Patent Application
20150132829
Algae are unicellular organisms, producing oxygen by photosynthesis. One group, the microalgae, are useful for biotechnology applications for many reasons, including their high growth rate and tolerance to varying environmental conditions. The use of microalgae in a variety of industrial processes for commercially important products is known and/or has been suggested. For example, microalgae have uses in the production of nutritional supplements, pharmaceuticals, natural dyes, a food source for fish and crustaceans, biological control of agricultural pests, production of oxygen and removal of nitrogen, phosphorus and toxic substances in sewage treatment, and pollution controls, such as biodegradation of plastics or uptake of carbon dioxide.
Microalgae, like other organisms, contain lipids and fatty acids as membrane components, storage products, metabolites and sources of energy. Some algal strains, diatoms, and cyanobacteria have been found to contain proportionally high levels of lipids (over 30%). Microalgal strains with high oil or lipid content are of great interest in the search for a sustainable feedstock for the production of biofuels.
Some wild-type algae are suitable for use in various industrial applications. However, it is recognized that by modification of algae to improve particular characteristics useful for the aforementioned applications, the relevant processes are more likely to be commercially viable. To this end, algal strains can be developed which have improved characteristics over wild-type strains. Such developments have been made by traditional techniques of screening and mutation and selection. Further, recombinant DNA technologies have been widely suggested for algae. Such approaches may increase the economic validity of production of commercially valuable products.
For the production of some commercial products of interest, organisms producing the products may be grown in liquid environments. It is sometimes beneficial to remove the organisms from the liquid environments. One method of removing organisms is to flocculate or aggregate the organisms to facilitate removal. Flocculants or flocculating agents promote flocculation by causing colloids and other suspended particles (e.g., cells) in liquids to aggregate, forming a floc. Flocculants are used in water treatment processes to improve the sedimentation of small particles. For example, a flocculant may be used in swimming pool or drinking water filtration to aid removal of microscopic particles which would otherwise cause the water to be cloudy and which would be difficult to remove by filtration alone.
Many flocculants are multivalent cations such as aluminium, iron, calcium or magnesium. These positively charged molecules interact with negatively charged particles and molecules to reduce the barriers to aggregation. In addition, many of these chemicals, under appropriate pH and other conditions such as temperature and salinity, react with water to form insoluble hydroxides which, upon precipitating, link together to form long chains or meshes, physically trapping small particles into the larger floe.
Flocculation of microalgae using chemical flocculants is known. Long-chain polymer flocculants, such as modified polyacrylamides, are manufactured and sold by the flocculant producing business. These can be supplied in dry or liquid form for use in the flocculation process. The most common liquid polyacrylamide, for example, is supplied as an emulsion with 10-40% actives and the rest is a carrier fluid, surthetants and latex.
Use of chemical flocculants, however, has multiple drawbacks. For instance, use in water treatment and other processes require subsequent removal of flocculants. The addition and removal of flocculants adds to the cost of commercial production of a product of interest, thus decreasing the economic feasibility of production.
One aspect of the present disclosure provides for vectors comprising a nucleic acid encoding a flocculating moiety and a regulatory element linked to express the flocculating moiety in the nucleus of a photosynthetic organism. The flocculating moieties employed in this invention may comprise carbohydrate binding proteins, antibodies binding to a cell surface antigen of C. reinhardtii, D. salina, D. tertiolecta, or H. pluvialis, lectin, FhuA protein, pb5 protein, or other proteins that form a tight protein-protein pairing where the pairing leads to flocculation of microalgae. The regulatory elements utilized in the vectors may comprise a constitutive promoter, light-inducible promoter, quorum-sensitive promoter, temperature-sensitive promoter, or nitrogen-starvation responsive promoter. The vectors disclosed herein may also contain nucleic acids capable of targeting the flocculation moiety to the cell surface and to anchor a portion of the flocculation moiety to be tightly bound on the cell surface. The vectors are utilized to transform non-vascular photosynthetic organisms, such as microalgae, for flocculation, in certain cases, the anchoring moiety can be optional as secreting the flocculation moiety into the culture, depending on the property of a flocculation moiety, is preferable method for flocculation.
Another aspect provides methods of flocculation utilizing non-vascular photosynthetic organisms. Examples of non-vascular photosynthetic organisms are C. reinhardtii, D. salina, D. tertiolecta, or H. pluvialis. The flocculation methods may comprise creating two distinctly different photosynthetic organisms by transforming with different flocculation moieties and causing flocculation by allowing the two flocculation moieties to interact each other. Examples of flocculation moieties comprise protein-protein interaction between FhuA and pb5, interactions between mating-type specific agglutinins, an antibody-antigen pairing, and other proteins capable of recognizing each other as a pair and form a stable protein-protein complex. Examples of methods for the induction of flocculation of non-vascular photosynthetic organisms are changing light condition, culture density, culture temperature, or nutrient availability; adding lectin, protein, heavy metal, cationic or chemical flocculant in the culture; or attaching the aforementioned flocculation agents to a solid support.
Another aspect provides methods to recycle the culture liquid from a liquid environment. Non-vascular photosynthetic organisms can be cultured in a media with defined media known in the art, such as min-70, M-medium, or TAP medium. After flocculation, it is often possible and economically beneficial to recycle the liquid portion of the culture. Disclosed herein are methods to enable the recycling of the liquid comprising growing microorganisms encoding a flocculation moiety; contacting the organism with flocculating moiety and thereby flocculating the organism; and removing the aggregated microorganism from the liquid environment.
One particular aspect provides a method for flocculating a non-vascular photosynthetic organism comprising expressing an exogenous nucleic acid encoding a first flocculation moiety on the external surface of a non-vascular photosynthetic organism and contacting the organism with a second flocculation moiety. The organism can be a member of the genus Chlamydomonas, Dunaliella, Scenedesmus or Hematococcus, for example C. reinhardtii, D. salina, D. tertiolecta, S. dimorphus or H. pluvialis, although members of other genera may be used. The first flocculation moiety can be a carbohydrate binding protein such as a lectin and in particular a c-type lectin. Other non-limiting examples of carbohydrate binding proteins include CD-SIGN, lectin-1, dectin-2, HECL, langerin, layilin, mincle, MMGL, E-selection, P-selectin, L-selectin, DEC-205, Endo 180, mannose receptor, phospholipase A2 receptor, sialoadhesin (siglec-1), siglec-2, siglec-3, siglec-4, siglec-5, -siglec-6, siglec-8, siglec-9, siglec-10, siglec-11, and galectins. In other embodiments, the first flocculation moiety may be a univalent, monovalent or polyvalent antibody, for example an say antibody, that binds to an antigen on the surface of an organism of interest. Examples of such organisms include members of the genus Chlamydomonas, Dunaliella, Scenedesmus or Hematococcus, such as C. reinhardtii, D. salina, D. tertiolecta, or H. pluvialis. Some embodiments may also include the use of a second flocculation moiety. The second flocculation moiety may be on the surface of a solid support or on the surface of a second organism, which may or may not be a non-vascular photosynthetic organism. When the second flocculation moiety is expressed on the surface of a second organism, the second flocculation moiety may be naturally occurring or may be the result of expression of an exogenous nucleotide sequence. In some embodiments, the first and second organisms are the same species, while in other embodiments, the first and second organisms are of different strains, different genera or different kingdoms. For example, in one embodiment the first and second organism are both algae while in another embodiment one organism is an alga while the other is a bacterium or yeast. When the second flocculation moiety is on the surface of a second organism or solid support, the method further comprises combining the first organism with the second organism, with the solid support, or both.
Another aspect provides a non-vascular photosynthetic organism comprising an exogenous protein on the organism's external cell surface, such as a cell membrane or cell wall; the exogenous protein comprising one member of a binding pair. The exogenous protein can be an antibody such as a single chain variable fragment (scFv) antibody or a carbohydrate binding protein such as a lectin, for example a c-type lectin. In other embodiments the exogenous protein is an antigen which is the target of an antibody, and in one embodiment an antibody specific to that antigen. In some embodiments the exogenous protein is a fusion or chimeric protein in which part of the protein serves to anchor the protein to the cell surface. The non-vascular photosynthetic organism may be an alga, for example a green alga, more particularly a member of the genus Chlamydomonas Scenedesmus or Hematococcus and more particularly one of C. reinhardtii, D. salina, D. tertiolecta, or H. pluvialis. In one embodiment, the non-vascular photosynthetic organism is a halophile such as D. salina or D. tertiolecta. In still other embodiments, the carbohydrate binding protein is at least one of CD-SIGN, dectin-1, dectin-2, HECL, langerin, layilin, mincle, MMGL, E-selection, P-selectin, L-selectin, DEC-205, Endo 180, mannose receptor, phospholipase A2 receptor, sialoadhesin (siglec-1), siglec-2, siglec-3, siglec-4, siglec-5, siglec-8, siglec-9, siglec-10, siglec-11, or galectins.
Yet another aspect provides a flocculation method comprising transforming a first non-vascular photosynthetic organism with a nucleic acid encoding a first flocculation moiety, transforming a second organism, which may or may not be a non-vascular photosynthetic organism, with a nucleic acid encoding a second flocculation moiety or a protein necessary for the production of a second flocculation moiety that interacts with the first flocculation moiety, expressing the first and second flocculation moieties and contacting the two moieties such that flocculation occurs. The two organisms may be of the same or different genera, the same or different species or the same or different kingdoms. For example, the organisms may be the same species of algae, different species of algae, or an alga and a bacterium or yeast. The first organism, the second organism or both can be members of the genus Chlamydomonas, Dunaliella Scenedesmus or Hematococcus such that one or both of the organisms can be C. reinhardtii, D. salina, D. tertiolecta, or S. dimorphus or H. pluvialis. The first or second flocculation moieties can be antibodies carbohydrate binding proteins or mating-type specific agglutinins.
