WO2006038035A2 - In vitro evolution in microfluidic systems - Google Patents
In vitro evolution in microfluidic systems Download PDFInfo
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- WO2006038035A2 WO2006038035A2 PCT/GB2005/003889 GB2005003889W WO2006038035A2 WO 2006038035 A2 WO2006038035 A2 WO 2006038035A2 GB 2005003889 W GB2005003889 W GB 2005003889W WO 2006038035 A2 WO2006038035 A2 WO 2006038035A2
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Definitions
- the present invention relates to methods for use in in vitro evolution of molecular libraries.
- the present invention relates to methods of selecting nucleic acids encoding gene products in which the nucleic acid and the activity of the encoded gene product are linked by compartmentation, using microfluidic systems to create and/or handle the compartments.
- nucleic acids that encode them.
- the selected nucleic acids can subsequently be cloned for further analysis or use, or subjected to additional rounds of mutation and selection.
- Molecules having the desired characteristics can be isolated through selection regimes that select for the desired activity of the encoded gene product, such as a desired biochemical or biological activity, for example binding activity.
- phage display relies upon the creation of nucleic acid libraries in vivo in bacteria.
- the practical limitation on library size allowed by phage display technology is of the order of 1O? to K)H, even taking advantage of D phage vectors with excisable filamentous phage replicons.
- the technique has mainly been applied to selection of molecules with binding activity. A small number of proteins with catalytic activity have also been isolated using this technique, however, selection was not directly for the desired catalytic activity, but either for binding to a transition-state analogue (Widersten and Mannervik, 1995) or reaction with a suicide inhibitor (Soumillion et al., 1994; Janda et al., 1997).
- “evolution” systems which can evolve both nucleic acids and proteins to effect the full range of biochemical and biological activities (for example, binding, catalytic and regulatory activities) and that can combine several processes leading to a desired product or activity.
- the desired activity of a gene product results in a modification of the genetic element which encoded it (and is present in the same microcapsule).
- the modified genetic element can then be selected in a subsequent step.
- a method for isolating one or more genetic elements encoding a gene product having a desired activity comprising the steps of:
- a genetic element may be expressed to form its gene product before or after compartmentalisation; where the gene product is expressed before compartmentalisation, it is linked to the genetic element such that they are compartmentalised together.
- At least one step is performed using electronic control of fluidic species.
- the method of the invention comprises the steps of:
- the method of the invention comprises the steps of:
- microcapsules according to the present invention compartmentalise genetic elements and gene products such that they remain physically linked together.
- a genetic element is a molecule or molecular construct comprising a nucleic acid.
- the genetic elements of the present invention may comprise any nucleic acid (for example, DNA, RNA or any analogue, natural or artificial, thereof).
- the nucleic acid component of the genetic element may moreover be linked, covalently or non- covalently, to one or more molecules or structures, including proteins, chemical entities and groups, and solid-phase supports such as beads (including nonmagnetic, magnetic and paramagnetic beads), and the like.
- these structures or molecules can be designed to assist in the sorting and/or isolation of the genetic element encoding a gene product with the desired activity.
- expression is used in its broadest meaning, to signify that a nucleic acid contained in the genetic element is converted into its gene product.
- expression refers to the transcription of the DNA into RNA; where this RNA codes for protein, expression may also refer to the translation of the RNA into protein.
- expression may refer to the replication of this RNA into further RNA copies, the reverse transcription of the RNA into DNA and optionally the transcription of this DNA into further RNA molecule(s), as well as optionally the translation of any of the RNA species produced into protein.
- expression is performed by one or more processes selected from the group consisting of transcription, reverse transcription, replication and translation.
- microcapsule is used herein in accordance with the meaning normally assigned thereto in the art and further described hereinbelow.
- a microcapsule is an artificial compartment whose delimiting borders restrict the exchange of trie components of the molecular mechanisms described herein which allow the sorting of trie genetic elements according to the function of the gene products which they encode.
- the microcapsules used in the method of the present invention will be capable of being produced in very large numbers, and thereby to compartmentalise a library of genetic elements which encodes a repertoire of gene products.
- a change in optical properties refers to any change in absorption or emission of electromagnetic radiation, including changes in absorbance, luminescence, phosphorescence or fluorescence. All such properties are included in the term "optical”.
- Microcapsules and/or genetic elements can be sorted, for example, by luminescence, fluorescence or phosphorescence activated sorting, hi a preferred embodiment, flow cytometry is employed to sort microcapsules and/or genetic elements, for example, light scattering (Kerker, 1983) and fluorescence polarisation (Rolland et al., 1985) can be used to trigger flow sorting.
- Changes in optical properties may be direct or indirect.
- the change may result in the alteration of an optical property in the microcapsule or genetic element itself, or may lead indirectly to such a change.
- modification of a genetic element may alter its ability to bind an optically active ligand, thus indirectly altering its optical properties.
- the sorting of genetic elements may be performed in one of essentially seven techniques.
- T the microcapsules are sorted according to an activity of the gene product or derivative thereof which makes the microcapsule detectable as a whole. Accordingly, a gene product with the desired activity induces a change in the microcapsule, or a modification of one or more molecules within the microcapsule, which enables the microcapsule containing the gene product and the genetic element encoding it to be sorted.
- trie microcapsules are physically sorted from each other according to the activity of the gene product(s) expressed from the genetic element(s) contained therein, which makes it possible selectively to enrich for microcapsules containing gene products of the desired activity.
- the genetic elements are sorted following pooling of the microcapsules into one or more common compartments.
- a gene with a desired activity induces a change in the microcapsule containing the gene product and the genetic element encoding it. This change, when detected, triggers the modification of the gene within the compartment. The reactions are stopped and the microcapsules are then broken so that all the contents of the individual microcapsules are pooled. Selection for the modified genetic elements enables enrichment of the genetic elements encoding the gene product(s) having the desired activity. Accordingly the gene product having the desired activity induces a change in the compartment which is detected and triggers the modification of the genetic element within the compartment so as to allow its isolation. It is to be understood that the detected change in the compartment may be caused by the direct action of the gene product, or indirect action, in "which a series of reactions, one or more of which involve the gene product having the desired activity leads to the detected change.
- the genetic elements may be sorted by a multi-step procedure, which involves at least two steps, for example, in order to allow the exposure of the genetic elements to conditions which permit at least two separate reactions to occur.
- the first microencapsulation step of the invention must result in conditions which permit the expression of the genetic elements - be it transcription, transcription and/or translation, replication or the like. Under these conditions, it may not be possible to select for a particular gene product activity, for example because the gene product may not be active under these conditions, or because the expression system contains an interfering activity.
- the selection is for optical changes in the genetic elements, the selection may be performed as follows:
- the genetic elements are sorted following pooling of the microcapsules into one or more common compartments.
- a gene product having the desired activity modifies the genetic element which encoded it (and which resides in the same microcapsule) so as to make it selectable as a result of its modified optical properties in a subsequent step.
- the reactions are stopped and the microcapsules are then broken so that all the contents of the individual microcapsules are pooled.
- the modification of the genetic element in the microcapsule may result directly in the modification of the optical properties of the genetic element. Alternatively, the modification may allow the genetic elements to be further modified outside the microcapsules so as to induce a change in their optical properties.
- the method therefore comprises expressing the genetic elements to produce their respective gene products within the microcapsules, linking the gene products to the genetic elements encoding them and isolating the complexes thereby formed.
- This allows for the genetic elements and their associated gene products to be isolated from the capsules before sorting according to gene product activity takes place.
- the complexes are subjected to a further compartmentalisation step prior to isolating the genetic elements encoding a gene product having the desired activity.
- This further compartmentalisation step which advantageously takes place in microcapsules, permits the performance of further reactions, under different conditions, in an environment where the genetic elements and their respective gene products are physically linked.
- Eventual sorting of genetic elements may be performed according to embodiment (V) above.
- the "secondary encapsulation" may also be performed with genetic elements linked to gene products by other means, such as by phage display, polysome display, RNA-peptide fusion or lac repressor peptide fusion, optionally where expression takes place prior to encapsulation; or even by the encapsulation of whole cells containing the desired genetic element.
- the selected genetic element(s) may also be subjected to subsequent, possibly more stringent rounds of sorting in iteratively repeated steps, reapplying the method described above either in its entirety or in selected steps only.
- genetic elements encoding gene products having a better optimised activity may be isolated after each round of selection.
- the genetic elements isolated after a first round of sorting may be subjected to mutagenesis before repeating the sorting by iterative repetition of the steps of the method of the invention as set out above. After each round of mutagenesis, some genetic elements will have been modified in such a way that the activity of the gene products is enhanced.
- the microcapsules may be sorted using microfluidic approaches.
- the microcapsules may be produced using microfluidic droplet formation techniques, such as those described herein, or by other techniques, for example conventional emulsification by forcing together two fluid phases. Sorting using microfluidics is applicable to embodiments I to VI above, and provides enhanced processing of microcapsules leading to improved sorting.
- Microcapsules may be split or fused according to methods described herein, or the contents thereof mixed. Moreover, the contents of the microcapsules may be analysed and the microcapsules sorted using detectors in microfluidic systems.
- the invention provides a product when selected according to the first aspect of the invention.
- a product may refer to a gene product, selectable according to the invention, or the genetic element (or genetic information comprised therein).
- the invention provides a method for preparing a gene product, the expression of which may result, directly or indirectly, in the modification the optical properties of a genetic element encoding it, comprising the steps of:
- steps (b) and (d) is performed under microfluidic control.
- step (a) preferably comprises preparing a repertoire of genetic elements, wherein each genetic element encodes a potentially differing gene product.
- Repertoires may be generated by conventional techniques, such as those employed for the generation of libraries intended for selection by methods such as phage display.
- Gene products having the desired activity may be selected from the repertoire, according to the present invention, according to their ability to modify the optical properties of the genetic elements in a manner which differs from that of other gene products. For example, desired gene products may modify the optical properties to a greater extent than other gene products, or to a lesser extent, including not at all.
