon
`United Kingdom
`Hungary
`Japan
`
`,
`
`Democratic People‘s Republic of Korea
`Liechtenstein
`Sri Lanka
`Luxembourg
`Monaco
`Madagascar
`Malawi
`Netherlands
`Norway
`Romania
`Sweden
`Senegal
`Soviet Union
`Chad
`Togo
`
`l.)
`
`4/V
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`

`

`W0 82/02562
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`PCT/USS2/00 124
`
`PROCESS FOR ISOLATING
`
`MICROBIOLOGICALLY ACTIVE MATERIAL
`
`Technical Field
`This invention relates to a process for isolating micro—
`biologically active material such as bacteria.
`
`Background of the Invention
`Presently,
`there are available a wide variety of
`apparatus for isolating relatively large microbiologically
`
`suspended in a liquid wherein the cells are sufficiently
`diluted and the stream is sufficiently small such that the
`cells pass by a point along the flow stream path one by one.
`Means are provided along the flow path to sense a physical
`characteristic of the cell which provides a measure of a
`particular desired biological characteristic of the cell.
`Examples of such physical measurements include light scatter-
`ing, electrical impedance, fluorescence intensity,
`light
`absorption or fluorescence polarization.
`The stream of cells
`passes by the means for measuring the cell physi3al char—
`acteristic which measurement means is adapted to control flow
`of the cell stream so that the individual cells characterized
`by a physical measurement within a desired range can be
`separated and isolated from the stream.
`In one class of
`such apparatus, a portion of the cell stream is diverted into
`a secondary stream such as with a syringe.
`In another class
`of such apparatus,
`the cell stream is divided into droplets
`each of which has a high probability for containing none or
`a single cell.
`The droplets are formed by any conventional
`means such as a piezoelectric crystal and the droplets
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`containing the desired cells are charged electrically
`while the remaining droplets are uncharged so that the
`charged droplets can be isolated from the remaining drop-
`lets such as with a set of conventional deflection plates.
`The charged droplets so recovered contain the desired cells
`at a far higher concentration that in the original stream.
`'
`Rotman, Proceedings National Academy of Sciences, Vol.
`47,
`gs. 1981-1991, 1961 discloses the formation of water
`droplets in oil which droplets contain a small number, often
`one, of enzymes. However, this procedure is very tedious,
`difficult to replicate and the enzymes are susceptible to
`migration from the droplets to the oil-water interface.
`While the present procedures have been highly satis-
`factory for concentrating animal cells of desired character-
`istics,
`these procedures have not been as satisfactory for
`isolating biological material of a smaller size than animal
`cells, such as bacteria or enzymes. This is because the
`physical characteristics of these smaller volume particles
`or molecules are much more difficult to measure accurately,
`
`particularly at the flow rates available with present cell
`sorters. Accordingly, it would be highly desirable to pro-
`vide a means for concentrating and isolating materials
`having microbiological activity which materials have a size
`much smaller than the normal size of eucaryote cells even
`
`25
`
`on a molecular size basis. Furthermore, it would be desir-
`
`able to provide such a process wherein a microbiologically
`active molecule cell or the like having an activity of
`
`interest can be isolated alone from a large population of
`
`a
`
`similar microbiologically active materials.
`
`30
`
`Summary of the Invention
`In accordance with this invention, microbiologically
`
`active materials are suspended at a dilute concentration in
`
`a medium which can be subsequently converted to a gel micro
`
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`“O 82/025562
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`”Lanaiv.‘._..-.
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`droplet medium being a size between about O-Zu to lOOOu, Pre-
`'The suspending medium is
`ferably being about 5p and lOOu.
`of the micro-
`capable of substantially preventing degradation
`biologically active molecules, cells or the like, or of
`supporting growth of the microbiologically active matter such
`as in the case of bacteria.
`The dilute suspension then is
`formed into small droplets such as be being forced through a
`nozzle to form a liquid stream which then is sheared to form
`small liquid droplets, each of which has a high probability
`of containing a desired small number of molecules of cells
`or less. Thus, for example, each droplet can contain zero
`or one microbiologically active molecule or cell of interest
`with or without microbiologically active molecules or cells
`which coact with the molecule or cell of interest by regula-
`ting the degree of dilution of the liquid composition is pro-
`cessed and the average size of the GMD produced.
`The drop—
`lets formed then are changed in temperature or directed
`into a second liquid or vapor medium wherein the droplets
`rapidly gel.
`The change in temperature or second liquid or
`vapor medium is capable of converting the droplets to gel
`form either by temperature change or by contact with a vapor,
`while preventing degradation of the microbiologically active
`material.’ In the case that the liquid droplets are caused
`to gel before encountering liquid medium, gel micro-droplets
`(GMDs) can be directed onto a solid surface.
`The GMDs are
`treated in a manner to effect a desirable alteration of the
`microbiologically active material such as by incubation or
`by exposure to conventional marker molecules such as to a
`fluorescent stain or by exposure to a mutagenic environment
`or the like.
`The suspension of gel micro-droplets suitably
`diluted then is processed through an apparatus which forms
`a stream of the micro-droplets such that the micro-droplets
`pass an analyzer one by one so that each micro-droplet can
`
`
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`