Still another aspect provides a vector comprising an isolated nucleic acid encoding a flocculation moiety, a regulatory element for expressing the flocculation moiety the photosynthetic organism, for example in the nucleus, and a targeting element that allows the flocculating moiety to be directed to the surface of the organism or be secreted by the organism. The flocculation moiety can be an antibody or any of the carbohydrate binding proteins described herein. Specific examples of flocculation moieties include antibodies, lectins. FluA protein and pb5 protein. When the nucleic acid encodes an antibody, it may be to an antigen of the surface of a member of the genus Chlamydomonas, Dunaliella, Scenedesmus or Hematococcus, for example, C. reinhardtii, D. salina, D. tertiolecta, or S. dimorphus or H. pluvialis. In certain embodiments, the vector further comprises a nucleic acid encoding an anchoring element that anchors the flocculation moiety to the surface of the organism.
Still another aspect provides a host cell comprising any of the preceding vectors. The host cell may or may not be a non-vascular photosynthetic organism. In some embodiments, the host cell is a member of the genus Chlamydomonas, Dunaliella, Scenedesmus or Hematococcus, for example C. reinhardtii, D. salina, D. tertiolecta, S. dimorphus or H. pluvialis. In another embodiment, the host cell is a bacterium or a yeast.
Yet another method provides a method for recycling liquid, such as water or a water based growth medium, comprising growing a non-vascular photosynthetic organism comprising an exogenotes nucleic acid encoding a flocculation moiety in a liquid environment, contacting the organism with a second flocculation moiety that binds directly or indirectly to the first flocculation moiety resulting in flocculation of the organism and separating at least a portion of the liquid in the liquid environment from, the organism. The first and second flocculation moieties can be any of the moieties described herein, including, but not limited to, antibodies, carbohydrate binding proteins, carbohydrates, heavy metal flocculants, chemical flocculants, cationic flocculants, and mating type agglutinins. The second flocculation moiety may be attached to the surface of a second organism, which may or may not be a non-vascular photosynthetic organism, or may be attached to a solid support.
Still another aspect provides a method of flocculating a non-vascular photosynthetic organism comprising contacting the non-vascular photosynthetic organism with a second organism comprising an exogenous nucleic acid encoding an antibody that binds to an antigen on the surface of the non-vascular photosynthetic organism thereby causing flocculation. In one embodiment, the antibody is expressed on the surface of the second organism. In another embodiment, the antibody is a single chain variable fragment (scFv) antibody. In still another embodiment, the exogenous nucleic acid encodes a fusion or chimeric protein that comprises the antibody and an anchoring component that anchors the antibody to the external surface of the cell membrane or cell wall of the second organism. In one embodiment, the second organism is a bacterium, while in another embodiment, the second organism is a yeast. The non-vascular photosynthetic organism may a green alga for example a member of the genus Chlamydomonas, Dunaliella, Scenedesmus or Hematococcus and more particularly C. reinhardtii, D. salina, D. tertiolecta, S. dimorphus or H. pluvialis.
Another aspect provides a method of flocculating a non-vascular photosynthetic organism comprising contacting the non-vascular photosynthetic organism with a second organism comprising an exogenous nucleic acid encoding a flocculation moiety that binds to a surface component of the non-vascular photosynthetic organism thereby causing flocculation of the non-vascular photosynthetic organism. The flocculation moiety can be secreted by the second organism, expressed on the surface of the second organism, or both. The second organism can be a yeast, a bacterium, a non-photosynthetic alga or a photosynthetic alga. In some embodiments, the second organism is E. coli or P. pastoris. The carbohydrate moiety can be at least one of the members of the group selected from CD-SIGN, dectin-1, dectin-2, HECL, langerin, layilin, mincle, MMGL, E-selection, P-selectin, L-selectin, DEC-205, Endo 180, mannose receptor, phospholipase A2 receptor, sialoadhesin (siglec-1), siglec-2, siglec-3, siglec-4, siglec-7, siglec-8, siglec-9, siglec-10, siglec-11, and galectins. In particular embodiments, the carbohydrate binding protein is a lectin, for example, a c-type lectin.
In any of the embodiments described herein, an exogenous nucleic acid encoding a flocculation moiety, for example an antibody or a carbohydrate binding protein, may further comprise one or more regulatory elements such as a promoter. Promoters may be constitutive or inducible. Inducible promoters include, but are not limited to light-inducible promoters, quorum-sensitive promoters, temperature-sensitive promoters or nitrogen sensitive promoters.
it should be understood that, unless stated otherwise, when reference is made to the expression or presence of a flocculation moiety on the surface of an organism or solid support such reference refers to the external surface of the organism or solid support, that is the surface facing the environment in which the organism to be flocculated is present.
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
The disclosure herein provides novel approaches to initiating flocculation in photosynthetic organisms, and particularly in non-vascular photosynthetic organisms (NVPO). Growth of photosynthetic organisms, particularly single-celled organisms such as microalgae or cyanobacteria, for industrial or agricultural purposes often involves harvesting the cells of the organisms from liquid environments. Currently, much of this harvesting is performed by centrifugation, belt-press processing, adding chemical flocculants to the liquid environment or some combination of methods. Traditional methods of flocculation may lead to additional costs cost of the flocculant, cost for removal of the flocculant from the liquid and/or organisms, etc.) and other problems (e.g., pollution, product purification, etc.). The present disclosure provides novel compositions, host cells and methods for flocculating photosynthetic organisms which can overcome the detriments of traditional flocculation methodologies.
The present disclosure takes advantage of strong molecular interactions between different molecules, for example, antibodies and antigens, proteins and carbohydrates, protein-protein binding pairs, etc. Flocculation pairs (two component complexes) or flocculation complexes (more than two component complexes) may be utilized. In some instances, one member of a flocculation pair or complex will be naturally expressed on an organism which will be flocculated, but the level of production may be genetically enhanced. In other instances, one member of a flocculation pair or complex will be recombinantly expressed on an organism to be flocculated. The organism is then contacted with the second (and/or subsequent) flocculation moiety to induce flocculation. The second or subsequent flocculation moieties may be added extrinsically (e.g., on a solid phase such as a bead or sieve), expressed on a separate organism (which may be the same or different species as the first organism) which is then contacted with the first organism, or expressed on the same organism (e.g., under control of an inducible regulatory element).
One approach utilized involves genetic manipulation of a photosynthetic organism (e.g., an NVPO) to express one or more flocculation moieties. Genetic manipulation may involve transient or integrative transformation of a photosynthetic organism (e.g., Chlamydomonas reinhardtii, Dunaliella salina, Dunaliella tertiolecta, Scenedesmus dimorphus, Hematococcus phrvalis) or a non-photosynthetic organism (e.g. E. coli) with a nucleic acid encoding a flocculation moiety. In some instances, the flocculation moiety is a protein capable of binding to another protein, a carbohydrate or another molecule of interest. For example, C. reinhardtii may be transformed with a gene encoding a C. reinhardtii-cell surface protein fused to an antibody, such that expression of the antibody on the surface of the cell will cause flocculation of the transformed cells or any other target organisms. Antibodies have been produced in C. Reinhardtii chloroplasts, but such expression does not yield antibody on the surface of the cell. See, e.g., Mayfield et al., PNAS 100(2):438-42 (2003). Alternatively the antibody can be expressed on the surface of a non-photosynthetic organism. In one embodiment, the non-photosynthetic organism is a prokaryote such as a bacterium. In another embodiment, the non-photosynthetic organism is a eukaryote, for example a fungi, and more particularly a yeast. The cell surface antibodies on the surface of the non-photosynthetic organism then bind to multiple photosynthetic organisms resulting in flocculation.
The present disclosure also contemplates a host cell transformed with one or more of the nucleic acids described herein. In certain embodiments, the host cell is photosynthetic. In some cases, the host cell is photosynthetic and non-vascular. In other cases, the host cell is photosynthetic and vascular. In still other cases the host cell is non-photosynthetic. The host cell can be eukaryotic or prokaryotic. Some host cells may be transformed with multiple genes encoding one or more flocculation moieties. For example, a single transformed cell may contain exogenous nucleic acids encoding one, two, three or more proteins or subunits thereof. For example, an alga such as C. reinhardtii, a bacterium such as E. coli or a cyanobacterium, or a yeast such as Pichia pastoris, may be transformed with a gene encoding an antibody which recognizes a cell-surface protein of the organism to be flocculated, where the antibody is produced as a fusion protein or in two or more subunits Which are assembled internally. The organism to be flocculated may be the same or different as the organism expressing the surface protein. Constructs may contain multiple copies of the same gene, and/or multiple genes encoding the same protein, and/or multiple genes with mutations in one or more parts of the coding sequences.
The host cell is transfected with a vector described herein (e.g., a vector comprising one or more flocculation moiety encoding genes). The vector may contain a plastid promoter or a nuclear promoter for transforming the nucleus or a chloroplast or other plastid of the host cell. The vector may also encode a fusion protein or agent that selectively targets the vector product to nucleus or the chloroplast or other plastid. Transfection of a host cell can occur using any method known in the art.
A host organism is an organism comprising a host cell. In certain embodiments, the host organism is photosynthetic. A photosynthetic organism is one that naturally photosynthesizes (has a plastid) or that is genetically engineered or otherwise modified to be photosynthetic. In some instances, a photosynthetic organism may be transformed with a construct which renders all or part of the photosynthetic apparatus inoperable. In some instances it is non-vascular and photosynthetic. The host cell can be prokaryotic. Examples of some photosynthetic prokaryotic organisms of the present invention include, but are not limited to cyanobacteria (e.g., Synechococcus, Synechocystis, Athrospiro). The host organism can be unicellular or multicellular. In several embodiments, the host organism is eukaryotic (e.g. green algae). Examples of organisms contemplated herein include, but are not limited to, rhodophyta, chlorophyta, heterokontophyta, tribophyta, glaucophyta, chlorarachniophytes, euglenoids, haptophyta, cryptomonads, dinoflagellata, and phytoplankton. In other embodiments the host organism is non-photosynthetic. Non-photosynthetic host organisms include bacteria and yeast. Examples of suitable bacterial host organisms can be found in the phylum proteobaeteria and include, but are not limited to Escherichia coli. Examples of suitable yeast organism can be found in the phylum Ascomycota and include, but are not limited to Pichiapasioris and Saccharomyces cerevisiae.