- the invention provides a method for screening a compound or compounds capable of modulation the activity of a gene product, the expression of which may result, directly or indirectly, in the modification of the optical properties of a genetic element encoding it, comprising the steps of:
- steps (e) and (d) are performed under microfluidic control.
- the method further comprises the step of:
- This selection system can be configured to select for RNA, DNA or protein molecules with catalytic, regulatory or binding activity.
- FIGS. IA and IB illustrate the splitting of droplets in accordance with one embodiment of the invention
- FIGS. 2A and 2B illustrate an apparatus in accordance with an. embodiment of the invention, before the application of an electric field thereto;
- Figures 3 A and 3B illustrate the apparatus of Figs. 2A and 2B after the application of an electric field thereto;
- Figures 4A and 4B illustrate the apparatus of Figs. 2A and 2B after the application of a reversed electric field thereto;
- FIG. 5 is a schematic diagram of droplet splitting, in accordance with one embodiment of the invention.
- Figures 6 A and 6B are schematic diagrams of additional embodiments of the invention.
- FIGS. 7a and 7b are schematic diagrams of the formation of microfiuidic droplets in accordance with the present invention.
- FIGS 8a-f illustrate the splitting of droplets in accordance with the invention
- FIG. 9a-d illustrate the induction of dipoles in droplets in accordance with the invention.
- FIG 17 Charged droplet generation.
- A Oil and water streams converge at a 30 micron orifice.
- E indium-tin-oxide
- a “desired activity”, as referred to herein, is the modulation of any activity of a target, or an activity of a molecule which is influenced by the target, which is modulatable directly or indirectly by a genetic element or genetic elements as assayed herein.
- the activity of the target may be any measurable biological or chemical activity, including binding activity, an enzymatic activity, an activating or inhibitory activity on a third enzyme or other molecule, the ability to cause disease or influence metabolism or other functions, and the like.
- Activation and inhibition denote the increase or decrease of a desired activity 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 100 fold or more. Where the modulation is inactivation, the inactivation can be substantially complete inactivation.
- the desired activity may moreover be purely a binding activity, which may or may not involve the modulation of the activity of the target bound to.
- a “selectable change” is any change which can be measured and acted upon to identify or isolate the genetic element which causes it. The selection may take place at the level of the microcapsule, the microbead, or the genetic element itself, optionally when complexed with another reagent.
- a particularly advantageous embodiment is optical detection, in which the selectable change is a change in optical properties, which can be detected and acted upon for instance in a flow sorting device to separate microcapsules or microbeads displaying the desired change.
- a change in optical properties refers to any change in absorption or emission of electromagnetic radiation, including changes in absorbance, luminescence, phosphorescence or fluorescence. All such properties are included in the term "optical”.
- Microcapsules or microbeads can be identified and, optionally, sorted, for example, by luminescence, fluorescence or phosphorescence activated sorting.
- flow sorting is employed to identify and, optionally, sort microcapsules or microbeads.
- optical properties can be used for analysis and to trigger sorting, including light scattering (Kerker, 1983) and fluorescence polarisation (Rolland et al., 1985).
- microcapsules or on beads can be identified using a variety of techniques familiar to those skilled in the art, including mass spectroscopy, chemical tagging or optical tagging.
- microfluidic control refers to the use of a microfmidic system comprising microfluidic channels as defined herein to direct or otherwise control the formation and/or movement of microcapsules (or “droplets") in order to carry out the methods of the present invention.
- microfluidic control of microcapsule formation refers to the creation of microcapsules using a microfluidic device to form “droplets" of fluid within a second fluid, thus creating a microcapsule.
- Microcapsules sorted under microfluidic control are sorted, as described herein, using a microfluidic device to perform one or more of the functions associated with the sorting procedure.
- Microfluidic control of fluidic species therefore, refers to the handling of fluids in a microfluidic system as defined in order to carry out the methods of the present invention.
- a "cell” is given its ordinary meaning as used in biology.
- the cell may be any cell or cell type.
- the cell may be a bacterium or other single-cell organism, a plant cell, or an animal cell. If the cell is a single-cell organism, then the cell may be, for example, a protozoan, a trypanosome, an amoeba, a yeast cell, algae, etc.
- the cell may be, for example, an invertebrate cell (e.g., a cell from a fruit fly), a fish cell (e.g., a zebrafish cell), an amphibian cell (e.g., a frog cell), a reptile cell, a bird cell, or a mammalian cell such as a primate cell, a bovine cell, a horse cell, a porcine cell, a goat cell, a dog cell, a cat cell, or a cell from a rodent such as a rat or a mouse.
- the cell is from a multicellular organism, the cell may be from any part of the organism.
- the cell may be a cardiac cell, a fibroblast, a keratinocyte, a heptaocyte, a chondrocyte, a neural cell, a osteocyte, a muscle cell, a blood cell, an endothelial cell, an immune cell (e.g., a T-cell, a B-cell, a macrophage, a neutrophil, a basophil, a mast cell, an eosinophil), a stem cell, etc.
- the cell may be a genetically engineered cell.
- the cell may be a Chinese hamster ovarian ("CHO") cell or a 3T3 cell.
- Microfluidic refers to a device, apparatus or system including at least one fluid channel having a cross-sectional dimension of less than 1 mm, and a ratio of length to largest cross-sectional dimension of at least 3:1.
- a "microfluidic channel,” as used herein, is a channel meeting these criteria.
- the "cross-sectional dimension" of the channel is measured perpendicular to the direction of fluid flow.
- Most fluid channels in components of the invention have maximum cross- sectional dimensions less than 2 mm, and in some cases, less than 1 mm.
- all fluid channels containing embodiments of the invention are microfluidic or have a largest cross sectional dimension of no more than 2 mm or 1 mm.
- the fluid channels may be formed in part by a single component (e.g. an etched substrate or moulded unit).
- larger channels, tubes, chambers, reservoirs, etc. can be used to store fluids in bulk and to deliver fluids to components of the invention.
- the maximum cross-sectional dimension of the channel(s) containing embodiments of the invention are less than 500 microns, less than 200 microns, less than 100 microns, less than 50 microns, or less than 25 microns.
- a “channel,” as used herein, means a feature on or in an article (substrate) that at least partially directs the flow of a fluid.
- Th.e channel can have any cross-sectional shape (circular, oval, triangular, irregular, square or rectangular, or the like) and can be covered or uncovered. In embodiments where it is completely covered, at least one portion of the channel can have a cross-section that is completely enclosed, or the entire channel may be completely enclosed along its entire lengtfci with the exception of its inlet(s) and outlet(s).
- a channel may also have an aspect ratio (length to average cross sectional dimension) of at least 2:1, more typically at least 3:1, 5: 1, or 10:1 or more.
- An open channel generally will include characteristics that facilitate control over fluid transport, e.g., structural characteristics (an elongated indentation.) and/or physical or chemical characteristics (hydrophobicity vs. hydrophilicity) or other characteristics tt ⁇ at can exert a force (e.g., a containing force) on a fluid.
- the fluid within the channel may partially or completely fill the channel, hi some cases where an open channel is used, the fluid may be held within the channel, for example, using surface tension (i.e., a concave or convex meniscus).
- the channel may be of any size, for example, having a largest dimension perpendicular to fluid flow of less than about 5 mm or 2 mm, or less than about 1 mm, or less than about 500 microns, less than about 200 microns, less than about 1OO microns, less than about 60 microns, less than about 50 microns, less than about 40 microns, less than about 30 microns, less than about 25 microns, less than about 10 microns, less than about 3 microns, less than about 1 micron, less than about 300 nm, less than about 100 ran, less than about 30 nm, or less than about 10 nm.
- ttxe dimensions of the channel may be chosen such that fluid is able to freely flow through the article or substrate.
- the dimensions of the channel may also be chosen, for example, to allow a certain volumetric or linear flowrate of fluid in the channel.
- the number of channels and the shape of the channels can be varied by any method known to those of ordinary skill in the art.
- more than one channel or capillary may be used.
- two or more channels may be used, where they are positioned inside each other, positioned adjacent to each other, positioned to intersect with each other, etc.
- integral means that portions of components are joined in such a way that they cannot be separated from each other without cutting or breaking the components from each other.
- the fluids need not be as immiscible.
- Those of ordinary skill in the art can select suitable miscible or immiscible dfluids, using contact angle measurements or the like, to carry out the techniques of the invemtion.
- hydrophobic liquid and a hydrophiLic liquid are substantially immiscible with respect to each other, where the hydrophilic liquid has a greater affinity to water than does the hydrophobic liquid.
- hydrophilic liquids include, but are not limited to, water and other aqueous solutions comprising water, such as cell or biological media, ethanol, salt solutions, etc.
- hydrophobic liquids include, but are not limited to, oils such as hydrocarbons, silicon oils, fluorocarbon oils, organic solvents etc.
- determining generally refers to the analysis or measurement of a species, for example, quantitatively or qualitatively, or the detection of the presence or absence of the species. “Determining” may also refer to the analysis or measurement of an interaction between two or more species, for example, quantitatively or qualitatively, or by detecting the presence or absence of the interaction.
- microcapsules of the present invention require appropriate physical properties to allow the working of the invention.
- the contents of each microcapsule are preferably isolated from the contents of the surrounding microcapsules, so that there is no or little exchange of the genetic elements and gene products between the microcapsules over the timescale of the experiment.
- the permeability of the microcapsules may be adjusted such that reagents maybe allowed to diffuse into and/or out of the microcapsules if desired.
- the method of the present invention requires that there are only a limited number of genetic elements per microcapsule. This ensures that the gene product of an individual genetic element will be isolated from other genetic elements. Thus, coupling between genetic element and gene product will be highly specific. The enrichment factor is greatest with on average one or fewer genetic elements per microcapsule, the linkage between nucleic acid and the activity of the encoded gene product being as tight as is possible, since the gene product of an individual genetic element will be isolated from the products of all other genetic elements.
- a ratio of 5, 10, 50, 100 or 1000 or more genetic elements per microcapsule may prove beneficial in sorting a large library. Subsequent rounds of sorting, including renewed encapsulation with differing genetic element distribution, will permit more stringent sorting of the genetic elements.