`
`
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`
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`W0 82/02562
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`PCT/US82/00124
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`can analyzed for a desired chemical or physical character-
`istic. Furthermore,
`the apparatus is capable of separating
`gel micro-droplets having the desired characteristics from
`the remainder of the stream in response to a signal from the
`onstream analyzer. Alternatively, measurements can be made
`on GMDs on a surface by scanning with a suitable light source
`or other means,
`in order to measure and distinguish desir-
`Subsequent to such scanning,
`
`able physical characteristics.
`
`the GMDs with desired physical
`
`characteristics can be
`
`10
`
`mechanically removed and thereby isolated.
`
`15
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`25
`
`Description of Specific Embodiments
`In accordance with this invention, microbiologically
`
`active materials having a small subcellular size such as
`yeast, bacteria, mold, enzymes or the like are suspended in
`an aqueous medium capable of gelation upon subsequent treat-
`ment of the suspending medium. Suitable suspending mediums
`include water soluble polymers. Representative suitable
`
`natural materials include kappa-carrageenan,
`
`iota—carrageenan,
`
`sodium alginate, furcellaran,
`
`rein, succinylated rein,
`
`succinylated cellulose, ethyl
`like. Representative suitable synthetic water soluble poly—
`mers include those formed from vinyl pyrolidone,
`2 - methyl
`S - vinyl pyrridine - metnyl acrylate - methacrylic acid
`1 pyrridine, vinyl pyrridine -
`
`copolymer, vinyl alcohol, viny
`
`succinylated cellulose or the
`
`styrene copolymer or the like.
`material is suspended in the suspending medium at a dilution
`which is selected using knowledge of the volume of the GMD
`
`The microbiologically active
`
`to be produced and the density of size of cells or molecules
`
`—
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`!
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`in the first liquid medium.
`
`30
`
`The GMDs are formed so that there is a high probability
`
`that each GMD contains a desired number or less of miCTO‘
`
`biologically active material. This can be effected by
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`regulating the dilution of the liquid composition to be pro-
`cessed to GMDs, a knowledge of the size of the microbiolog-
`ically active material and the size of the GMDs
`to be produced.
`The reaulation of these factors can be determined by con-
`ventional Poisson statistical analysis so that the number
`of GMDs containing more than the desired number of micro~
`biologically active materials is more than two standard
`deviations from the mean.
`»
`Gel material and nutrients can be incorporated in the
`suspending medium,
`in which case very little dilution may
`be desired.
`Then, for example, when it is desired to have
`a high probability of zero or one microbiologically active_
`material for GMDs, it is only necessary to dilute the sample
`by more than about a factor of about 1.1, usually up to
`about 10 or larger if desired if the unknown cell or enzyme
`concentration is believed to be large.
`For exazple, if the
`average volume CVGMD)
`is about 10'7 ml, corresponding to a
`GMD with diameter about
`5 x 10-3 cm (SOum),
`is spherical,
`dilution is generally not needed until the initial cell con-
`centration reaches about 10+? cells-ml-l.
`For smaller
`spherical GMDs, for example,
`loam diameter, dilution is not
`needed until the sample concentration reaches about 109
`cells‘ml-l.
`It is desirable, for example,
`to isolate zero
`or one microbiologically active cell per GMD in recombinant
`DNA research whereas it is desired to isolate a particularly
`active genetically altered bacteria such as E. coli capable
`of producing a desired molecule, e.g: interfere: from a
`large population of such bacterium.
`For purposes of sorting or isolating microbiologically
`active material, it is desirable to utilize dilutions such
`that the suspension can be subsequently formed into droplets
`each of which have a high probability of containing none of
`the microbiologically active material or only a single cell
`