A host organism may be grown under conditions which permit photosynthesis, however, this is not a requirement (e.g., a host organism may be grown in the absence of light). In some instances, the host organism may be genetically modified in such a way that photosynthetic capability is diminished and/or destroyed. In growth conditions where a host organism is not capable of photosynthesis (e.g., because of the absence of light and/or genetic modification), typically, the organism will be provided with the necessary nutrients to support growth in the absence of photosynthesis. For example, a culture medium in (or on) which an organism is grown, may be supplemented with any required nutrient, including an organic carbon source, nitrogen source, phosphorous source, vitamins, metals, lipids, nucleic acids, micronutrients, or an organism-specific requirement. Organic carbon sources include any source of carbon which the host organism is able to metabolize including, but not limited to, acetate, simple carbohydrates (e.g., glucose, sucrose, lactose), complex carbohydrates (e.g., starch, glycogen), proteins, and lipids. One of skill in the art will recognize that not all organisms will be able to sufficiently metabolize a particular nutrient and that nutrient mixtures may need to be modified from one organism to another in order to provide the appropriate nutrient mix.
A host organism can be grown on land, e.g., in ponds, aqueducts, landfills, or in closed or partially closed bioreactor systems. The host organisms can also be grown directly in water, e.g., oceans, seas, lakes, rivers, reservoirs, etc. In embodiments where algae are mass-cultured, the algae can be grown in high density photobioreactors. Methods of mass-culturing algae are known in the art. For example, algae can be grown in high density photobioreactors (see, e.g., Lee et al, Biotech. Bioengineering 44:1161-1167, 1994) and other bioreactors (such as those for sewage and waste water treatments) (e.g., Sawayama et al, Appl. Micro. Biotech., 41:729-731, 1994). Additionally, algae may be mass-cultured to remove heavy metals (e.g., Wilkinson, Biotech. Letters, 11:861-864, 1989), hydrogen (e.g., U.S. Patent Application Publication No. 20030162273), and pharmaceutical compounds
An exemplary group of organisms are the green algae. One example, Chlamydomonas, is a genus of unicellular green algae (Chlorophyta). These algae are found in soil, fresh water, oceans, and even in snow on mountaintops. Algae in this genus have a cell wall, a chloroplast, and two anterior flagella allowing mobility in liquid environments. More than 500 different species of Chlamydomonas have been described.
The most widely used laboratory species is C. reinhardtii. Cells of this species are haploid, and can grow on a simple medium of inorganic salts, using photosynthesis to provide energy. They can also grow in total darkness if acetate is provided as a carbon source. When deprived of nitrogen, C. reiniardtii cells can differentiate into isogametes. Two distinct mating types, designated mt+ and mt−, exist. These fuse sexually, thereby generating a thick-walled zygote which forms a hard outer wall that protects it from various environmental conditions. The controlled expression of mating type agglutinins can be used to aid in flocculation. When restored to nitrogen culture medium in the presence of light and water, the diploid zygospore undergoes meiosis and releases four haploid cells that resume the vegetative life cycle. In mitotic growth the cells double as fast as every eight hours. C. reinhardtii cells can grow under a wide array of conditions. While a dedicated, temperature-controlled space can result in optimal growth, C reinhardtii can be readily grown at room temperature under standard fluorescent lights. The cells can be synchronized by placing them on a light-dark cycle and depriving them of acetate.
The nuclear genetics of C. reinhardtii is well established. There are a large number of mutants that have been characterized and the C. reinhardtii center (www.chlamy.org) maintains an extensive collection of mutants, as well as annotated genomic sequences of Chlamydomonas species. A large number of chloroplast mutants as well as several mitochondrial mutants have been developed in C. reinhardtii.
The term “plant” is used broadly herein to refer to a eukaryotic organism containing plastids, particularly chloroplasts, and includes any such organism at any stage of development, or to part of a plant, including a plant cutting, a plant cell, a plant cell culture, a plant organ, a plant seed, and a plantlet. A plant cell is the structural and physiological unit of the plant, comprising a protoplast and a cell wall. A plant cell can be in the form of an isolated single cell or a cultured cell, or can be part of higher organized unit, for example, a plant tissue, plant organ, or plant. Thus, a plant cell can be a protoplast, a gamete producing cell, or a cell or collection of cells that can regenerate into a whole plant. As such, a seed, which comprises multiple plant cells and is capable of regenerating into a whole plant, is considered plant cell for purposes of this disclosure. A plant tissue or plant organ can be a seed, protoplast, callus, or any other groups of plant cells that is organized into a structural or functional unit. Particularly useful parts of a plant include harvestable parts and parts useful for propagation of progeny plants. A harvestable part of a plant can be any useful part of a plant, for example, flowers, pollen, seedlings, tubers, leaves, stems, fruit, seeds, roots, and the like. A part of a plant useful for propagation includes, for example, seeds, fruits, cuttings, seedlings, tubers, rootstocks, and the
The vectors described herein may be capable of stable transformation of multiple photosynthetic organisms, including, but not limited to, photosynthetic bacteria (including cyanobacteria), cyanophyta, prochlorophyta, rhodophyta, chlorophyta, heterokontophyta, tribophyta, glaucophyta, chlorarachniophytes, euglenophyta, eugienoids, haptophyta, chrysophyta, cryptophyta, cryptomonads, dinophyta, dinoflagellate, pyrmnesiophyta, bacillariophyta, xanthophyta, eustigmatophyta, raphidophyta, phaeophyta, and phytoplankton. Other vectors are capable of stable transformation of C. reinhardtii, D. salina, D. tertiolecta, S. dimorphus or H. pluvialis. Still other vectors are capable of stable transformation of non-photosynthetic organisms such as yeast and bacteria. Vectors for stable transformation of bacteria and yeast are well known in the art and can be obtained from commercial vendors. Expression vectors can be engineered to produce heterologous and/or homologous protein(s) of interest (e.g., antibodies, mating type agglutinins, etc.). Such vectors are useful for recombinantly producing the protein of interest. Such vectors are also useful to modify the natural phenotype of host cells (e.g., expressing a flocculation moiety).
An expression cassette can be constructed in an appropriate vector. In some instances, the cassette is designed to express one or more protein-coding sequences in a host cell. Such vectors can be constructed using standard techniques known in the art. In a typical expression cassette, the promoter or regulatory element is positioned on the 5′ or upstream side of a coding sequence whose expression is desired. In other cassettes, a coding sequence may be flanked by sequences which allow for expression upon insertion into a target genome (e.g., nuclear or plastid). For example, a nucleic acid encoding a flocculation moiety may be inserted into a nuclear genome of a host cell, such that the flocculation moiety expression is controlled by a naturally occurring regulatory element. In the present disclosure, any regulatory element which provides expression under appropriate conditions such that the mRNA or protein product is expressed to a level sufficient to produce flocculation of the transformed NVPO can be used.
One or more additional protein coding sequences can be operatively fused downstream or 3′ of a promoter. Coding sequences for single proteins can be used, as well as coding sequences for fusions of two or more proteins. Coding sequences may also contain additional elements that would allow the expressed proteins to be targeted to the cell surface and either be anchored on the cell surface or be secreted to the environment. A selectable marker is also employed in the design of the vector for efficient selection for green algae transformed by the vector. Both a selectable marker and another sequence which one desires to introduce may be introduced fused to and downstream of a single promoter. Alternatively, two protein coding sequences can be introduced, each under the control of a promoter.
A regulatory element, as the term is used herein, broadly refers to a nucleotide sequence that regulates the transcription or translation of a polynucleotide or the localization of a polypeptide to which it is operatively linked. A regulatory element may be native or foreign to the nucleotide sequence encoding the polypeptide. Such elements include, but are not limited to, a ribosome binding site (RBS), a leader, a polyadenylation sequence, a pro-peptide sequence, a promoter, a signal peptide sequence, and a transcription terminator, an enhancer, an initiation (start) codon, a splicing signal for intron excision and maintenance of a correct reading frame, a STOP codon, an amber or ochre codon, and/or an internal ribosome entry site (IRES). Typically, a regulatory element includes a promoter and transcriptional and translational stop signals. Elements may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleotide sequence encoding a polypeptide. In some instances, such vectors include promoters. Additionally, a cell compartmentalization signal (i.e., a sequence that targets a polypeptide to the cytosol, nucleus, chloroplast membrane or cell membrane). Such signals are well known in the art and have been widely reported (see, e.g., U.S. Pat. No. 5,776,689). Promoters usefill for the present invention may come from any source (e.g., viral, bacterial, fungal, protist, animal). The promoters contemplated herein can be specific to photosynthetic organisms, non-vascular photosynthetic organisms, vascular photosynthetic organisms (e.g., algae, flowering plants), yeast and bacteria. As used herein, the term “non-vascular photosynthetic organism,” refers to any macroscopic or microscopic organism, including, but not limited to, algae, cyanobacteria and photosynthetic bacteria, which do not have a vascular system such as that found in higher plants. In some instances, the nucleic acids above are inserted into a vector that comprises a promoter of a photosynthetic organism, e.g., algae. The promoter can be a promoter for expression in the nucleus or in a chloroplast and/or other plastid. Examples of promoters contemplated for insertion with any of the nucleic acids herein into the chloroplast include those disclosed in US Application No. 2004/0014174. Promoters suitable for use in bacteria, include but are not limited to, promoters from Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV) promoter, the HIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV. Cytomegalovirus (CMV) such as the CMV immediate early promoter, Epstein Barr Virus (EBV), and Rous Sarcoma Virus (RSV). The promoter can be a constitutive promoter or an inducible promoter. A promoter typically includes necessary nucleic acid sequences near the start site of transcription, (e.g., a TATA element).