- the formation and the composition of the microcapsules advantageously does not abolish the function of the machinery the expression of the genetic elements and the activity of the gene products.
- any microencapsulation system used preferably fulfils these three requirements.
- the appropriate system(s) may vary depending on the precise nature of the requirements in each application of the invention, as will be apparent to the skilled person.
- Microcapsules can also be generated by interfacial polymerisation and interfacial complexation (Whateley, 1996). Microcapsules of this sort can have rigid, nonpermeable membranes, or semipermeable membranes. Semipermeable microcapsules bordered by cellulose nitrate membranes, polyamide membranes and lipid-polyamide membranes can all support biochemical reactions, including multienzyme systems (Chang, 1987; Chang,
- Non-membranous microencapsulation systems based on phase partitioning of an aqueous environment in a colloidal system, such as an emulsion, may also be used.
- the microcapsules of the present invention are formed from emulsions; heterogeneous systems of two immiscible liquid phases with one of the phases dispersed in the other as droplets of microscopic or colloidal size (Becher, 1957; Sherman, 1968; Lissant, 1974; Lissant, 1984).
- Emulsions may be produced from any suitable combination of immiscible liquids.
- the emulsion of the present invention has "water” (an aqueous liquid containing the biochemical components) as the phase present in the form of finely divided droplets (the disperse, internal or discontinuous phase) and a hydrophobic, immiscible liquid (an 'oil') as the matrix in which these droplets are suspended (the nondisperse, continuous or external phase).
- water an aqueous liquid containing the biochemical components
- an 'oil' hydrophobic, immiscible liquid
- W/O water-in-oiP
- the emulsion may be stabilised by addition of one or more surface-active agents (surfactants). These surfactants are termed emulsifying agents and act at the water/oil interface to prevent (or at least delay) separation of the phases.
- surfactants are termed emulsifying agents and act at the water/oil interface to prevent (or at least delay) separation of the phases.
- Many oils and many emulsifiers can be used for the generation of water-in-oil emulsions; a recent compilation listed over 16,000 surfactants, many of which are used as emulsifying agents (Ash and Ash, 1993). Suitable oils include light white mineral oil and decane.
- stirrers such as magnetic stir-bars, propeller and turbine stirrers, paddle devices and whisks
- homogenisers including rotor-stator homogenisers, high-pressure valve homogenisers and jet homogenisers
- colloid mills ultrasound and 'membrane emulsification' devices (Becher, 1957; Dickinson, 1994), and microfluidic devices (Umbanhowar et al., 2000).
- Complicated biochemical processes notably gene transcription and translation are also active in aqueous microcapsules formed in water-in-oil emulsions. This has enabled compartmentalisation in water-in-oil emulsions to be used for the selection of genes, which are transcribed and translated in emulsion microcapsules and selected by the binding or catalytic activities of the proteins they encode (Doi and Yanagawa, 1999;
- aqueous microcapsules formed in the emulsion were generally stable with little if any exchange of nucleic acids, proteins, or the products of enzyme catalysed reactions between microcapsules.
- the preferred microcapsule size will vary depending upon the precise requirements of any individual selection process that is to be performed according to the present invention. In all cases, there will be an optimal balance between gene library size, the required enrichment and the required concentration of components in the individual microcapsules to achieve efficient expression and reactivity of the gene products.
- the processes of expression occurs within each individual microcapsule provided by the present invention. Both in vitro transcription and coupled transcription-translation become less efficient at sub-nanomolar DNA concentrations. Because of the requirement for only a limited number of DNA molecules to be present in each microcapsule, this therefore sets a practical upper limit on the possible microcapsule size.
- the mean volume of the microcapsules is less that 5.2 x 10 ⁇ 16 m 3, (corresponding to a spherical microcapsule of diameter less than lO ⁇ m, more preferably less than 6.5 x 10 ⁇ 17 m ⁇ (5 ⁇ m diameter), more preferably about 4.2 x 10" ⁇ m ⁇ (2 ⁇ m diameter) and ideally about 9 x 10" I ⁇ m ⁇ (2.6 ⁇ m diameter).
- thermostable for example, the coupled transcription-translation systems can be made from a thermostable organism such as Thermus aquaticus).
- microcapsule volume 5.2 x 10 "16 m3 (corresponding to a sphere of diameter lO ⁇ m).
- RNA molecules of nucleoside triphosphate per microcapsule 8.33 x 10 ⁇ 22 moles.
- this number of molecules is contained within a microcapsule of volume 4.17 x 10" ⁇ litres (4.17 x 10 ⁇ 22 m ⁇ which if spherical would have a diameter of 93nm.
- the ribosomes necessary for the translation to occur are themselves approximately 20nm in diameter.
- the preferred lower limit for microcapsules is a diameter of approximately O.l ⁇ m (lOOnm).
- compartments droplets of 2.6 ⁇ m mean diameter
- Escherichia are 1.1-1.5 x 2.0-6.0 ⁇ m rods
- Azotobacter are 1.5-2.0 ⁇ m diameter ovoid cells.
- Darwinian evolution is based on a 'one genotype one phenotype' mechanism.
- the concentration of a single compartmentalised gene, or genome drops from 0.4 nM in a compartment of 2 ⁇ m diameter, to 25 pM in a compartment of 5 ⁇ m diameter.
- the prokaryotic transcription/translation machinery has evolved to operate in compartments of ⁇ l-2 ⁇ m diameter, where single genes are at approximately nanomolar concentrations.
- the size of emulsion microcapsules may be varied simply by tailoring the emulsion conditions used to form the emulsion according to requirements of the selection system.
- the size of the microcapsules is selected not only having regard to the requirements of the transcription/translation system, but also those of the selection system employed for the genetic element.
- the components of the selection system such as a chemical modification system, may require reaction volumes and/or reagent concentrations which are not optimal for transcription/translation.
- such requirements may be accommodated by a secondary re-encapsulation step; moreover, they may be accommodated by selecting the microcapsule size in order to maximise transcription/translation and selection as a whole.
- Empirical determination of optimal microcapsule volume and reagent concentration is preferred.
- the polypeptide or other molecular group or construct is a ligand or a. substrate which directly or indirectly binds to or reacts with the gene product in order to alter the optical properties of the genetic element. This allows the sorting of the genetic element on the basis of the activity of the gene product.
- the ligand or substrate can be connected to the nucleic acid by a variety of means that will be apparent to those skilled in the art (see, for example, Hermanson, 1996).
- a biotinylated nucleic acid may be coupled to avidin or streptavidin complexed to a large protein molecule such as thyxoglobulin (669 Kd) or ferritin (440 Kd).
- This complex can be derivatised with substrate or ligand, for example by covalent coupling to the E-amino group of lysines or througli a non-covalent interaction such as biotin-avidin.
- an alternative is to couple the nucleic acid to a product-specific antibody (or other product-specific molecule).
- the sixbstrate (or one of the substrates) is present in each microcapsule unlinked to the genetic element, but has a molecular "tag” (for example biotin, DIG or DNP or a fluorescent group).
- a molecular "tag” for example biotin, DIG or DNP or a fluorescent group.
- Mutations may be introduced into the genetic elements(s) as set forth above.
- a repertoire size of at least K)H can be selected using ImI aqueous phase in a 20 ml emulsion.
- the internal environment of a microcapsule may be altered by addition of reagents to the oil phase of the emulsion.
- the reagents diffuse through the oil phase to the aqueous microcapsule environment.
- the reagents are at least partly water-soluble, such that a proportion thereof is distributed from the oil phase to the aqueous microcapsule environment.
- the reagents are substantially insoluble in the oil phase.
- Reagents are preferably mixed into the oil phase by mechanical mixing, for example vortexing.
- the reagents which may be added via the oil phase include substrates, buffering components, factors and the like.
- the internal pH of microcapsules may be altered in situ by adding acidic or basic components to the oil phase.
- the invention relates to a method for optimising a production process which involves at least one step which is facilitated by a polypeptide.
- the step may be a catalytic step, which is facilitated by an enzyme.
- the invention provides a method for preparing a compound or compounds comprising the steps of:
- Water-in-oil emulsions can be re-emulsified to create water-in-oil-in water double emulsions with an external (continuous) aqueous phase. These double emulsions can be analysed and, optionally, sorted using a flow cytometer (Bernath et al., 2004).
- Electric charge may be created in the fluid within the liquid using any suitable technique, for example, by placing the fluid within an electric field (which may be AC, DC, etc.), and/or causing a reaction to occur that causes the fluid to have an electric charge, for example, a chemical reaction, an ionic reaction, a photocatalyzed reaction, etc.
- the fluid is an electrical conductor.
- a "conductor" is a material having a conductivity of at least about the conductivity of 18 megohm (MOhm or M ⁇ ) water.
- the liquid surrounding the fluid may have a conductivity less than that of the fluid.
- the liquid may be an insulator, relative to the fluid, or at least a "leaky insulator,” i.e., the liquid is able to at least partially electrically insulate the fluid for at least a short period of time.
- the fluid may be substantially hydrophilic, and the liquid surrounding the fluid may be substantially hydrophobic.
- the charge created on the fluid maybe at least about 10 "22 C/micrometer 3 .
- the charge may be at least about 10 "21 C/micrometer 3 , and in other cases, the charge maybe at least about 10 "20 C/micrometer 3 , at least about 10 "19 C/micrometer 3 , at least about 10 "18 C/micrometer 3 , at least about 10 "17 C/micrometer 3 , at least about 10 ⁇ 16 C/micrometer 3 , at least about lO "15 C/micrometer 3 , at least about 10 "14 C/micrometer 3 , at least about 10 ⁇ 13 C/micrometer 3 , at least about 10 ⁇ 12 C/micrometer 3 , at least about ICT 11 C/micrometer 3 , at least about 10 "10 C/micrometer 3 , or at least about 1O '9 C/micrometer 3 or more.
- the charge created on the fluid may be at least about 10 ⁇ 21 C/micrometer 2 , and in some cases, the charge may be at least about 10 "20 C/micrometer 2 , at least about 10 "19 C/micrometer 2 , at least about 10 "18 C/micrometer 2 , at least about 10 '17 C/micrometer 2 , at least about 10 "16 C/micrometer 2 , at least about 10 "15 C/micrometer 2 , at least about 10 "14 C/micrometer 2 , or at least about 10 "13 C/micrometer 2 or more.