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`By
`or molecule of the microbiologically active material.
`separating and localizing the biologically active material
`in this manner, it is possible to isolate materials of desired
`activity which activity is not diluted by the presence of
`other biologically active material not having the desired
`activity.
`For example, it may be desirable to isolate
`
`bacteria such as E. coli, altered by conventional recombinant
`
`to produce a desired material such as a
`DNA techniques,
`hormone,
`insulin,
`interferon, etc.
`In these techniques, DNA
`is altered on a batch basis and bacteria also is processed
`
`on a batch basis to effect implanation of a plasmid, said
`bacteria are then suspended in a liquid capable of forming
`a gel.upon subsequent treatment and then converted into drop-
`let such that there is a high probability that each droplet
`
`The thus-pro-
`contains none or only one of such bacteria.
`duced liquid droplets are directed into a liquid medium
`Capable of effecting gelation of the droplets. Alternatively,
`the initially liquid droplets are changed in temperature or
`contacted with suitable gel-inducing vapors before entering
`
`the gel droplets
`In addition,
`the second liquid medium.
`also can, but are not required to, contain a conventional
`bacterial growth supporting composition which permits the
`bacteria to replicate within the droplets.
`The suspension
`of such gel droplets then is treated in a conventional manner
`such as with a fluorescent dye, a fluorescent labeled anti-
`
`body or fluorescent labeled antigen or the like in order to
`mark the
`gel droplets having the desired bacteria while.
`preventing or greatly reducing the marking of the gel drop-
`
`lets not containing the desired bacteria.
`
`(Al O
`
`This invention is useful for isolating a wide variety
`
`of materials having microbiological activity,
`
`interaction
`
`or suppression including parasites, virus, yeasts, cells,
`
`bacteria mole enzymes,
`
`interactions between variant cells,
`
`cell-virus interactions, hydrodomis or the like.
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`Representative bacteria which can be processed in
`accordance with this invention include E. coli, Bacillis
`subtilus, Pseudomonas species, Clostridium,
`thermocellum,
`Zymonanas mobilis, Methane bacterium sochngenii, Salmonella
`typhimurium, Serratia marcens, Clostridium botulinum,
`Bacillia sterothermophilis. Any conventional
`tagging means
`can be utilized in order to identify the gel droplets con-
`taining the bacteria having the desired characteristics
`including radioactively labeled antibody, fluorescent anti-
`body labeling, use of fluorescent dyes, viable stains,
`magnetic labeling or the like.
`These procedures are well
`known to be selective in labeling and/or staining products
`produced by bacteria. Similarly,
`the same techniques can
`be utilized for selectively identifying desired products of
`
`yeast, mold, parasites, etc.
`In the case of molecular size biologically active
`molecules such as enzyme activity, or the accumulation or
`depletion of NADH or other fluorescent products or cofactors
`can be measured,
`the following procedure can be utilized in
`accordance with this invention.
`A sample containing unknown
`quantity of a particular enzyme is suspended, with modest
`dilution,
`in a first liquid medium which contains buffering
`compounds, substrates, cofactors and a gelling agent. When
`the approximate upper limit of the concentration of enzymes
`to be measured can be estimated, a GMD volume is selected
`so that there is a high probability the GMDs will contain
`either none or one enzyme molecule.
`'When a linked enzyme
`assay is used,
`the appropriate additional enzymes, sub-
`strates and cofactors are also included. Similarly, if an
`assay is to be based on cell-cell interactions, one type of
`cell is also provided at relatively high concentrations.
`The resulting diluted sample is then passed through a vibrat-
`ing orifice or nozzle or other means to cause formation of
`
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`the liquid drop-
`liquid droplets. As described previously,
`lets are caused to enter the gel state by cooling, contact-
`ing with a suitable vapor or solid substrate or entering a
`second liquid medium.