To construct the vector, the upstream DNA sequences of a gene expressed under control of a suitable promoter may be restriction mapped and areas important for the expression of the protein characterized. The exact location of the start codon of the gene is determined and, making use of this information and the restriction map, a vector may be designed for expression of a heterologous protein by removing the region responsible for encoding the gene's protein but leaving the upstream region found to contain the genetic material responsible for control of the gene's expression. A synthetic oligonucleotide is preferably inserted in the location where the protein sequence once was, such that any additional gene could be cloned in using restriction endonuclease sites in the synthetic oligonucleotide (i.e., a multicloning site). An unrelated gene (or coding sequence) inserted at this site would then be under the control of an extant start codon and upstream regulatory region that will drive expression of the foreign (i.e., not normally present) protein encoded by this gene. Once the gene for the foreign protein is put into a cloning vector, it can be introduced into the host organism using any of several methods, some of which might be particular to the host organism. Variations on these methods are described in the general literature. Manipulation of conditions to optimize transformation for a particular host is within the skill of the art.
Regarding the type of plasmid used for the insertion of the various DNA expression cassette, any convenient plasmid may be employed. The plasmids, or vectors, may include such vectors as pUC or its derivatives, pBR322, pBluescript, or pGEM. Other vectors included pPIC3.5K, pPIC9K and pAO815 available from Invitrogen (Carlsbad, Calif. USA). A particular plasmid is chosen based on the nature of the markers, availability of convenient restriction sites, copy number and the like. The DNA sequence encoding the polypeptide of interest may be synthetic, naturally derived, or any combination thereof. Depending upon the nature of the DNA sequence of interest, it may be desirable to synthesize the sequence with codons preferred in NVPO. The DNA sequence encoding the polypeptide of interest may be a structural gene or a portion thereof which provides for a desired expression product. The gene may be any gene, whether native, mutant of the native, or foreign to the host. The term foreign is intended to denote a gene not endogenous to the host cell, but may include indigenous sequences, such as viral sequences and bacterial sequences which are naturally associated with the NVPO.
A vector utilized can also contain one or more additional nucleotide sequences that confer desirable characteristics on the vector, including, for example, sequences such as cloning sites that facilitate manipulation of the vector, regulatory elements that direct replication of the vector or transcription of nucleotide sequences contain therein, sequences that encode a selectable marker, and the like. As such, the vector can contain, for example, one or more cloning sites such as a multiple cloning sites, which can, but need not, be positioned such that a heterologous polynucleotide can be inserted into the vector and operatively linked to a desired element. The vector also can contain a prokaryote origin of replication (ori), for example, an E. coli ori or a cosmid ori, thus allowing passage of the vector in a prokaryote host cell, as well as in a plant chloroplast, as desired. Such features, combined with appropriate selectable markers, allows for the vector to be “shuttled” between the target host cell and the bacterial and/or yeast cell. The ability to passage a shuttle vector in a secondary host may allow for more convenient manipulation of the features of the vector. For example, a reaction mixture containing the vector and putative inserted polynucleotides of interest can be transformed into prokaryote host cells such as E. coli, amplified and collected using routine methods, and examined to identify vectors containing an insert or construct of interest. If desired, the vector can be further manipulated, for example, by performing site directed mutagenesis of the inserted polynucleotide, then again amplifying and selecting vectors having a mutated polynucleotide of interest. A shuttle vector then can be introduced into plant cell chloroplasts, wherein a polypeptide of interest can be expressed and, if desired, isolated.
A vector or other recombinant nucleic acid molecule may include a nucleotide sequence encoding a reporter polypeptide or other selectable marker. The term “selectable marker” refers to a polynucleotide (or encoded polypeptide) that confers a detectable phenotype. A reporter generally encodes a detectable polypeptide, for example, a green fluorescent protein or an enzyme such as luciferase, which, when contacted with an appropriate agent (a particular wavelength of light or luciferin, respectively) generates a signal that can be detected by eye or using appropriate instrumentation (Giacomin, Plant Sci. 116:59-72, 1996; Scikantha, J. Bacteriol. 178:121, 1996; Gerdes, FEBS Lett. 389:44-47, 1996; see, also, Jefferson, EMBO J. 6:3901-3907. 1997, f1-glucuronidase). A selectable marker generally is a molecule that, when present or expressed in a cell, provides a selective advantage (or disadvantage) to the cell containing the marker, for example, the ability to grow in the presence of an agent that otherwise would kill the cell.
A selectable marker can provide a means to obtain prokaryotic cells or plant cells or both that express the marker and, therefore, can be useful as a component of a vector (see, for example, Bock, supra, 2001). Examples of selectable markers include, but are not limited to, those that confer antimetabolite resistance, for example, dihydrofolate reductase, which confers resistance to methotrexate (Reiss, Plant Physiol. (Life Sci. Adv.) 13:143-149, 1994); neomycin phosphotransferase, which confers resistance to the aminoglycosides neomycin, kanamycin and paromycin (Herrera-Estrella, EMBO 2:987-995, 1983), hygro, which confers resistance to hygromycin (Marsh, Gene 32:481-485, 1984), trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman, Proc. Natl. Acad. Sci., USA 85:8047, 1988); mannose-6-phosphate isomerase which allows cells to utilize mannose (WO 94/20627); ornithine decarboxylase, which confers resistance to the ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine (DFMO; McConlogue, 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.); and deaminase from Aspergillus terreus, which confers resistance to Blasticidin S (Tamura, Biosci. Biotechnol. Biochem. 59:2336-2338, 1995). Additional selectable markers include those that confer herbicide resistance, for example, phosphinothricin acetyltransferase gene, which confers resistance to phosphinothricin (White et al., Nucl. Acids Res. 18:1062, 1990; Spencer et al., Theor. Appl. Genet. 79:625-631, 1990), a mutant EPSPV-synthase, which confers glyphosate resistance (Hinchee et al., BioTechnology 91:915-922, 1998), a mutant acetolactate synthase, which confers imidazolione or sulfonylurea resistance (Lee et al., EMBO J. 7:1241-1248, 1988), a mutant psbA, which confers resistance to atrazine (Smeda et al., Plant Physiol. 103:911-917, 1993), or a mutant protoporphyrinogen oxidase (see U.S. Pat. No. 5,767,373), or other markers conferring resistance to an herbicide such as glufosinate. Selectable markers include polynucleotides that confer dihydrofolate reductase (DHFR) or neomycin resistance for eukaryotic cells and tetracycline; ampicillin resistance for prokaryotes such as E. coli; and bleomycin, gentamycin, glyphosate, hygromycin, kanamycin, methotrexate, phleomycin, phosphinotricin, spectinomycin, streptomycin, sulfonamide and sulfonylurea resistance in plants (see, for example, Maliga et al., Methods in Plant Molecular Biology, Cold Spring Harbor Laboratory Press, 1995, page 39).
One or more codons of an encoding polynucleotide can be biased to reflect the preferred codon usage of the host cell, or an organelle thereof (e.g. chloroplasts). Most amino acids are encoded by two or more different (degenerate) codons, and it is well recognized that various organisms utilize certain codons in preference to others. The codon bias of C. reinhardtii has been reported (See U.S. Application 2004/0014174) as has codon bias in bacteria and yeast (See e.g. Gene 18:199-209 (1982); FEBS Lett., 285:165-169 (1991); J. Biol. Chem., 257:3026-3031 (1982)).
The term “biased,” when used in reference to a codon, means that the sequence of a codon in a polynucleotide has been changed such that the codon is one that is used preferentially in the target which the bias is for, e.g., alga cells, chloroplasts, bacteria or yeast. A polynucleotide that is biased for a particular codon usage can be synthesized de novo, or can be genetically modified using routine recombinant DNA techniques, for example, by a site directed mutagenesis method, to change one or more codons such that they are biased for chloroplast codon usage. Codon bias can be variously skewed in different plants, including, for example, in alga as compared to tobacco. Generally, the codon bias selected reflects codon usage of the plant (or organelle therein) which is being transformed with the nucleic acids of the present invention. For example, where C. reinhardtii is the host, the chloroplast codon usage may be biased to reflect nuclear or chloroplast codon usage (e.g., about 74.6% AT bias in the third codon position for sequences targeting the chloroplast). Alternatively, when bacteria or yeast are used as the host organism, codon usage may be biased for those organisms.
Disclosed herein are vectors employed in various flocculation methods. These vectors or plasmids may comprise regulatory elements recognized by the gene-transcription machinery of the host organism used, including for nuclear or plastid genes, coding sequences operably linked to the regulatory elements, selection markers allowing the selection for host cells transformed by the vector, and other elements allowing the vector to be stably integrated into the nuclear or plastid genome of a host organism. Examples of regulatory elements include, but are not limited to, constitutive promoters, light-inducible promoters, quorum-sensing promoters, temperature-sensitive promoters, or nitrogen-starvation responsive promoters. An example of light-inducible promoter is described in U.S. Pat. No. 6,858,429. Examples of flocculation moieties which may be encoded by polynucleotides used in the present disclosure include, but are not limited to, FhuA, pb5, Chlamydomonas male gamete agglutinin (mt+), Chlamydomonas female gamete agglutinin (mt−), lectin, carbohydrate binding proteins, antibodies (e.g., anti-carbohydrate antibody, anti-flagella antibody, anti-Fus1 antibody, anti-cell surface protein antibodies). Examples of selection markers may include, but are not limited to, kanamycin, phleomycin, bleomycin, hygromycin, or zeocin.
Transformed cells are produced by introducing homologous and/or heterologous DNA into a population of target cells and selecting the cells which have taken up the DNA. For example, transformants containing exogenous DNA with a selectable marker which confers resistance to kanamycin may be grown in an environment containing kanamycin.