- the charge may be at least about 10 "14 C/droplet, and, in some cases, at least about 10 "13 C/droplet, in other cases at least about 10 "12 C/droplet, in other cases at least about 10 "11 C/droplet, in other cases at least about 10 "10 C/droplet, or in still other cases at least about 10 "9 C/droplet.
- the electric field is generated from an electric field generator, i.e., a device or system able to create an electric field that can be applied to the fluid.
- the electric field generator may produce an AC field (i.e., one that varies periodically with respect to time, for example, sinusoidally, sawtooth, square, etc.), a DC field (i.e., one that is constant with respect to time), a pulsed field, etc.
- the electric field generator may be constructed and arranged to create an electric field within a fluid contained within a channel or a microfluidic channel.
- the electric field generator may be integral to or separate from the fluidic system containing the channel or microfluidic channel, according to some embodiments.
- integral means that portions of the components integral to each other are joined in such a way that the components cannot be manually separated from each other without cutting or breaking at least one of the components.
- an electric field is produced by applying voltage across a pair of electrodes, which may be positioned on or embedded within the fluidic system (for example, within a substrate defining the channel or microfluidic channel), and/or positioned proximate the fluid such that at least a portion of the electric field interacts with the fluid.
- the electrodes can be fashioned from any suitable electrode material or materials known to those of ordinary skill in the art, including, but not limited to, silver, gold, copper, carbon, platinum, copper, tungsten, tin, cadmium, nickel, indium tin oxide (“ITO”), etc., as well as combinations thereof.
- the electric field generator can be constructed and arranged (e.g., positioned) to create an electric field applicable to the fluid of at least about 0.01 V/micrometer, and, in some cases, at least about 0.03 V/micrometer, at least about 0.05 V/micrometer, at least about 0.08 V/micrometer, at least about 0.1 V/micrometer, at least about 0.3 V/micrometer, at least about 0.5 V/micrometer, at least about 0.7 V/micrometer, at least about 1 V/micrometer, at least about 1.2 V/micrometer, at least about 1.4 V/micrometer, at least about 1.6 V/micrometer, or at least about 2 V/micrometer.
- even higher electric field intensities may be used, for example, at least about 2 V/micrometer, at least about 3 V/micrometer, at least about 5 V/micrometer, at least about 7 V/micrometer, or at least about 10 V/micrometer or more.
- an electric field may be applied to fluidic droplets to cause the droplets to experience an electric force.
- the electric force exerted on the fluidic droplets may be, in some cases, at least about 10 '16 N/micrometer 3 . In certain cases, the electric force exerted on the fluidic droplets may be greater, e.g., at .
- the electric force exerted on the fluidic droplets, relative to the surface area of the fluid may be at least about 10 "15 N/micrometer 2 , and in some cases, at least about 10 "14 N/micrometer 2 , at least about 10 '13 N/micrometer 2 , at least about 10 "12 N/micrometer 2 , at least about 10 "11 N/micrometer 2 , at least about 10 "10 N/micrometer 2 , at least about 10 "9 N/micrometer 2 , at least about 10 "8 N/micrometer 2 , at least about 10 '7 N/micrometer 2 , or at least about 10 "6 N/micrometer 2 or more.
- the electric force exerted on the fluidic droplets may be at least about 10 "9 N, at least about 10 "8 N, at least about 10 '7 N 5 at least about 10 '6 N, at least about 10 "5 N, or at least about 10 '4 N or more in some cases.
- systems and methods are provided for at least partially neutralizing an electric charge present on a fluidic droplet, for example, a fluidic droplet having an electric charge, as described above.
- the fluidic droplet may be passed through an electric field and/or brought near an electrode, e.g., using techniques such as those described herein.
- the fluidic droplet may become electrically neutralized, and/or have a reduced electric charge.
- Channel 5 IO narrows at location 501, causing fluid 500 to form a series of individual fluidic droplets 515.
- internal obstructions may also be used to cause droplet formation to occur.
- baffles, ridges, posts, or the like may be used to disrupt liquid flow in a manner that causes the fluid to coalesce into fluidic droplets.
- the channel dimensions may be altered with respect to time (for example, mechanically or electromechanically, pneumatically, etc.) in such a manner as to cause the formation of individual fluidic droplets to occur.
- the channel may be mechanically contracted ("squeezed") to cause droplet formation, or a fluid stream may be mechanically disrupted to cause droplet formation, for example, through the use of moving baffles, rotating blades, or the like.
- fluid 500 flows through channel 510 in a downward direction. Fluid 500 is surrounded by liquid 505. Piezoelectric devices 520 positioned near or integral to channel 510 may then mechanically constrict or "squeeze" channel 510, causing fluid 500 to break up into individual fluidic droplets 515.
- individual fluidic droplets can be created and maintained in a system comprising three essentially mutually immiscible fluids (i.e., immiscible on a time scale of interest), where one fluid is a liquid carrier, and the second fluid and the third fluid alternate as individual fluidic droplets within the liquid carrier.
- surfactants are not necessarily required to ensure separation of the fluidic droplets of the second and third fluids.
- a first fluid 701 and a second fluid 702 are each carried within liquid carrier 705.
- First fluid 701 and second fluid 702 alternate as a series of alternating, individual droplets, each carried by liquid carrier 705 within channel 700.
- any two of the fluids can come into contact without causing droplet coalescence to occur.
- a photomicrograph of an example of such a system is shown in Fig. 14F3, illustrating first fluid 701 and second fluid 702, present as individual, alternating droplets, each contained within liquid carrier 705.
- a system involving three essentially mutually immiscible fluids is a silicone oil, a mineral oil, and an aqueous solution (i.e., water, or water containing one or more other species that are dissolved and/or suspended therein, for example, a salt solution, a saline solution, a suspension of water containing particles or cells, or the like).
- aqueous solution i.e., water, or water containing one or more other species that are dissolved and/or suspended therein, for example, a salt solution, a saline solution, a suspension of water containing particles or cells, or the like.
- Another example of a system is a silicone oil, a fluorocarbon oil, and an aqueous solution.
- the fluidic droplets may each be substantially the same shape and/or size.
- the shape and/or size can be determined, for example, by measuring the average diameter or other characteristic dimension of the droplets.
- determining generally refers to the analysis or measurement of a species, for example, quantitatively or qualitatively, and/or the detection of the presence or absence of the species. “Determining” may also refer to the analysis or measurement of an interaction between two or more species, for example, quantitatively or qualitatively, or by detecting the presence or absence of the interaction. Examples of suitable techniques .
- the invention provides for the production of droplets consisting essentially of a substantially uniform number of entities of a species therein (i.e., molecules, compounds, cells, genetic elements, particles, etc.). For example, about 90%, about 93%, about 95%, about 97%, about 98%, or about 99%, or more of a plurality or series of droplets may each contain the same number of entities of a particular species.
- the droplets of fluid in a liquid containing droplets of fluid, some of which contain a species of interest and some of which do not contain the species of interest, the droplets of fluid may be screened or sorted for those droplets of fluid containing the species as further described below (e.g., using fluorescence or other techniques such as those described above), and in some cases, the droplets may be screened or sorted for those droplets of fluid containing a particular number or range of entities of the species of interest, e.g., as previously described.
- a plurality or series of fluidic droplets may be enriched (or depleted) in the ratio of droplets that do contain the species, for example, by a factor of at least about 2, at least about 3, at least about 5, at least about 10, at least about 15, at least about 20, at least about 50, at least about 100, at least about 125, at least about 150, at least about 200, at least about 250, at least about 500, at least about 750, at least about 1000, at least about 2000, or at least about 5000 or more in some cases.
- the enrichment may be in a ratio of at least about 10 4 , at least about 10 5 , at least about 10 6 , at least about 10 7 , at least about 10 8 , at least about 10 9 , at least about 10 10 , at least about 10 ⁇ , at least about 10 12 , at least about 10 13 , at least about 10 14 , at least about 10 15 , or more.
- a fluidic droplet containing a particular species may be selected from a library of fluidic droplets containing various species, where the library may have about 10 5 , about 10 6 , about 10 7 , about 10 8 , about 10 9 , about 10 10 , about l ⁇ ", about 10 12 , about 10 13 , about 10 14 , about 10 15 , or more items, for example, a DNA library, an RNA library, a protein library, a combinatorial chemistry library, a library of genetic elements, etc.
- the droplets carrying the species may then be fused, reacted, or otherwise used or processed, etc., as further described below, for example, to initiate or determine a reaction.
- the use of microfluidic handling to create microcapsoules according to the invention has a number of advantages:
- fluidic droplets 215 contained in channel 230 are carried by a surrounding liquid, which flows towards intersection 240, leading to channels 250 and 255.
- the surrounding liquid flows through channels 250 and 255 at equal flowrates.
- fluidic droplets 215 do not have a preferred orientation or direction, and move into exit channels 250 and 255 with equal probability due to the surrounding liquid flow.
- fluidic droplets 215 are split into two droplets at intersection 240, forming new droplets 216 and 217. Droplet 216 moves to the left in channel 250, while droplet 217 moves to the right in channel 255.
- Microcapsules can be split into two or more smaJler microdroplets allowing the reagents contained therein to be reacted with a series o f different molecules in parallel or assayed in multiplicate.
- Reagents can be mixed very rapidly (in ⁇ 2 ms) in microcapsules using chaotic advection, allowing fast kinetic measurements and very high throughput.
- Stable streams of microcapsules can be formed in microchannels and identified by their relative positions.
- control of the flow of liquids in microfluidic systems is not used to direct the flow of fluidic droplets therein, but that an alternative method is used.
- the microcapsules are not sorted by altering the direction of the flow of a carrier fluid in a microfluidic system.
- fluidic droplet 600 is surrounded by a liquid 605 in channel 610.
- Channel 610 divides into channels 611, 612.
- liquid reservoirs 617 and 618 Positioned in fluidic communication with channels 611 and 612 are liquid reservoirs 617 and 618, which may be expanded and/or contracted, for instance, by piezoelectric components 615 and 616, by a piston (not shown), etc.