`The resulting GMDs are coated with a
`thin layer impermeable or having controlled permeability to
`the substrates, products and/or cofactors of the enzyme
`catalyzed reaction, such as phosphatidyl ethanolamine or
`phosphatidyl choline or the like.
`For example,
`the permea-
`bility of the coating can be controlled to allow entry into
`the GMD of a reagent that effects marking,
`lysing or the like
`within the GMD and restricts outflow from the GMD of such
`
`The coated GMDs are maintained at a suit-
`marked product.
`able temperature such that the enzyme reactions are carried
`out, not necessarily to completion, and fluorescent product
`
`is accumulated and retained in GMDs containing an enzyme
`molecule. Alternatively, a fluorescent substrate can be
`putilized,
`in which case the fluorescent substrate decreases
`or disappears in GMDs containing an enzyme molecule.
`After the biologically active material Within the gel
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`micro-droplets has been treated in order to effect the
`
`desired change in the material, such as by incubation, muta-
`
`tion, staining with fluorescent stains,
`
`labeled with magnet-
`
`ically tagged or other immunological agents,'the suspension
`
`of the gel micro-droplets then is processed in an apparatus
`having the capability of sensing a physical characteristic
`
`I‘J U1
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`of individual gel micro-droplets to determine the presence
`or absence of a desired physical characteristic and there-
`after isolating the gel micro-droplets having the desired
`
`physical characteristic.
`
`For example,
`
`the desired gel
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`I}
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`30
`
`micro-droplets may be selectively stained with a fluorescent
`
`dye and can be passed one by one in a liquid stream by
`an optical analyzer capable of sensing the concentrated
`
`fluorescent dye on the gel micro-droplet.
`
`The analyzer
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`controls means for isolating that portion of the liquid stream
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`For example, a por-
`which satisfies the sensing criteria.
`tion of the liquid stream can be diverted into a secondary
`stream for subsequent recovery of the gel micro—droplets
`such as is disclosed by Kamentskyj United States Patent
`No. 3,560,754. Alternatively,
`the mainstream can be con-
`
`nozzle which is vibrated such as by a piezoelectric crystal
`by the means disclosed, for example, by Fylwyler, United
`The
`States Patent Nos. 3,710,933; 3,790,492 and 4,162,282.
`drops containing the gel micro-droplets having the desired
`characteristics then can be electrically charged selectively
`and then passed between a pair of deflecting plates in order
`to seleCtively divert the electrically charged droplets so
`that they can be recovered.
`V
`The process of this invention provides substantial
`advantages over the prior art processes in that microbio—
`logically active material having a size much smaller than
`5
`that of the normal cell, usually within the range of about
`to 0.5 microns and even as small as molecular size materials,
`can be isolated to recover microbiologically active materials
`having a desired characteristic from a large population of
`such microbiologically active materials,
`the majority of
`which do not have the desired microbiological characteristic.
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`EXAMPLE I
`Any of a variety of protocols for genetic manipula-
`tion is carried out on a large, for example about 109 to
`about 1012 cells, population of bacteria such as E. coli
`or the like, with the result that a small number, perhaps
`only one, or the cells of the post manipulation population
`have a new, desired feature such as the production of a
`specific biomolecule such as a specific protein or hormone
`or the like. Frequently, such biomolecules are not secret-
`ed, but are retained within the desired modified bacterium.
`Further,
`the desired modified bacterium typically obtains
`no selective growth characteristic uhich would allow use of
`conventional microbiological selection procedures.
`Instead,
`the desired modified bacterium must be sorted from the very