The basic techniques used for transformation and expression in photosynthetic organisms are similar to those commonly used for E. coli, Saccharomyces cerevisiae and other species and include calcium phosphate transfection, DEAE-dextran mediated transfection, Polyhrene, protoplast fusion, liposomes, direct microinjection into the nuclei, scrape loading, and electroporation. Transformation methods customized for an NVPO, e.g., the chloroplast of a strain of algae, are known in the art. These methods have been described in a number of texts for standard molecular biological manipulation (see Packer & Glaser, 1988, “Cyanobacteria”, Meth. Enzymol., Vol. 167; Weissbach & Weissbach, 1988, “Methods for plant molecular biology,” Academic Press, New York, Sambrook, Fritsch & Maniatis, 1989, “Molecular Cloning: A laboratory manual,” 2nd edition Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; and Clark M S, 1997, Plant Molecular Biology, Springer, N.Y.). These methods include, for example, biolistic devices (See, for example, Sanford, Trends in Biotech. (1988) 6: 299-302, U.S. Pat. No. 4,945,050; electroporation (Fromm et al., Proc. Nat'l, Acad. Sci. (USA) (1985) 82: 5824-5828); use of a laser beam, electroporation, microinjection or any other method capable of introducing DNA into a host cell (e.g., an NVPO).
Plastid transformation is a routine and well known method for introducing a polynucleotide into a plant cell chloroplast (see U.S. Pat. Nos. 5,451,513, 5,545,817, and 5,545,818; WO 95/16783; McBride et al., Proc. Natl. Acad. Sci., USA 91:7301-7305, 1994). In some embodiments, chloroplast transformation involves introducing regions of chloroplast DNA flanking a desired nucleotide sequence, allowing for homologous recombination of the exogenous DNA into the target chloroplast genome. In some instances one to 1.5 kb flanking nucleotide sequences of chloroplast genornic DNA may be used. Using this method, point mutations in the chloroplast 16S rRNA and rps12 genes, which confer resistance to spectinomycin and streptomycin, can be utilized as selectable markers for transformation (Svab et al., Proc. Natl. Acad. USA 87:8526-85:30, 1990), and can result in stable hornoplasmic transformants, at a frequency of approximately one per 100 bombardments of target leaves.
Microprojectile mediated transformation also can be used to introduce a polynucleotide into a plant cell (Klein et al., Nature 327:70-73, 1987). This method utilizes microprojectiles such as gold or tungsten, which are coated with the desired polynucleotide by precipitation with calcium chloride, spermidine or polyethylene glycol. The microprojectile particles are accelerated at high speed into a plant tissue using a device such as the BIOLISTIC PD-1000 particle gun (BioRad; Hercules Calif. USA). Methods for the transformation using biolistic methods are well known in the art (see, e.g.; Christou, Trends in Plant Science 1:423-431, 1996). Microprojectile mediated transformation has been used, for example, to generate a variety of transgenic plant species, including cotton, tobacco, corn, hybrid poplar and papaya. Important cereal crops such as wheat, oat, barley, sorghum and rice also have been transformed using microprojectile mediated delivery (Duan et al. Nature Biotech. 14:494-498, 1996; Shimamoto, Curr. Opin. Biotech. 5:158-162, 1994). The transformation of most dicotyledonous plants is possible with the methods described above. Transformation of monocotyledonous plants also can be transformed using, for example, biolistic methods as described above, protoplast transformation, electroporation of partially permeabilized cells, introduction of DNA using glass fibers, the glass bead agitation method, and the like. Transformation frequency may be increased by replacement of recessive rRNA or r-protein antibiotic resistance genes with a dominant selectable marker, including, but not limited to the bacterial aadA gene (Svab and Maliga, Proc. Natl. Acad. Sci., USA 90:913-917, 1993).
To harvest a product of interest from a host cell of the present invention (e.g., an NVPO), efficient separation of the host cell from the liquid in which it is grown is often useful. Various methods of separating the organism from the liquid have been used in the past. Centrifugation, for example, has been successfully used in separating cells in small scale culture from liquid media. For large scale applications, however, which usually involve growing an organism in large quantities of water (e.g., a pond, a lake, a bioreactor, etc.), centrifugation can be time-consuming and/or cost-prohibitive. Flocculation is an alternative approach to the separation of cells for large and small scale applications. Certain chemical flocculants, such as heavy metals, pose a challenge as it the metal may need to be removed from the flocculated organism for downstream processes (e.g., enzyme purification, nutriceutical production, transesterification of triglycerides, etc.). Strains of organisms (e.g., NVPOs) engineered to express flocculation moieties, on the other hand, do not present the problem of removing a dangerous, costly, or polluting flocculant from the organisms and/or the liquid environment. Thus, one novel aspect of the present disclosure is the production of an organism engineered to produce one or more flocculation moieties.
The flocculation moiety can be incorporated into an organism, either photosynthetic or non-photosynthetic, by transformation with vectors, such as those described herein. Techniques involved in such processes include, but are not limited to, the development of suitable expression cassette, insertion (i.e. transformation) of the expression cassette into a host cell, and screening the host cell expressing the desired flocculation moiety. Depending on the design of the vector, the flocculation moiety can be of constitutively expressed (e.g., at all times) or can inducibly expressed (e.g., temperature-induced, quorum-induced, etc.). Engineered photosynthetic organisms capable of expressing one or more flocculation moieties can be used for flocculation with or without the addition of other compounds. For example, a non-photosynthetic organisms such as E. coli can be transformed so as to produce a carbohydrate-binding protein, for example a lectin, or an antibody fused to a cell surface protein (for example LppOrnpA, beta-autotransporter, etc.) such that when expressed, the fusion protein is present on the cell surface and binds to multiple organisms of interest, such as a green algae, to cause flocculation. Alternatively, a green algae such as C. reinhardtii, may be transformed to produce a carbohydrate-binding protein, for example a lectin, or an antibody fused to a cell surface protein, for example GP1, such that the fusion protein is expressed at the cell surface and binds to multiple organisms of interest, such as green algae, to cause flocculation.
Alternately, one host organism (e.g., C. reinhardtii) may be transformed so as to produce the FhuA protein from E. coli and a second host organism—the same or different species than the first organism—may be transformed so as to produce the T5 phage tail protein, pb5. FhuA and pb5 form a very stable 1:1 stoichiometric complex, thus, by combining the two transformed host cells at a desired time, or by controlling expression of the two flocculation moieties in the different strains to only express the flocculation moieties at a desired time, binding between the two moieties will cause flocculate via the interaction of the two moieties.
A flocculating moiety will typically be expressed such that it is present on the outer surface of the host cell (e.g., cell wall and/or cell membrane). In some instances, a flocculation moiety is secreted by the host cell into the surrounding environment. Standard molecular biology techniques allow the targeting of a recombinantly expressed protein to various subcellular compartments of a host cell. Examples of such subcellular compartments include Golgi, lysosome, secretion system, cell wall or plasma membrane. Thus, in some instances, the vectors useful in the present disclosure will contain DNA sequences encoding one or more specialized polypeptides (e.g., signal peptides) that are capable of directing recombinant proteins to subcellular compartments if the signal peptide is operably linked to the recombinant protein.
Candidates for genes expressing flocculation moieties can be obtained from a variety o :organisms including eukaryotes, prokaryotes, or viruses. In some instances, a flocculation moiety is one member of a protein binding pair. Protein pairs forming strong protein-protein complexes are useful as flocculation moieties. Self-aggregating proteins, proteins capable of forming multimeric complexes are also useful as flocculants. Carbohydrate moieties of glycoproteins (e.g., arabinosyl, galactosyl, mannosyl, and rhamnosyl residues), membrane and/or cell wall carbohydrates (e.g., alginic acid, xylanes, mannanes, agarose, carrageenan, porphyran, furcelleran, etc.), proteins increasing the production of certain carbohydrates or glycolipids may also be used to induce flocculation as certain classes of carbohydrates are known components of protein complex formation.
Flocculation moieties can also be recombinantly expressed in host cells and purified to a useful level (e.g., homogeneity). The purified flocculants can be added to a target cell culture to cause flocculation. Such flocculants typically will not pose the same challenges as the use of heavy metal flocculants. For example, a recombinant lectin can be produced by a host cell (e.g. secreted or produced on the surface), collected, and introduced into a culture of an organism to be flocculated.
One of skill in the art will recognize that multiple types of molecules can be utilized in the practice of the present methods. Disclosed herein are various exemplary proteins, carbohydrates, antibodies, and other moieties which may be utilized as a flocculation moiety, either when expressed by a microorganism or by incorporation in the liquid culture media. These examples are meant to provide illustration of the types of molecules which can be utilized and are not intended to be limiting.
Proteins, as a category, are known to bind and/or complex with other macromolecules and compounds. As such, proteins may be utilized as flocculation moieties. Carbohydrate binding proteins (e.g., lectins), for example, are one category of protein which is useful for a flocculation moiety. Carbohydrate binding proteins recognize and bind to a variety of cell surface carbohydrates, carbohydrate moieties, polysaccharide side chains of glycoproteins, glycosylated proteins, glycopeptides, and/or cell surface proteins. Thus, in one embodiment, c-type lectin is expressed on the cell wall of C. reinhardtii, which induces flocculation by binding to a glycoprotein on the surface of C. reinhardtii Examples of these flocculation moieties include, but are not limited to, lysophosphatidic acid, c-type lectin, Gal/GalNAc. O-linked sugars, O-linked polysaccharides, GlcNAc, phospholipase A2, GalNAc-SO4, sialic acid, glycosphingolipids, glucose monomycolate, lipoarabinomannan, phosphatidyl inositols, hexosyl-1-phosphoisoprenoids, mannosyl-phosphodolicols, α-galactosylceramide, or terminal galactoside. Examples of carbohydrate binding proteins include, but are not limited to, CD-SIGN, dectin-1, dectin-2, HECL, langerin, layilin, mincle, MMGL, E-selection, P-selectin, L-selectin, DEC-205, Endo 180, mannose receptor, phospholipase A2 receptor, sialoadhesin (siglec-1), siglec-2, siglec-3, siglec-4, siglec-5, siglec-6, siglec-7, siglec-8, siglec-9, siglec-10, siglec-11, or galectins.