- liquid reservoir 617 has been expanded, while liquid reservoir 618 has been contracted.
- the effect of the expansion/contractions of the reservoirs is to cause a net flow of liquid towards channel 611, as indicated by arrows 603.
- fluidic droplet 600 upon reaching the junction between the channels, is directed to channel 611 by the movement of liquid 605.
- a fluidic droplet may be sorted and/or split into 5 two or more separate droplets, for example, depending on the particular application. Any of the above-described techniques may be used to spilt and/or sort droplets. As a non- limiting example, by applying (or removing) a first electric field to a device (or a portion thereof), a fluidic droplet may be directed to a first region or channel; by applying (or removing) a second electric field to the device (or a portion thereof), the droplet may be 0 directed to a second region or channel; by applying a third electric field to the device (or a portion thereof), the droplet may be directed to a third region or channel; etc., where the electric fields may differ in some way, for example, in intensity, direction, frequency, duration, etc.
- each droplet may be independently sorted and/or split; for example, some droplets may be directed to one location or another, while other 5 droplets may be split into multiple drop
- fluidic droplet 550, surrounding liquid 555 in channel 560 may be directed to channel 556, channel 557, or be split in some fashion between channels 562 and 564.
- fluidic droplet 550 may be directed towards the left into channel 562; in Fig. O 8C, by directing surrounding liquid 555 towards channel 564, fluidic droplet 550 may be directed towards the right into channel 564,
- an electric field may be applied, in combination with control of the flow of liquid 555 surrounding fluidic droplet 550, that causes the droplet to impact junction 561, which may cause the droplet to split into two separate fluidic droplets 565, 566.
- Fluidic droplet 565 is directed to channel 562, while fluidic droplet 566 is directed to channel 566.
- a high degree of control of the applied electric field may be achieved to control droplet formation; thus, for example, after fluidic droplet 565 has been split into droplets 565 and 566, droplets 565 and 566 may be of substantially equal size, or either of droplets 565 and 566 may be larger, e.g., as is shown in Figs. 8E and 8F, respectively.
- channel 540 carrying fluidic droplet 530 and liquid 535, divides into cliannel 542 and 544.
- Fluidic droplet 530 may be electrically charged, or it may uncharged.
- Electrode 526 is positioned near channel 542, while electrode 527 is positioned near channel 544.
- Electrode 528 is positioned near the junction of channels 540, 542, and 544.
- fluidic droplet 530 When fluidic droplet 530 reaches the junction, it may be subjected to an electric field, and/or directed to a channel or other region, for example, by directing the surrounding liquid into the channel. As shown in Fig.
- fluidic droplet 530 may be split into two separate droplets 565 and 566 by applying an electric field 525 to the droplet using electrodes 526 and 527.
- a dipole can be induced in droplet 530 by applying an electric field 525 to the droplet using electrodes 527 and 528. Due to the strength of the applied electric field, the droplet maybe strongly attracted to the right, into channel 544.
- a dipole may be induced in droplet 530 by applying an electric field 525 to the droplet using electrodes 526 and 528, causing the droplet to be attracted into channel 542.
- one or more fluidic droplets within channel 540 may be sorted and/or split into two droplets, and each droplet may independently be sorted and/or split.
- the system can be configured to select for RNA, DNA or protein gene product molecules with catalytic, regulatory or binding activity.
- the genetic element may be linked to the gene product in the microcapsule via the ligand. Only gene products with affinity for the ligand will therefore bind to the genetic element and only those genetic elements with gene product bound via the ligand will acquire the changed optical properties which enable them to be retained in the selection step.
- the genetic element will thus comprise a nucleic acid encoding the gene product linked to a ligand for the gene product.
- the change in optical properties of the genetic element after binding of the gene product to the ligand may be induced in a variety of ways, including:
- the optical properties of the ligand may be modified on binding of the gene product, for example, the fluorescence of the ligand is quenched or enhanced on binding (Voss, 1993; Masui and Kuramitsu, 1998).
- the optical properties of both ligand and gene product are modified on binding, for example, there can be a fluorescence resonance energy transfer (FRET) from ligand to gene product (or vice versa) resulting in emmission at the "acceptor” emmission wavelength when excitation is at the "donor” absoption wavelength (Heim & Tsien, 1996; Mahajan et al, 1998; Miyawaki et al, 1997).
- FRET fluorescence resonance energy transfer
- the invention provides a method according to the first aspect of the invention, wherein in step (b) the gene products bind to genetic elements encoding them.
- the gene products together with the attached genetic elements are then sorted as a result of binding of a ligand to gene products having the desired binding activity.
- all gene products can contain an invariant region which binds covalently or non-covalently to the genetic element, and a second region which is diversified so as to generate the desired binding activity.
- the ligand for the gene product is itself encoded by the genetic element and binds to the genetic element.
- the genetic element encodes two (or indeed more) gene products, at least one of which binds to the genetic element, and which can potentially bind each other. Only when the gene products interact in a microcapsule is the genetic element modified in a way that ultimately results in a change in a change in its optical properties that enables it to be sorted.
- This embodiment for example, isused to search gene libraries for pairs of genes encoding pairs of proteins which bind each other.
- TSA Tyramide Signal Amplification
- TSA may be configured such that it results in a direct increase in the fluorescence of the genetic element, or such that a ligand is attached to the genetic element which is bound by a second fluorescent molecule, or a sequence of molecules, one or more of which is fluorescent.
- the genetic element in each microcapsule may comprise the substrate of the reaction. If the genetic element encodes a gene product capable of acting as a catalyst, the gene product will catalyse the conversion of the substrate into the product. Therefore, at the end of the reaction the genetic element is physically linked to the product of the catalysed reaction.
- the substrate may also be desirable, in some cases, for the substrate not to be a component of the genetic element.
- the substrate would contain an inactive "tag:” that requires a further step to activate it such as photoactivation (e.g. of a "caged" biotin analogue, (Sundberg et al, 1995; Pirrung and Huang, 1996)).
- the catalyst to be selected then converts the substrate to product.
- the "tag” is then activated and the "tagged" substrate and/or product bound by a tag-binding molecule (e.g. avidin or streptavidin) complexed with the nucleic acid.
- a tag-binding molecule e.g. avidin or streptavidin
- optical properties of genetic elements with product attached and which encode gene products with the desired catalytic activity can be modified by either:
- the substrate and product having different optical properties are available commercially (see for example Haugland, 1996) including substrates for glycosidases, phosphatases, peptidases and proteases (Craig et al., 1995; Huang et al, 1992; Brynes et al, 1982; Jones et al., 1997; Matayoshi et al., 1990; Wang et al., 1990)), or
- (b) optionally bind both substrate and product if only the product, and not the substrate binds to, or reacts with, the genetic element.
- the pooled genetic elements encoding catalytic molecules can then be enriched by selecting for the genetic elements with modified optical properties.
- nucleic acid to a product-specific antibody (or other product-specific molecule).
- the substrate or one of the substrates
- the substrate is present in each microcapsule unlinked to the genetic element, but has a molecular "tag"
- selection may be performed indirectly by coupling a first reaction to subsequent reactions that takes place in the same microcapsule.
- the product of the first reaction is reacted with, or bound by, a molecule which does not react with the substrate of the first reaction.
- a second, coupled reaction will only proceed in the presence of the product of the first reaction.
- a genetic element encoding a gene product with a desired activity can then be purified by using the properties of the product of the second reaction to induce a change in the optical properties of the genetic element as above.
- the product of the reaction being selected may be the substrate or cofactor for a second enzyme-catalysed reaction.
- the enzyme to catalyse the second reaction can either be translated in situ in the microcapsules or incorporated in the reaction mixture prior to microencapsulation. Only when the first reaction proceeds will the coupled enzyme generate a product which can be used to induce a change in the optical properties of the genetic element as above.
- This concept of coupling can be elaborated to incorporate multiple enzymes, each using as a substrate the product of the previous reaction. This allows for selection of enzymes that will not react with an immobilised substrate. It can also be designed to give increased sensitivity by signal amplification if a product of one reaction is a catalyst or a cofactor for a second reaction or series of reactions leading to a selectable product (for example, see Johannsson and Bates, 1988; Johannsson, 1991). Furthermore an enzyme cascade system can be based on the production of an activator for an enzyme or the destruction of an enzyme inhibitor (see Mize et al, 1989). Coupling also has the advantage that a common selection system can be used for a whole group of enzymes which generate the same product and allows for the selection of complicated chemical transformations that cannot be performed in a single step.
- Such a method of coupling thus enables the evolution of novel "metabolic pathways" in vitro in a stepwise fashion, selecting and improving first one step and then the next.
- the selection strategy is based on the final product of the pathway, so that all earlier steps can be evolved independently or sequentially without setting up a new selection system for each step of the reaction.
- Genetic elements encoding enzymes with substrate specificity or selectivity can be specifically enriched by carrying out a positive selection for reaction with one substrate and a negative selection for reaction with another substrate.
- Such combined positive and negative selection pressure should be of great importance in isolating regio-selective and stereo-selective enzymes (for example, enzymes that can distinguish between two enantiomers of the same substrate).
- two substrates e.g. two different enantiomers
- tags e.g. two different fluorophores
- the substrate specificity of the enzyme can be determined from the optical properties of the genetic element and those genetic elements encoding gene products with the wrong (or no) specificity rejected.
- Tags conferring no change in optical activity can also be used if tag- specific ligands with different optical properties are added (e.g. tag-specific antibodies labelled with different fluorophores).
- a similar system can be used to select for regulatory properties of enzymes.
- the components of the biochemical process can either be translated in situ in each microcapsule or can be incorporated in the reaction mixture prior to microencapsulation.
- the genetic element being selected is to encode an activator, selection can be performed for the product of the regulated reaction, as described above in connection with catalysis. If an inhibitor is desired, selection can be for a chemical property specific to the substrate of the regulated reaction.
- the gene product binds back to the genetic element, for example through a common element of the gene product which binds to a ligand which is part of the genetic element. After pooling the genetic elements they can then be sorted using the optical properties of the bound gene products.