`large post manipulation population.
`An exemplary process
`for isolation begins by suspending the post manipulation
`population of E. coli in a conventional medium such as YPD
`(containing yeast extract, potato starch and dextrose) but
`also 2% height of sodium alginate, which suspension is in
`the pre gel state.
`The suspension is then passed through a
`vibrating orifice nozzle such that the liquid stream breaks
`up into liquid droplets.
`Said liquid droplets are directed
`into a second liquid medium, typically stirred gently,
`0. 5% CaClZ at pH7, where said liquid droplets rapidly
`exchange Na ions for Ca ions and enter the gel state. Gel
`formation typically occurs first at the outer surface of
`said liquid droplets,
`forming a temporary deformable skin,
`which deformable skin allons interfacial forces to direct_
`the droplet
`into an approximately spherical or ellipsoid
`shape before the droplet is completely gelled. Within less
`tian l to 15 minutes, depending on liquid droplet siz e and
`the KCl concentration,
`the transition into the gel state
`is complete.
`The G}IDS are maintained in suspension by
`
`I?
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`repelled from
`
`gentle stirring and do not adhere to each other even after
`Additional nutrients such as glucose, ammonia,
`many hours,
`supplied to the stirred
`growth or medium such as YPD are
`medium such that the individual bacteria in each occupied
`GMD can grow and divide to form a microcolony. This incuba-
`tion period is continued for several hours,
`typically about
`5
`to 10 hours,
`the exact hours depending on the volume of
`the GMDs which are used, until each initially occupied GMD
`is saturated or fully occupied. Any bacteria which are
`released into the liquid medium during said incubation are
`GMDs when the bacterial surface charge is the
`f the outer surface of the GMDs and con-
`same sign as that o
`tamination and cross——contamination of GMDs
`is avoided.
`When the bacterial and GMD surface do not have the same
`surface charge,
`the GMDs are instead placed on the surface
`of a suitable filter such as a Nuclepore filter and gently
`washed, with flow through the filter such that any released
`bacteria are carried through the filter and do not contact
`other GMDs.
`In this case, it is often advantageous to select
`the original cell suspension concentration with respect to
`GMD volume such that there are many more unoccupied GMDS
`than GMDs occupied by one cell, which relationship greatly
`decreases the probability of occupied GMDs contacting each
`other on the surface of the filter.
`Following incubation
`either in suspension or retained on a filter surface,
`the
`GMDs contain a microcolony of 103 to 105 cells and are
`contacted typically for 1 second 10 lbO seConds, depending
`on GMD size with a chemical
`lysing agent such as Trition—X,
`sodium dodecyl sarcosinlate or sodium lauryl sarcosinlate.
`The brief exposure of GMDs
`to a chemical
`lysing agent
`results in lysing of cells mainly near the outer surface of
`the GMDs, with increasingly femer lysed cells tonard the
`center of the EYES, and therefore increasing numbers of
`viable cells toward the center.
`The GMDs are now, or at
`
`the
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`same time as the lysing agent,‘exposed to a fluorescent
`labeled antibody (PA);
`the antibody being preselected to
`be specific toward the desired protein or hormone.
`How-
`ever,
`the exposure to the PA is usually different than to
`the lysing agent, said FA exposure usually being longer.
`A preferential staining of GMDs with releaSed specific pro-
`tein or hormone thereby occurs.
`The suspension of GMDs,
`'now containing a few preferentially stained GMDs with
`viable cells near the center, is passed through a flow
`
`microfluorometer/cell.sorter, such that the preferentially
`stained GMDs with viable cells near the center, is passed
`through a flow microfluorometer/cell sorter, such that the
`preferentially stained GMDs are isolated or sorted by the
`usual means now employed for isolating or sorting animal
`cells. Further incubation of the thuseisolated GMDs allows
`large and useful quantities of the rare, desirable modified
`
`E. coli to be obtained and used.
`
`5
`
`10
`
`15
`
`
`
`
`
`
`
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`
`