Another category of proteins that may be utilized as flocculation moieties is antibodies. For example, antibodies against known cell surface antigens expressed on an organism can be used. Expression of such antibodies may lead antigen-antibody complex formation which can subsequently lead to flocculation of an organism expressing the antibody where the antibody specifically binds to a cell-surface antigen on the transformed strain, or a different strain or organism. For example, C. reinhardtii may be transformed to inducibly express a single chain variable fragment (scFv) of an antibody, which detects an antigen on the external surface of an organism. The surface antigen may be naturally occurring on the surface of C. reinhardtii or another organism or may be the result transformation of the organism to produce the cell surface antigen. The antibodies used may be univalent, multivalent, or polyvalent. Other antibodies against various glycoproteins are known in the art (See, e.g., Matsuda et. al., J. Plant. Res., 100:373-384, 1987; Musgrave et al., Planta, 170:328-335, 1987). The antibodies can be anchored on the cell surface by creating a fusion protein comprising a protein expressed at the cell surface, for example the cell membrane or cell wall. Alternately, the antibody can be engineered to be secreted by the host cell into the surrounding environment to cause flocculation. Antibodies recognizing cell wall components or engineered antibodies of the host cell can be added to the media or other growth environment to induce flocculation. Antibodies known for forming stable complexes with specific epitopes can be utilized as flocculation moieties. In some instances, antibodies may be utilized which target a naturally occurring epitope.
A first organism expressing such antibodies can be paired with another organism expressing the corresponding epitope so as to induce flocculation upon combination. The corresponding epitope may be naturally occurring or may be genetically engineered. Alternately, the corresponding epitope may be added to the media or other growth environment when flocculation is desired. Epitopes may be coupled to solid state supports such as beads, meshes, sieves, etc, to facilitate flocculation. Examples of antibodies useful for the present invention include, but are not limited to, anti-HA tag antibody, anti-His tag antibody, anti-Myc tag antibody, anti-AU1 tag antibody, anti-GST tag antibody, anti-KT3 tag antibody, anti-MAT tag antibody, anti-MBP tag antibody, anti-S tag antibody, anti-S1 tag antibody, anti-SNAP tag antibody, anti-SRT tag antibody, anti-V5 tag antibody, anti-VSV-G tag antibody, anti-TAP tag antibody, and anti-Trx antibody. One of skill in the art will recognize that a single host cell may express more than one flocculation moiety from one or more categories. For example an organism may be transformed to express two different antibodies or an antibody and a carbohydrate-binding protein.
In some embodiments, a flocculation moiety is an antibody to a cell surface epitope of a cell of interest. The particular cell surface epitope may be present on a cell surface protein. Examples of such proteins include, but are not limited to HRP-2, PHC21, ISG-C1, LMR3, VSP (1, 2, 3, 4), HRP3, GP1, FAS1, FAS2, FAS3, E_GWH.1280.7, GAS30, MMP14, ZAP2-2, MIN1, or GOX1 from C. reinhardtii and p150 from D. salina. Methods of producing antibodies to a given protein are well known in the art and may comprise culturing a photosynthetic organism; fractionating the membrane and/or cell wall portion of the microorganism; injecting the fraction to an appropriate host animal for immunization; withdrawing sera from the immunized animal; and screening, selecting and purifying one or more antibodies reacting to the fraction. Also, for example where the immunized animal is a mouse, immune cells producing the antibody of interest can be subjected to routine hybridoma formation and culture techniques to produce monoclonal antibodies of interest. In another embodiment, proteins are further purified from the membrane fraction and are utilized in high-throughput screening system as a target of phage display technique whereby DNA sequences expressing a portion of the antibody polypeptide interacting with the target are identified. Phage display techniques are known in the art. See, e.g., U.S. Pat. No. 5,855,885; McCafferty et al., Nature, 348:552-54, 1990.
Another category of proteins which may be utilized as flocculation moieties include proteins which form a binding pair. This category includes, among others, proteins forming multimeric complexes and proteins forming a complex with another class of protein. For example, the E. coli protein FhuA is a monomeric iron transporter channel with a single membrane spanning domain that can be overexpressed; pb5 is a T5 phage tail protein that uses FhuA as the phage receptor. The interaction between the two proteins is very stable. Such protein pairs can be utilized as flocculation moieties. For example, in one embodiment, FhuA is expressed in one strain of D. salina and pb5 is expressed in a second strain of a D. salina. Upon combination of the two strains, flocculation occurs due to interaction of the two proteins. One of skill in the art will recognize that many other protein binding pairs exist and can be utilized. To allow for interaction between some proteins, it may be necessary to engineer the proteins such that they are inserted into the cell membrane and/or cell wall.
Having control over the timing of flocculation may be advantageous in some instance to control flocculation. For example, by controlling the time of flocculation, an operator of a target culture system may ensure efficient use of nutrients, time of harvesting, quantity of harvested organism, and/or maximal product production. One of skill in the art will recognize that optimal conditions may vary according to multiple factors, but such determinations of culture medium preparation are routine. For flocculation moieties expressed by a given host organism, the timing of flocculation can be controlled by incorporating one or more regulatory elements (e.g., promoters) into the design of an expression cassette. Such an approach may allow for expression of a flocculation moiety only at a desired time. As is apparent from the present disclosure, multiple routes are available to control timing of flocculation. Although exemplary approaches to controlling the timing of flocculation, this discussion is not meant to limit these approaches to the particular approaches mentioned herein.
For expression of some flocculation moieties, regulatory elements resulting in constitutive expression can be used. Thus, the flocculation moiety under the control of such regulatory elements is expected to be produced throughout the life of the host cell. Examples of regulatory elements are 35S or 19S region of cauliflower mosaic virus (CaMV), the coat promoter of TMV, promoters of nopaline synthase, mannopine synthase, or octopine synthase of Agrobacterium, cell promoter, chalcone synthase promoter, actin promoter, adhI promoter, ubiquitin promoter, or patatin promoter. To induce flocculation, two separate strains constitutively expressing one part of as flocculation pair (e.g., antibody-antigen, protein binding pair, etc.) may be combined when flocculation is desired. In such instances, the flocculation moiety on one strain may be naturally occurring (e.g., the first strain produces a recombinant lectin which recognizes a glycoprotein on the second strain). Alternately, flocculation of a single strain may be induced by adding the second part of a flocculation pair to the culture or environment of the strain when flocculation is desired. In still another approach, host cells containing one part of a flocculation pair which is constitutively expressed may be grown in combination with another strain which expresses the second part of the flocculation pair under inducible conditions. In such instances, inducing expression of the second part of the flocculation pair in the second strain would lead to flocculation.
Inducible regulatory elements may be used to control expression of a flocculation moiety. In such embodiments, the organism, for example a non-vascular photosynthetic organism, may be kept in culture without the expression of the flocculation moiety until flocculation is desired. Induction of expression may be performed by introducing an extrinsic factor (e.g., exposed to light, where a light-inducible promoter controls expression of the flocculation moiety) or by the occurrence of a factor necessary for expression (e.g., reaching a particular cell density where a quorum-sensing promoter controls expression of a flocculation moiety). Extrinsic factors which may be used to induce expression of a flocculation moiety include, but are not limited to, chemicals, light, culture density, temperature, hormones, or nutrients. Non-limiting examples of inducible promoters responding to cell extrinsic factors which may be utilized for the present invention include: the soybean Gmhsp 17.3-B heat shock promoter (Prandl et al., Plant Mol. Physiol. 31:157-62, 2004); chemically inducible promoters (e.g., 1P′T′Glac promoter); phosphatephosphate starvation inducible promoter (e.g., U.S. Pat. No. 6,175,060); L-arabinose/ara B promoter; 3-β-indoylacrylic acid/Tip promoter, salicylic acid/PR-1 promoter (e.g., U.S. Pat. No. 5,689,044); galactose inducible promoters such as GAL1, GAL7, GAL10 (U.S. Pat. No 5,972,664); metal/metallothionine promoter; FUS1 promoter which is inducible by a pheromone (e.g., U.S. Pat. No. 5,063,154); light inducible promoters (e.g., subunit of ribulose bisphosphate carboxylase promoter, see also U.S. Pat. No. 6,858,429); quorum sensing promoters, which sense cell density in a liquid environment and can be utilized to automatically induce flocculation as the culture reaches a certain density (e.g., Whiteley et al., Journal of Bacteriology, 183:529-5534, 2001); a temperature sensing promoter system utilizing PL promoter has also been described (see e.g., U.S. Pat. No. 4,711,845.). One of skill in the art will recognize that one or more endogenous regulatory elements may be utilized to control expression of a flocculation moiety by, for example, including an endogenous promoter in an expression cassette or inserting a nucleic acid encoding a flocculation moiety into a target genome such that it will be controlled by a native regulatory element (e.g., homologous recombination).
Another type of promoter includes nitrogen starvation induced promoters. Nitrogen starvation results in the formation of gametes in certain NVPOs, such as C. reinhardtii. By withdrawing nitrogen or by limiting the amount of available nitrogen, flocculation can be induced by using nitrogen starvation responsive promoters to control expression of a flocculation moiety. Mating type agglutinins, which are naturally expressed by some NVPOs under nitrogen starvation conditions may be utilized, although not normally at levels which would yield flocculation in large culture. Thus, in one embodiment, increased production (e.g., through transformation of a host strain) of one or more agglutinins may yield flocculation.