- This embodiment can be used, for example, to select variants of green fluorescent protein (GFP) (Cormack et ah, 1996; Delagrave et al, 1995; Ehrig et ah, 1995), with improved fluorescence and/or novel absoption and emmission spectra.
- GFP green fluorescent protein
- HTS in vitro Mgh-throughput screening
- the effect of a compound(s) on a target can be determined by compartmentalising a cell (or cells) in a microcapsule together with a genetic element(s) and using an appropriate cell-based assay to identify those compartments containing genetic elements with the desired effect on the cell(s).
- a cell or cells
- an appropriate cell-based assay to identify those compartments containing genetic elements with the desired effect on the cell(s).
- the use of water-in-fluorocarbon emulsions may be particularly advantageous: the high gas dissolving capacity of fluorocarbons can support the exchange of respiratory gases and has been reported to be beneficial to cell culture systems (Lowe, 2002) .
- microcapsules will be analysed and, optionally, sorted by flow cytometry. Many formats of microcapsule can be analysed and, optionally, sorted directly using flow cytometry.
- microfluidic devices for flow analysis and, optionally, flow sorting (Fu, 2002) of microcapsules will be used.
- a sorting device can be integrated directly on the microfluidic device, and can use electronic means to sort the microcapsules and/or genetic elements.
- Optical detection also integrated directly on the microfluidic device, can be used to screen the microcapsules to trigger the sorting.
- Other means of control of the microcapsules, in addition to charge, can also be incorporated onto the microfluidic device.
- optical properties can be used for analysis and to trigger sorting, including light scattering (Kerker, 1983) and fluorescence polarisation (Rolland et al., 1985).
- the difference in optical properties of the microcapsules or microbeads will be a difference in fluorescence and, if required, the microcapsules or microbeads will be sorted using a microfluidic or conventional fluorescence activated cell sorter (Norman, 1980; Mackenzie and Pinder, 1986), or similar device.
- Flow cytometry has a series of advantages:
- fluorescence activated cell sorting equipment from established manufacturers (e.g. Becton-Dickinson, Coulter, Cytomation) allows the analysis and sorting at up to 100,000 microcapsules or microbeads per second.
- the fluorescence signal from each microcapsule or microbead corresponds tightly to the number of fluorescent molecules present. As little as few hundred fluorescent molecules per microcapsules or microbeads can be quantitatively detected;
- the wide dynamic range of the fluorescence detectors (typically 4 log units) allows easy setting of the stringency of the sorting procedure, thus allowing the recovery of the optimal number microcapsules or microbeads from the starting pool (the gates can be set to separate microcapsules or microbeads with small differences in fluorescence or to only separate out microcapsules or microbeads with large differences in fluorescence, dependant on the selection being performed);
- fluorescence-activated cell sorting equipment can perform simultaneous excitation and detection at multiple wavelengths (Shapiro, 1995). allowing positive and negative selections to be performed simultaneously by monitoring the labelling of the microcapsules or microbeads with two to thirteen (or more) fluorescent markers, for example, if substrates for two alternative targets are labelled with different fluorescent tags the microcapsules or microbeads can labelled with different fluorophores dependent on the target regulated.
- microcapsules or microbeads are optically tagged, flow cytometry can also be used to identify the genetic element or genetic elements in the microcapsule or coated on the microbeads (see below).
- Optical tagging can also be used to identify the concentration of reagents in the microcapsule (if more than one concentration is used in a single experiment) or the number of compound molecules coated on a microbead (if more than one coating density is used in a single experiment).
- optical tagging can be used to identify the target in a microcapsule (if more than one target is used in a single experiment). This analysis can be performed simultaneously with measuring activity, after sorting of microcapsules containing rnicrobeads, or after sorting of the microbeads.
- the invention provides for the identification and, optionally, the sorting of intact microcapsules where this is enabled by the sorting techniques being employed.
- Microcapsules may be identified and, optionally, sorted as such when the change induced by the desired genetic element either occurs or manifests itself at the surface of the microcapsule or is detectable from outside the microcapsule.
- the change may be caused by the direct action of the gene product, or indirect, in which a series of reactions, one or more of which involve the gene product having the desired activity leads to the change.
- the microcapsule may be so configured that a component or components of the biochemical system comprising the target are displayed at its surface and thus accessible to reagents which can detect changes in the biochemical system regulated by the gene product within the microcapsule.
- microcapsule identification and, optionally, sorting relies on a change in the optical properties of the microcapsule, for example absorption or emission characteristics thereof, for example alteration in the optical properties of the microcapsule resulting from a reaction leading to changes in absorbance, luminescence, phosphorescence or fluorescence associated with the microcapsule. All such properties are included in the term "optical”.
- microcapsules can be identified and, optionally, sorted by luminescence, fluorescence or phosphorescence activated sorting.
- flow cytometry is employed to analyse and, optionally, sort microcapsules containing gene products having a desired activity which result in the production of a fluorescent molecule in the microcapsule.
- microcapsules can, optionally, be separated using a microfluidic flow sorter to allow recovery and further analysis or manipulation of the molecules they contain.
- the flow sorter would be an electronic flow sorting device.
- a sorting device can be integrated directly on the microfluidic device, and can use electronic means to sort the microcapsules.
- Optical detection also integrated directly on the microfluidic device, can be used to screen the microcapsules to trigger the sorting.
- Ottier means of control of the microcapsules, in addition to charge, can also be incorporated onto the microfluidic device.
- a change in microcapsule fluorescence when identified, is used to trigger the modification of the microbead within the compartment.
- microcapsule identification relies on a change in the optical properties of the microcapsule resulting from a reaction leading to luminescence, phosphorescence or fluorescence within the microcapsule. Modification of the microbead within the microcapsules would be triggered by identification of luminescence, phosphorescence or fluorescence. For example, identification of luminescence, phosphorescence or fluorescence can trigger bombardment of the compartment with photons (or other particles or waves) which leads to modification of the microbead or molecules attached to it.
- microbeads A similar procedure has been describ ed previously for the rapid sorting of cells (Keij et al, 1994). Modification of the microbead may result, for example, from coupling a molecular "tag", caged by a photolabile protecting group to the microbeads: bombardment with photons of an appropriate wavelength leads to the removal of the cage. Afterwards, all microcapsules are combined and the microbeads pooled together in one environment. Genetic elements exhibiting the desired activity can be selected by affinity purification using a molecule that specifically binds to, or reacts specifically with, the "tag".
- the genetic elements will be sorted by flow cytometry.
- a variety of optical properties can be used to trigger sorting, including light scattering (Kerker, 1983) and fluorescence polarisation (Rolland et al., 1985).
- the difference in optical properties of the genetic elements will be a difference in fluorescence and the genetic elements will be sorted using a fluorescence activated cell sorter (Norman, 1980; Mackenzie and Pinder, 1986), or similar device.
- a sorting device can be integrated directly on the microfluidic device, and can use electronic means to sort the genetic elements.
- Optical detection also integrated directly on the microfluidic device, can be used to screen the genetic elements to trigger the sorting.
- the genetic element comprises of a nonfluorescent nonmagnetic (e.g. polystyrene) or paramagnetic microbead (see Fornusek and Vetvicka, 1986), optimally 0.6 to 1 .0 ⁇ m diameter, to which are attached both the gene and the groups involved in generating a fluorescent signal:
- a nonfluorescent nonmagnetic e.g. polystyrene
- paramagnetic microbead see Fornusek and Vetvicka, 1986
- the fluorescence signal from each bead corresponds tightly to the number of fluorescent molecules attached to the bead. At present as little as few hundred fluorescent molecules per particle can be quantitatively detected;
- the wide dynamic range of the fluorescence detectors (typically 4 log units) allows easy setting of the stringency of the sorting procedure, thus allowing the recovery of the optimal number of genetic elements from the starting pool (the gates can be set to separate beads with small differences in fluorescence or to only separate out beads with large differences in fluorescence, dependant on the selection being performed;
- fluorescence-activated cell sorting equipment can perform simultaneous excitation at up to two different wavelengths and detect fluorescence at up to four different wavelengths (Shapiro, 1983) allowing positive and negative selections to be performed simultaneously by monitoring the labelling of the genetic element with two (or more) different fluorescent markers, for example, if two alternative substrates for an enzyme (e.g. two different enantiomers) are labelled with different fluorescent tags the genetic element can labelled with different fluorophores dependent on the substrate used and only genes encoding enzymes with enantioselectivity selected.
- highly uniform derivatised and non-derivatised nonmagnetic and paramagnetic microparticles are commercially available from many sources (e.g. Sigma, and Molecular Probes) (Fornusek and Vetvicka, 1986).
- the selection procedure may comprise two or more steps.
- transcription/replication and/or translation of each genetic element of a genetic element library may take place in a first microcapsule.
- Each gene product is then linked to the genetic element which encoded it (which resides in the same microcapsule), for example via a gene product-specific ligand such as an antibody.
- the microcapsules are then broken, and the genetic elements attached to their respective gene products optionally purified.
- genetic elements can be attached to their respective gene products using methods which do not rely on encapsulation.
- phage display Smith, G.P.,1985), polysome display (Mattheakkis et al., 1994), RNA-peptide fusion (Roberts and Szostak, 1997) or lac repressor peptide fusion (Cull, et al., 1992).
- each purified genetic element attached to its gene product is put into a second microcapsule containing components of the reaction to be selected. This reaction is then initiated. After completion of the reactions, the microcapsules are again broken and the modified genetic elements are selected.
- one or more intervening steps may be performed between the initial step of creation and linking of gene product to genetic element, and the final step of generating the selectable change in the genetic element.
- release of the gene product from the genetic element within a secondary microcapsule can be achieved in a variety of ways, including by specific competition by a low-molecular weight product for the binding site or cleavage of a linker region joining the binding domain of the gene product from the catalytic domain either enzymatically (using specific proteases) or autocatalytically (using an integrin domain).