`

`W08202 2
`/
`56
`
`PCT/U582/00124
`
`
`
`The process of performing select microbiological
`1.
`studies upon a sample of a given material comprising the
`steps of:
`,
`forming mutually independent microsamples
`of said material by
`(i)
`forming a dilute suspension of said
`material in a liquid diluent capable
`of forming a gel upon subsequent
`
`a]
`
`(ii)
`
`(iii)
`
`treatment,
`converting said suspension into gel
`droplets having a size between 0.2
`and 1000 microns, and
`sensing and selectively separating
`said droplets containing micro-
`biologically active material having
`the desired characteristics from the
`remainder of said material,
`
`and
`
`b)
`
`conducting said studies on said droplets each
`in a confined substantially mutually inde-
`
`pendent fashion.
`The process of claim 1 wherein said droplets have
`2.
`a size between about
`5 and 100 microns.
`3.
`The process of claim 1 wherein said material is a
`
`bacterium.
`4.
`The process of claim 3 wherein said bacterium is
`E. coli.
`5.
`
`The process of claim 1 wherein said material
`
`is an
`
`enzyme.
`6.
`
`.
`The process of claim 1 wherein said material
`
`is a
`
`yeast.
`
`.
`
`
`
`

`

`35x
`
`W0 82/02562
`
`PCT/U882/00124
`
`-14..
`
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`
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`
`T.
`
`The process of claim 1 wherein said material is
`
`a mold.
`8.
`
`‘
`The process of claim 1 wherein said material
`
`comprises an animal cell.
`
`9.
`
`The process of claim 1 wherein said material
`
`- comprises a plant cell.
`
`10.
`
`The process of claim 4 wherein said bacterium
`
`includes a genetically modified plasmid.
`
`11.
`
`The process of any one of claims 1, 2, 3, 4, 5,
`
`6, 7, 8, 9, or 10 wherein the microbiologically active
`
`material having the desired characteristic is selectively
`
`tagged with a marker composition capable of being sensed and
`
`sensing said marker composition to effect isolation of gel
`
`droplets containing the microbiologically active material
`
`having the desired characteristic.
`
`12.
`
`The process of any one of claims 1, Z, 3, 4, 5,
`
`6, 7, 8,79 or 10 wherein the microbiologically active
`
`material having the desired characteristic produces a
`
`metabolite capable of being sensed and sensing said metabolite
`
`to effect isolation of gel micro-droplets containing the
`
`microbiologically active material having.the desired char-
`acteristic.
`
`13.
`
`'The process of any one of claims 1, Z, 3,
`
`4.3
`
`,-
`2 D!
`
`V.
`
`6, 7, 8, 9, or 10 wherein the microbiologically active
`
`material having the desired characteristic coacts with a
`reagent within said micro-droplet to produce a reaction
`product capable of being sensed and sensing said reactionr
`
`product to effect isolation of gel micro-droplets containing
`
`the microbiologically active material having the desired
`
`5
`
`characteristic.
`
`

`

`WO 82/02562
`
`PCT/U882/00124-
`
`- 15 -
`
`The process for isolating a microbiologically
`11.
`active material having a desired microbiological character—
`istic from a large population of microbiologically active
`materials lacking said desired characteristic which com—
`prises forming a dilute suspension of said population in
`a liquid diluent capable of forming a gel upon subsequent
`treatment, converting said suspension into gel droplets
`having a size between 0.5 and about 1000 microns, said
`droplets having a high probability of containing zero or
`one molecule or cell of said microbiologically active
`material, sensing the gel droplets containing the micro—
`biologically active material having the desired character-
`istic and selectively separating said gel droplets containing
`microbiologically active material having the desired
`characteristic.from said population.
`15.
`The process of claim 14 wherein said material is
`
`a bacterium.
`16;
`The process of claim 15 wherein said bacterium is
`
`E. coli.
`17.
`
`The process of claim 14 wherein said material is
`
`an enzyme.
`18.
`The process of claim 14 wherein said material is
`
`a yeast.
`19.
`
`a mold.
`20.
`
`The process of claim 14 wherein said material is
`
`The process of claim 14 wherein the material com-
`
`prises an animal cell.
`21.
`The process of claim 14 wherein the material com-
`
`prises a plant cell.
`22.
`The process of claim 16 wherein said bacterium
`includes a genetically modified plasmid.
`
`BU REA U
`(25!?!
`
`

`

`V: eggs; .1113.“ .'
`
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`
`W0 82/052562
`
`PCT/U882/00124
`
`-16-
`
`“p
`1“
`
`The process of any one of claims 14, 15, 16, 17,
`23;
`18, 19, 20, 21 or 22 wherein the microbiologically active
`material having the desired characteristic is selectively
`tagged with a marker composition capable of being sensed
`and sensing said marker composition to effect isolation of
`gel droplets containing the microbiologically active
`material having the desired characteristic.
`24.
`The process of any one of claims 14, 15, 16, 17,
`18, 19, 20, 21 or 22 wherein the microbiologically active
`
`material having the desired characteristic produces a meta-
`
`bolite capable of being sensed and sensing said metabolite
`to effect isolation of gel micrOcdroplets containing the
`
`microbiologically active material having the desired charac—
`
`teristic.
`
`'
`
`The process of any one of claims 14, 15, 16, 17,
`25.
`18, 19, 20, 21 or 22 wherein the microbiologically active
`material having the desired characteristic coacts with a
`
`reagent within Said micro—droplet to produce a reaction pro-
`
`duct capable of being sensed and sensing said reaction pro-
`
`duct to effect isolation of gel micro-droplets containing
`
`the microbiologically active material having the desired
`
`characteristic.
`
`