For many organisms, including non-vascular photosynthetic organisms (NVPOs), routine growth can occur at room temperature on 1.5% agar, either on plates or in tubes, while active growth is typically performed in liquid culture. Optimal growth is usually between 20-25° C., though the cells can survive exposure to 35° C. and may be grown at lower temperatures. Cell densities of 1-5×106 cells/ml are normal in liquid, with shaking or mixing, and a typical growth rate may yield a tenfold increase in cells per day, depending on growth conditions. Long term storage of cells can be achieved by streaking them onto plates, sealing the plates with PARAFILM™ and placing them in dim light at 10-15° C. Alternatively, cells may be streaked or stabbed into agar tubes, capped and grown as above. Both methods allow storage for several months. For longer storage, the cells can be grown in liquid culture to late log phase and then made to 7% with sterile DMSO and stored at −80° C. Freezing the container in liquid nitrogen in the presence of methanol is also recommended for long term storage.
NVPOs can typically be grown on a simple defined medium with light as the sole energy source. In most cases fluorescent light bulbs at a distance of 1-2 feet are adequate to supply energy for growth. Bubbling with air or 5% CO2 may improve the growth rate. If the lights are turned on and off at regular intervals (either 12:12 or 14:10 hours of light:dark) the cells of some NVPOs can be synchronized.
Because photosynthetic organisms such as algae require sunlight, CO2 and water for growth, they can be cultivated in open ponds and lakes. Due to the fact that these are open system, they are much more vulnerable to being contaminated. One challenge with using open systems is that the NVPO of interest may not necessarily be the quickest to reproduce. This creates a problem where other species colonize the liquid environment. In addition, in open systems there is relatively less control over water temperature, CO2 concentration and lighting conditions. These imply that the growing season is largely dependent on location and aside from tropical areas, is limited to the warmer months. While the above are the disadvantages with “open systems”, some of the benefits of this type of system are that it typically has lower production costs.
Another approach is to use a semi-closed system, such as covering the pond or pool with a greenhouse. While this usually results in a smaller system, it addresses many of the problems associated with an open system. It allows more species to be grown, it allows the species that are being grown to stay dominant, it extends the growing season, and if the greenhouse is heated, production can continue year round. It is also possible to increase the amount of CO2 in these semi-closed systems, thus again increasing the rate of growth of algae.
A variation of the pond system is an artificial pond e.g., a raceway pond. In these ponds, the algae, water and nutrients circulate around a “racetrack.” By providing water movement, for example by the use of paddlewheels, algae are kept suspended in the water, and are circulated back to the surface on a regular frequency. Raceway ponds are usually kept shallow because the algae need to be exposed to sunlight, and sunlight can only penetrate the pond water to a limited depth. However, depth can be varied according to the wavelength(s) utilized by an organism. The ponds can be operated in a continuous manner, with CO2 and nutrients being constantly fed to the ponds, while algae-containing water is removed at the other end.
Alternatively, algae could be grown in closed structures such as photobioreactors, where the environment is under stricter control than in open ponds. While the costs of setting up and operating a photobioreactor would be higher than for those for open ponds, the efficiency and higher yields from these photobioreactors could be significantly higher as well, thus offsetting the initial cost disadvantage in the medium and long run.
A photobioreactor is a bioreactor that incorporates some type of light source. A pond covered with a greenhouse could also be considered a photobioreactor. Because these systems are closed everything that the algae need to grow, (carbon dioxide, water and light) need to be introduced into the system.
Photobioreactors can be set up to be continually harvested (the majority of the larger cultivation systems), or by harvesting a batch at a time (like polyethylene bag cultivation). A batch photobioreactor is set up with nutrients and algal seed, and allowed to grow until the batch is harvested. A continuous photobioreactor is harvested either continually, as daily, or more frequently. Some types of photobioreactors include glass, plastic tubes, tanks, plastic sleeves or bags. Some sources that can be used to provide the light energy required to sustain photosynthesis include fluorescent bulbs LEDs, or natural sunlight.
Some of the organisms which may be used to practice the present invention are halophilic. For example, D. salina can grow in ocean water and salt lakes (salinity from 30-300 parts per thousand) and high salinity media (e.g., artificial seawater medium, seawater nutrient agar, brackish water medium, seawater medium, etc.). In some embodiments a host cell comprising a vector described herein can be grown in a liquid environment which is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 31., 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3 molar or higher concentrations of sodium chloride. One of skill in the art will recognize that other salts (sodium salts, calcium salts, potassium salts, etc.) may also be present in the liquid environments.
Where a halophilic organism is utilized, it may be transformed with any of the vectors described herein. For example, D. salina may be transformed with a vector which is capable of insertion into the nuclear genome and which contains nucleic acids which encode a flocculation moiety (e.g., an anti-cell-surface-protein antibody, a carbohydrate binding protein, etc.). Transformed halophilic organisms may then be grown in high-saline environments (e.g., salt lakes, salt ponds, high-saline media, etc.) to produce the products (e.g., isoprenoids, fatty acids, biomass degrading enzymes, etc.) of interest. In some instances, the flocculation moiety may be non-functional under high salinity conditions. In such embodiments, flocculation may be induced by any of the methods described herein and/or by lowering the salinity (e.g., by diluting the liquid environment). Alternately, the flocculation moiety may be functional under high salinity conditions and flocculation may be controlled by any of the methods described herein. Flocculation of the organisms may take place under high salinity conditions, or the liquid environment may be diluted to a lower salinity to allow binding. Isolation of any products of interest produced by the organism may involve removing a transformed organism from a high-saline environment prior to extracting the product from the organism. In instances where the product is secreted into the surrounding environment, it may be necessary to desalinate the liquid environment prior to any further processing of the product.
Provided herein are methods for flocculating a host organism. As discussed herein, flocculation moieties can be either extrinsic or intrinsic to a host organism. Extrinsic flocculation moieties may include recombinantly expressed and purified proteins, carbohydrates, or other biological molecules capable to binding the cell surface of an NVPO. Intrinsic flocculation moieties may include naturally occurring or recombinant proteins engineered to be expressed in the transformed NVPO, other biological material, such as naturally occurring carbohydrates, lipids, or glycolipids produced at natural levels or that are produced in increased quantity due to genetic modification of the transformed. Thus, any method which allows for interaction of at least two members of a flocculation pair may be utilized.
For example, flocculation may be induced by adding one part of a flocculation pair which binds to an intrinsic flocculation moiety (e.g, a lectin binding to a cell wall glycoprotein).
An alternate approach is shown in
In other instances, one part of the flocculation pair may he secreted by a genetically modified host cell and induce flocculation upon binding to the second part of the flocculation pair present on that cell or another cell. In still other instances, one member of a flocculation pair may be attached to a solid phase (e.g., a bead, a mesh, a sieve), to induce flocculation of a host strain expressing the other member of the flocculation pair.
Flocculation may be induced by combining, separately grown cultures in any ratio ranging from 1:1 to 1:10 where the different cultures each express one part of a flocculation pair (two-part binding complex) or flocculation complex (three-or-more-part binding complex). A generalized approach is shown in
Flocculation may be induced by inducibly regulating expression of one member of a flocculation pair, for example by: 1) exposing a culture from dark to light where a flocculation moiety is controlled by an inducible regulatory element; 2) increasing the culture temperature to about 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 34° C., 35° C., 36° C., 37° C., 39° C., 40° C., 41° C., 42° C., or 43° C. for a brief period of time ranging from several seconds to several minutes to a few hours; 3) increasing the culture density to about 5×106, 6×106, 7×106, 8×106, 9×106, 1×107, 2×107, 3×107, 4×107, 5×107, 6×107, 7×107, 8×107, 9×107, 1×108, 2×108, 3×108, 4×108, 5×108, 6×108, 7×108, 8×108, 9×108, 1×109, 2×109, 3×109, 4×109, 5×109, 6×109, 7×109, 8×109, 9×109, 1×1010, 2×1010, 3×1010, 4×1010, 5×1010, 6×1010, 7×1010, 8×1010, 9×1010 or more cells/ml; 4) adding nitrogen, for example in the form of nitrate, to the culture for about 15 min, 30 min, 45 min, 60 min, 75 min, 90 min, 105 min, 120 min, 135 min, 150 min, 165 min, 180 min, 195 min, 210 min, 225 min, 240 min, 255 min, 270 min, 285 min, 300 min, 315 min, 330 min, 345 min, 360 min, 375 min, 390 min, 405 min, 420 min, 435 min, 450 min, 465 min, 480 min, 495 min, 510 min, 525 min, 540 min, 555 min, 570 min, 585 min, 600 min, 12 hours, 18 hours, 24 hours 36 hours, 24 hours or more; and/or 5) depriving nitrogen from the culture media for about 15 min, 30 min, 45 min, 60 min, 75 min, 90 min, 105 min, 120 min, 135 min, 150 min, 165 min, 180 min, 195 min, 210 min, 225 min, 240 min, 255 min, 270 min, 285 min, 300 min, 315 min, 330 min, 345 min, 360 min, 375 min, 390 min, 405 min, 420 min, 435 min, 450 min, 465 min, 480 min, 495 min, 510 min, 525 min, 540 min, 555 min, 570 min, 585 min, 600 min, 12 hours, 18 hours, 24 hours 36 hours, 24 hours or more.
An example of growing two strains in the same culture where both parts of a flocculation pair are inducibly controlled is shown in
One advantage of the use of engineered anchor naturally expressed flocculation moieties, as disclosed herein, is the recycling of the liquid environment. The recycling of media (e.g., laboratory media, pond water, lake water, bioreactor contents, etc.) is economically advantageous, especially in large scale operations. For example, in a controlled circulating pond system, the liquid environment can be recycled by allowing continuous flow of the liquid while nutrients are continuously added. In another embodiment, in a closed photobioreactor system, media recycling may comprise scooping out flocculated NVPO mass; measuring the pH of the media; measuring the level of each nutrient in the liquid; adjusting nutrients to optimal level; sterilizing the liquid by autoclaving; and/or returning the media for new culture.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, specific examples of appropriate materials and methods are described herein.