- the system can be configured such that the desired binding, catalytic or regulatory activity encoded by a genetic element leads, directly or indirectly to the activation of expression of a "reporter gene" that is present in all microcapsules. Only gene products with the desired activity activate expression of the reporter gene. The activity resulting from reporter gene expression allows the selection of the genetic element (or of the compartment containing it) by any of the methods described herein.
- activation of the reporter gene may be the result of a binding activity of the gene product in a manner analogous to the "two hybrid system” (Fields and Song, 1989).
- Activation can also result from the product of a reaction catalysed by a desirable gene product.
- the reaction product can be a transcriptional inducer of the reporter gene.
- arabinose may be used to induce transcription from the araBAD promoter.
- the activity of the desirable gene product can also result in the modification of a transcription factor, resulting in expression of the reporter gene.
- the desired gene product is a kinase or phosphatase the phosphorylation or dephosphorylation of a transcription factor may lead to activation of reporter gene expression.
- the method comprises the further step of amplifying the genetic elements.
- Selective amplification may be used as a means to enrich for genetic elements encoding the desired gene product.
- genetic material comprised in the genetic elements may be amplified and the process repeated in iterative steps.
- Amplification may be by the polymerase chain reaction (Saiki et al., 1988) or by using one of a variety of other gene amplification techniques including; Qb replicase amplification (Cahill, Foster and Mahan, 1991; Chetverin and Spirin, 1995; Katanaev, Kurnasov and Spirin, 1995); the ligase chain reaction (LCR) (Landegren et al., 1988; Barany, 1991); the self-sustained sequence replication system (Fahy, Kwoh and Gingeras, 1991) and strand displacement amplification (Walker et al., 1992).
- the amplification procedure can be performed in a micro fluidic device.
- the reagents contained in the fused microcapsule can be mixed rapidly using chaotic advection by passing the droplets through channels that disrupt the laminar flow lines of the fluid within the droplets, their contents can be rapidly mixed, fully initiating any chemical reactions.
- sensors are provided that can sense and/or determine one or more characteristics of the fluidic droplets, and/or a characteristic of a portion of the fluidic system containing the fluidic droplet (e.g., the liquid surrounding the fluidic droplet) in such a manner as to allow the determination of one or more characteristics of the fluidic droplets.
- Characteristics determinable with respect to the droplet and usable in the invention can be identified by those of ordinary skill in the art.
- Non-limiting examples of such characteristics include fluorescence, spectroscopy (e.g., optical, infrared, ultraviolet, etc.), radioactivity, mass, volume, density, temperature, viscosity, pH, concentration of a substance, such as a biological substance (e.g., a protein, a nucleic acid, etc.), or the like.
- fluorescence e.g., optical, infrared, ultraviolet, etc.
- radioactivity e.g., mass, volume, density, temperature, viscosity, pH, concentration of a substance, such as a biological substance (e.g., a protein, a nucleic acid, etc.), or the like.
- the senor may be connected to a processor, which in turn, causes an operation to be performed on the fluidic droplet, for example, by sorting the droplet, adding or removing electric charge from the droplet, fusing the droplet with another droplet, splitting the droplet, causing mixing to occur within the droplet, etc., for example, as previously described.
- a processor may cause the fluidic droplet to be split, merged with a second fluidic droplet, sorted etc.
- One or more sensors and/or processors may be positioned to be in sensing communication with the fluidic droplet.
- Sensor communication means that the sensor may be positioned anywhere such that the fluidic droplet within the fluidic system (e.g., within a channel), and/or a portion of the fluidic system containing the fluidic droplet may be sensed and/or determined in some fashion.
- the sensor may be in sensing communication with the fluidic droplet and/or the portion of the fluidic system containing the fluidic droplet fluidly, optically or visually, thermally, pneumatically, electronically, or the like.
- the sensor can be positioned proximate the fluidic system, for example, embedded within or integrally connected to a wall of a channel, or positioned separately from the fluidic system but with physical, electrical, and/or optical communication with the fluidic system so as to be able to sense and/or determine the fluidic droplet and/or a portion of the fluidic system containing the fluidic droplet (e.g., a channel or a microchannel, a liquid containing the fluidic droplet, etc.).
- a sensor may be free of any physical connection with a channel containing a droplet, but may be positioned so as to detect electromagnetic radiation arising from the droplet or the fluidic system, such as infrared, ultraviolet, or visible light.
- the electromagnetic radiation may be produced by the droplet, and/or may arise from other portions of the fluidic system (or externally of the fluidic system) and interact with the fluidic droplet and/or the portion of the fluidic system containing the fluidic droplet in such as a manner as to indicate one or more characteristics of the fluidic droplet, for example, through absorption, reflection, diffraction, refraction, fluorescence, phosphorescence, changes in polarity, phase changes, changes with respect to time, etc.
- a laser may be directed towards the fluidic droplet and/or the liquid surrounding the fluidic droplet, and the fluorescence of the fluidic droplet and/or the surrounding liquid may be determined.
- "Sensing communication,” as used herein may also be direct or indirect.
- light from the fluidic droplet may be directed to a sensor, or directed first through a fiber optic system, a waveguide, etc., before being directed to a sensor.
- Non-limiting examples of sensors useful in the invention include optical or electromagnetically-based systems.
- the sensor may be a fluorescence sensor (e.g., stimulated by a laser), a microscopy system (which may include a camera or other recording device), or the like.
- the sensor may be an electronic sensor, e.g., a sensor able to determine an electric field or other electrical characteristic.
- the sensor may detect capacitance, inductance, etc., of a fluidic droplet and/or the portion of the fluidic system containing the fluidic droplet.
- a "processor” or a “microprocessor” is any component or device able to receive a signal from one or more sensors, store the signal, and/or direct one or more responses (e.g., as described above), for example, by using a mathematical formula or an electronic or computational circuit.
- the signal may be any suitable signal indicative of the environmental factor determined by the sensor, for example a pneumatic signal, an electronic signal, an optical signal, a mechanical signal, etc.
- a device of the invention may contain fluidic droplets containing one or more cells.
- the desired activity of one or more gene products may result in the expression (or inhibition of expression) of a 'marker' gene, for example a gene for green fluorescent protein (GFP).
- GFP green fluorescent protein
- the cells may be exposed to a fluorescent signal marker that binds if a certain condition is present, for example, the marker may bind to a first cell type but not a second cell type, the marker may bind to an expressed protein, the marker may indicate viability of the cell (i.e., if the cell is alive or dead), the marker may be indicative of the state of development or differentiation of the cell, etc., and the cells may be directed through a fluidic system of the invention based on the presence/absence, and/or magnitude of the fluorescent signal marker.
- determination of the fluorescent signal marker may cause the cells to be directed to one region of the device (e.g., a collection chamber), while the absence of the fluorescent signal marker may cause the cells to be directed to another region of the device (e.g., a waste chamber).
- a population of cells may be screened and/or sorted on the basis of one or more determinable or targetable characteristics of the cells, for example, to select live cells, cells expressing a certain protein, a certain cell type, etc.
- various components of the invention can be formed from solid materials, in which the channels can be formed via micromachining, film deposition processes such as spin coating and chemical vapor deposition, laser fabrication, photolithographic techniques, etching methods including wet chemical or plasma processes, and the like. See, for example, Scientific American, 248:44-55, 1983 (Angell, et al).
- at least a portion of the fluidic system is formed of silicon by etching features in a silicon chip.
- various components of the systems and devices of the invention can be formed of a polymer, for example, an elastomeric polymer such as polydimethylsiloxane (“PDMS”), polytetrafluoroethylene (“PTFE” or Teflon ® ), or the like.
- PDMS polydimethylsiloxane
- PTFE polytetrafluoroethylene
- Teflon ® Teflon ®
- a base portion including a bottom wall and side walls can be fabricated from an opaque material such as silicon or PDMS, and a top portion can be fabricated from a transparent or at least partially transparent material, such as glass or a transparent polymer, for observation and/or control of the fluidic process.
- Components can be coated so as to expose a desired chemical functionality to fluids that contact interior channel walls, where the base supporting material does not have a precise, desired functionality.
- components can be fabricated as illustrated, with interior channel walls coated with another material.
- Material used to fabricate various components of the systems and devices of the invention may desirably be selected from among those materials that will not adversely affect or be affected by fluid flowing through the fluidic system, e.g., material(s) that is chemically inert in the presence of fluids to be used within the device.
- various components of the invention are fabricated from polymeric and/or flexible and/or elastomeric materials, and can be conveniently formed of a hardenable fluid, facilitating fabrication via molding (e.g. replica molding, injection molding, cast molding, etc.).
- the hardenable fluid can be essentially any fluid that can be induced to solidify, or that spontaneously solidifies, into a solid capable of containing and/or transporting fluids contemplated for use in and with the fluidic network.
- the hardenable fluid comprises a polymeric liquid or a liquid polymeric precursor (i.e. a "prepolymer").
- Suitable polymeric liquids can include, for example, thermoplastic polymers, thermoset polymers, or mixture of such polymers heated above their melting point.
- a suitable polymeric liquid may include a solution of one or more polymers in a suitable solvent, which solution forms a solid polymeric material upon removal of the solvent, for example, by evaporation.
- a suitable solvent such polymeric materials, which can be solidified from, for example, a melt state or by solvent evaporation.;, are well known to those of ordinary skill in the art.
- a variety of polymeric materials, many of which are elastomeric, are suitable, and are also suitable for forming molds or mold masters, for embodiments where one or both of the mold masters is composed of an elastomexic material.
- a non-limiting list of examples of such polymers includes polymers of the general classes of silicone polymers, epoxy polymers, and acrylate polymers.
- Epoxy polymers are characterized by the presence of a three- membered cyclic ether group commonly referred to as an epoxy group, 1,2-epoxide, or oxirane.
- diglycidyl ethers of bisphenol A can be used, in addition to compounds based on aromatic amine, triazine, and cycloaliphatic backbones.
- Another example includes the well-known Novolac polymers.
- Non-limiting examples of silicone elastomers suitable for use according to the invention include those formed from precursors including the chlorosilanes such as methylchlorosilanes, ethylchlorosilanes, phenylchlorosilan.es, etc.
- silicone polymers such as PDMS
- PDMS polymethyl methacrylate copolymer
- flexible (e.g., elastomeric) molds or masters can be advantageous in this regard.
- One advantage of forming structures such as microfluidic structures of the invention from silicone polymers, such as PDMS, is the ability of such polymers to be oxidized, for example by exposure to an oxygen-containing plasma such as an air plasma, so that the oxidized structures contain, at their surface, chemical groups capable of cross-linking to other oxidized silicone polymer surfaces or to the oxidized surfaces of a variety of other polymeric and non-polymeric materials.
- an oxygen-containing plasma such as an air plasma
- Droplet size and frequency are not independent; instead their product is determined by tfcie infusion rate of the dispersed phase Q d -f V d -
- the droplet size decreases with increasing field strength, as shown in Figs. 17, B to E.
- the dependence of the droplet size on applied voltage for three different flow rates is summarized in Fig. 17F.
- q is proportional to the applied voltage, V.
- Sorting module The contents of individual droplets must be probed, and selected droplets sorted into discreet streams.
- electrostatic charging of droplets provides a means for sorting that can be precisely controlled, can be switched at high frequencies, and requires no moving parts.
- Electrostatic charge on the droplets enables drop-by-drop sorting based on the linear coupling of charge to an external electric field.
- a T-junction bifurcation that splits the flow of carrier fluid equally will also randomly split the droplet population equally into the two streams, as shown in Fig. 24A.
- a small electric field applied at the bifurcation precisely dictates which channel the drops enter; a schematic of the electrode configuration is shown in Fig. 24B.
- lacZmut Full-length mutant lacZ (lacZmut), which has an internal frameshift and hence does not encode an active ⁇ -galactosidase, is obtained by cutting pIVEX2.2b-LacZ with restriction enzyme Sad (NEB). Digested DNA is blunted by incubation for 15 min at 12°C with T4 DMA polymerase (2 U) and dNTPs (500 ⁇ M final concentration). The reaction is quenched by adding EDTA to a final concentration of 10 mM and heating to 75 0 C for 20 minutes. Blunted DNA is purified and self-ligated with T4 DNA ligase (1 Weiss unit) in the presence of 5% PEG 4,000 by incubating for 2 hrs at 22°C.
- T4 DNA ligase (1 Weiss unit) in the presence of 5% PEG 4,000 by incubating for 2 hrs at 22°C.
- EcoProT7 A commercial in vitro translation system (EcoProT7, Novagen/EMD biosciences Ltd, Madison, Wi, USA) is used according to the manufacturer's protocol.
- EcoProT7 extract is compartmentalised into droplets (a) of mean 10 ⁇ m diameter (520 fl volume).
- Droplets (b), of mean 7.4 ⁇ m diameter (220 fl volume) are formed containing 0.67 mM L-methionine and 0.25 mM 7-hydroxycoumarin-3- carboxylic acid (Sigma Aldrich) (excitation 386 nm, emission 448 nm), and 0.75 pM DNTA (mixes of LacZ and lacZmut linear DNA at the ratios described above) in nuclease- free water.
- the droplets are formed in a carrier fluid consisting of perfiuorinated oil; the perfiuorinated oil can either consist of the mixture described in example 1 or alternatively one of the 3MTM FluorinertTM liquids.
- Each droplet (a) is fused with a droplet (b).
- the continuous wave emission from two diode lasers (363 nm and 488 nrn) is used for excitation dichroic beam splitters and band pass filters (450 ⁇ 20 nm and 530 ⁇ 20 nm) are used to isolate the fluorescent emission to detect the 7- hydroxycoumarin-3-carboxylic acid fluorescence and the fluorescein fluorescence as measured with photomultiplying tubes.
- Droplets with the highest fluorescein fluorescence (with a sorting gate set such that less than 0.05% of the population of droplets from a negative control without DNA) are sorted. For each sort, 10,000 droplets are collected.
- DNA from the sorted droplets is precipitated by adding 100 ⁇ l 0.3 M sodium acetate pH 5.2 and 70 ⁇ l isopropanol in the presence of 20 ⁇ g glycogen as carrier (Roche Biochemicals GmbH, Mannheim, Germany). DNA is pelleted by centrifugation at 20,000xg for 15 min at 4°C. Precipitated DNA is ished twice with 100 ⁇ l 70% ethanol and the DNA pellet is dried using a Speedvac (Eppendorf). DNA is resuspended into 10 ⁇ l nuclease-free water.
- Example 3 mutants with improved ⁇ -galactosidase activity can be selected from a random mutagenesis library of evolved ⁇ -galactosidase (Ebg) using compartmentalisation of genes in aqueous droplets in a microfluidic device
- the gene encoding for evolved- ⁇ -galactosidase is often used as a model to study the evolution of novel enzyme functions within an organism.
- the wild type ebgA gene of Escherichia coli encodes an enzyme " with feeble ⁇ -galactosidase activity, but ebgA has the potential to evolve sufficient activity to replace the lacZ gene for growth on the sugars lactose and lactulose. Genetic analysis of these mutants has revealed that only two amino acid replacements account for the drastic increase in ⁇ -galactosidase activity.
- a gene segment encoding for the A domain and the C domain of evolved ⁇ -galactosidase enzyme is amplified from genomic DNA of E. coli strain BL21 using primers EbgACFw (5'-CAGACTGCACCGCGGGATGAATCGCTGGGAAAACATTCAGC-S ') and EbgACBw (5'-GCGAGGAGCTCTTATTTGTTATGGAAATAACCATCTTCG-S').
- the PCR product is cloned into vector pIVEX2.2b using restriction endonucleases SacII and Sad (NEB). DNA is transfected into XLlO-gold cells and. single colonies are screened for the presence of the EbgAC gene construct with the right nucleotide sequence.
- pDNA from a single clone with the right EbgAC gene sequence is used as template to generate a random mutagenesis library using nucleoside analogues essentially as described by Zaccolo et al. (J MoI Biol 255(4): 589-603, 1996).
- a mixture of the 5 '-triphosphates of 6- (2-deoxy-b-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido-[4,5-C][l,2]oxazin-7-one (dPTP) and of 8-0X0-2 'deoxyguanosine (8-oxodG) is prepared in PCR grade water at 2 mM and 10 mM concentrations, respectively.
- This base analogue mix is diluted 167x and 333x in expand long template PCR buffer 1 (Roche), containing MgCl 2 (2 mM), dNTPs (500 ⁇ M), expand long template PCR polymerase enzyme mix (Roche), primer LMB2-9E (5'- GC ATTTATC AGGGTTATTGTC-3; 500 nM') and triple biotinylated primer PIVB-I (5'-3Bi-GCGTTGATGCAATTTCT-S'; 500 nM) in a total reaction volume of 50 ⁇ l.
- expand long template PCR buffer 1 containing MgCl 2 (2 mM), dNTPs (500 ⁇ M), expand long template PCR polymerase enzyme mix (Roche), primer LMB2-9E (5'- GC ATTTATC AGGGTTATTGTC-3; 500 nM') and triple biotinylated primer PIVB-I (5'-3Bi-GCGTTGATGCAATTTCT-S'; 500
- Streptavidin-coated magnetic beads (Dynabeads M-280 streptavidin, Dynal Biotech, Oslo, Norway) are rinsed in 2x binding buffer provided with the beads, resuspended into 50 ⁇ l 2x binding buffer and added to the purified DNA. Beads and DNA are incubated for 2.5 hrs at room temperature in a rotating device. Beads' are collected with a magnet and rinsed twice with ish buffer that is provided with the beads and twice with PCR-grade water. Finally, beads are resuspended into 25 ⁇ l water.
- PCR product is purified using a Qiaquick PCR purification kit and recovered in 50 ⁇ l of PCR-grade water. . Iterative rounds of in vitro selection using a microfluidic system
- the number of positive droplets within the Ebg library increased by at least 10-fold.
- DNA is recovered from the double emulsions by standard isopropanol precipitation and PCR amplified using primers LMB2-11 and PIVB-Il.
- Amplified DNA is digested with restriction endonucleases Sad and SacII and cloned into pIVEX2.2b that is digested with the same enzymes.
- the ligation product is transformed into ElectroBlue electrocompetent cells (Strategene) by electroporation (at 17 kV/cm, 600 ⁇ , 25 ⁇ F) and plated onto LB agar plates with ampicillin.
- Ebg gene constructs are amplified from single colonies by colony PCR using primers LMB2-10E and PIVB-4.
- PCR product is added to 14 ⁇ l of IVT mix (Novagen's EcoProT7 extract, supplemented with 200 ⁇ M L-methionine) and incubated for 90 min at 30°C.
- IVT mix Novagen's EcoProT7 extract, supplemented with 200 ⁇ M L-methionine
- Forty microlitres substrate solution 250 ⁇ M FDG 5 10 mM MgCl 2 , 50 mM NaCl, 1 mM DTT and 100 ⁇ g/ml BSA in 10 mM Tris-HCl, pH 7.9 is added and the conversion of FDG into fluorescein is monitored every 45 s for 90 min at 37 0 C .
- the screened clones show a broad variety of ⁇ -galactosidase activities. ⁇ 50% of colonies have ⁇ -galactosidase activities that are comparable to or lower than wild type Ebg. ⁇ 12.5% of clones show ⁇ -galactosidase activity that is comparable to the Class I and Class ⁇ mutants (single point mutations) described by Hall et al. (FEMS Microbiol Lett 174(1): 1-8, 1999; Genetica 118(2-3): 143-56, 2003).
- the system described here can be used for the selection of ebg variants with improved ⁇ -galactosidase activity from a large gene library.
- Hoogenboom, H. R. (1997). Designing and optimizing library selection strategies for generating high-affinity antibodies. Trends Biotechnol. 15, 62-70. Hoogenboom, H.R., et al, (1991) Nucl. Acids Res., 91, 4133-4137.
- catELISA a facile general route to catalytic antibodies. Proc Natl Acad Sd USA 90(2), 373-7.
- Microcapsules preparation by interfacial polymerisation and interfacial complexation and their applications.
- Microencapsulation methods and industrial applications (Benita, S., ed.), pp. 349-375. Marcel Dekker, New York.
Abstract
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