As used in this specification and the appended claims, the singular forms “a”, “an” and “the” are to be construed to cover both singular and plural referents unless the content or context clearly dictates otherwise. Thus, for example, reference to “polypeptide” includes two or more such polypeptides. The terms “comprising,” “having,” “including,” and “containing” arc to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted.
Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. The headings provided in the description of the invention are included merely for convenience and are not intended to be limiting in the scope of the disclosure.
Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, molecular biology, nucleic acid chemistry, and protein chemistry described below are those well known and commonly employed by those of ordinary skill in the art. In accordance with the present invention, common recombinant DNA techniques, molecular biology techniques, molecular genetics, and microbiology techniques may be used by one of skill in the art. For example, techniques such as those described in Sambrook, Goeddel, supra, and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1994, supplemented through 1999) (hereinafter “Ausubel”), DNA CLONING: A PRACTICAL APPROACH, Vols. I-II (Glover ed. 1985); Animal Cell Culture (Freshmey ed. 1986) may be used for recombinant nucleic acid methods, nucleic acid synthesis, cloning methods, cell culture methods, transfection and transformation, and transgene incorporation, e.g., electroporation, injection, gene gun, impressing through the skin, and lipofection. Generally, oligonucleotide synthesis and purification steps are performed according to specifications. The techniques and procedures are generally performed according to conventional methods in the art and various general references that arc provided throughout this document. The procedures therein are believed to be well known to those of ordinary skill in the art and are provided for the convenience of the reader.
While certain embodiments have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
Nucleic acids encoding lectins were introduced into E. coli. Transforming DNA is shown graphically in
The transforming DNA was introduced into BL21(DE3) pLysS competent cells (Invitrogen) according to the manufacturer's instructions. Briefly, colonies were lysed by BugBuster Protein Extraction Reagent (Novagen) and MAT-tagged proteins were separated using MagneHis Ni-particles (Promega), according to the manufacturer's instructions. Colonies were cultured in 5 mL of Luria Broth (LB) in the presence of 50 μ/mL kanamycin. 100 μL of the culture at O.D.600=1 was centrifuged and the supernatant was removed. The pellets were lysed by resuspending the cells in 50 μl of 1×SDS sample buffer with reducing agent (BioRad). Samples were then boiled and run on a 10% Bis-tris polyacrylamide gel (BioRad) and transferred to PVDF membranes using a Trans-blot semi-dry blotter (BioRad) according to the manufacturer's instructions. Membranes were blocked by Starting Block (TBS) blocking buffer (Thermo Scientific) and probed for one hour with mouse anti-FLAG antibody-horseradish peroxidase conjugate (Sigma) diluted 1:3000 in Starting Block buffer. After probing, membranes were washed four times with TBST, then developed with Supersignal West Dura chemiluminescent substrate (Thermo Scientific) and imaged using a CCD camera (Alpha Innotech).
To determine the activity of the recombinant lectins, 4 L of each strain were grown and harvested by centrifugation at 5000×g for 15 minutes. All subsequent manipulations are performed at 4° C. and in the presence of protease inhibitors. The pellet was resuspended in 35 mL 1× Tris Buffered Saline (TBS) and sonicated at 30% power using a macrotip for 3 cycles of 30 seconds. The solution was then centrifuged at 30,000×g for 30 minutes to remove the unlysed cells. The supernatant was transferred and 1 mL Ni-NTA resin (Qiagen) applied for 1 hour. The resin was collected, washed with 30 column volumes of 1× Tris Buffered Saline (TES) and eluted with 5 mL TBS and 400 mM imidazole pH 7.5. The eluate was then concentrated to a final concentration of 5 mg/mL using Amicon Ultra-15 Centrifugal Filter Units (Millipore). Purified protein was then conjugated to FITC (fluorescein isothiocyanate) using a FITC labeling kit (Thermo Scientific) according to the manufacturer's instructions. Chlamydomonas reinhardtii and Scenedesmus dimerphos cells were then mixed with the FITC labeled protein to show activity. Results from several representative lectins are shown in
Nucleic acids encoding lectins from H. pomatia and L. culinaris were fused to scaffold proteins lipoprotein-outer membrane protein A hybrid (Epp-OmpA) or the β-autotransporter from Neisseria gonorrhoea (SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5) and introduced into E. coli. Transforming DNA is shown graphically in
The transforming DNA was introduced into BL21(DE3) pLysS competent cells (Invitrogen) according to the manufacturer's instructions. Briefly, colonies were lysed by BugBuster Protein Extraction Reagent (Novagen) and MAT-tagged proteins were separated using MagneHis Ni-particles (Promega), according to manufacturer's instructions. Colonies were cultured in 5 mL of Luria Broth (LB) in the presence of 50 μg/mL kanamycin. 100 μL of the culture at O.D.600=1 was centrifuged and the supernatant was removed. The pellets were lysed by resuspending cells in 50 μl of 1× SDS sample buffer with reducing agent (BioRad). Samples were then boiled and run on a 10% Bis-tris polyacrylamide gel (BioRad) and transferred to PVDF membranes using a Trans-blot semi-dry blotter (BioRad) according to the manufacturer's instructions. Membranes were blocked by Starting Block (TBS) blocking buffer (Thermo Scientific) and probed for one hour with mouse anti-FLAG antibody-horseradish peroxidase conjugate (Sigma) diluted 1:3000 in Starting Block buffer. After probing, membranes were washed four times with TEST, then developed with Supersignal West Dura chemiluminescent substrate (Thermo Scientific) and imaged using a CCD camera (Alpha Innotech).
To determine whether the lectin-scaffold fusion is properly inserted in the plasma membrane, membrane isolations were performed. 25 mLs of a culture of at O.D.600=1 were centrifuged at 5000×g for 10 minutes. All subsequent manipulations were performed at 4′ C. and in the presence of protease inhibitors. The pellet was resuspended in 3 mL 1× Tris Buffered Saline (TBS) and sonicated at 30% power using a macrotip for 3 cycles of 10 seconds. The solution was then centrifuged at 30,000×g for 30 minutes to removed the unlysed cells. 1 mL of the supernatant (mixture of membranes and cytosolic proteins) was carefully removed and centrifuged at 540,000×g in a TLA100.3 rotor (Beckman) for 20 minutes to separate the insoluble (membranes) from the soluble (cytosolic proteins). Samples were then boiled and run on a 10% Bis-tris polyacrylamide gel (BioRad) and transferred to PVDF membranes using a Trans-blot semi-dry blotter (BioRad) according to the manufacturer's instructions. Membranes were blocked by Starting Block (TBS) blocking buffer (Thermo Scientific) and probed for one hour with mouse anti-FLAG antibody-horseradish peroxidase conjugate (Sigma) diluted 1:3000 in Starting Block buffer. After probing, membranes were washed four times with TEST, then developed with Supersignal West Dura chemiluminescent substrate (Thermo Scientific) and imaged using a CCD camera (Alpha Innotech). Results are shown in
In order to characterize the activity of the cell surface displayed sugar-binding protein, sedimentation experiments were employed. The E. coli transformed to produce a fusion protein composed of the lectin from L. culinaris and the scaffold protein β-autotransporter was grown to an O.D.600=1.6. The culture was centrifuged and the pellet was resuspended to an O.D.600=30 using TAP media. Varying amounts of culture (0 uL, 5 uL, 10 uL, 20 uL, 40 uL, 100 uL, 250 uL, and 500 uL corresponding to a final O.D.600=0, 0.02, 0.04, 0.08, 0.160, 0.400, 1.00, 2.00) were added to 7 mL of S. diniorphus grown in TAP media, 500 uL of BL21(DE3) was added to 7 mL of S. dimorphus as a negative control. The mixtures were shaken gently for 30 minutes before settling. Photos (
A single chain variable fragment (scFv) antibody to algal surface antigens were fused to scaffold proteins lipoprotein-outer membrane protein A hybrid (Lpp-OmpA) introduced into E. coli (SEQ ID NO. 16). Transforming DNA is shown graphically in
The transforming DNA was introduced into BL21(DE3) pLysS competent cells (Invitrogen) according to the manufacturer's instructions. Briefly, colonies were lysed by BugBuster Protein Extraction Reagent (Novagen) and MAT-tagged proteins were separated using MagneHis Ni-particles (Promega), according to manufacturer's instructions. Colonies were cultured in 5 ml of Luria Broth (LB) in the, presence of 50 μg/mL Kanamycin. 100 μL of the culture at O.D.600=1 was centrifuged and the supernatant was removed. The pellets were lysed by resuspending cells in 50 μl of 1× SDS sample buffer with reducing agent (BioRad). Samples were then boiled and run on a 10% Bis-tris polyacrylamide gel (BioRad) and transferred to PVDF membranes using a Trans-blot semi-dry blotter (BioRad) according to the manufacturer's instructions. Membranes were blocked by Starting Block (TBS) blocking buffer (Thermo Scientific) and probed for one hour with mouse anti-FLAG antibody-horseradish peroxidase conjugate (Sigma) diluted 1:3000 in Starting Block buffer. After probing, membranes were washed four times with TBST, then developed with Supersignal West Dura chemiluminescent substrate (Thermo Scientific) and imaged using a CCD camera (Alpha Innotech).
To determine whether the LppOmpA-scFv fusion is properly inserted in the plasma membrane, membrane isolations were performed. 25 mLs of a culture of at O.D.600=1 were centrifuged at 5000×g for 10 minutes. All subsequent manipulations were performed at 4° C. and in the presence of protease inhibitors. The pellet was resuspended in 3 mL 1× Tris Buffered Saline (TBS) and sonicated at 30% power using a macrotip for 3 cycles of 10 seconds. The solution was then centrifuged at 30,000×g for 30 minutes to remove the unlysed cells. 1 mL of the supernatant (mixture of membranes and cytosolic proteins) was carefully removed and centrifuged at 540,000×g in a TLA100.3 rotor (Beckman) for 20 minutes to separate the insoluble (membranes) from the soluble (cytosolic proteins). Samples were then boiled and run on a 10% Bis-tris polyacrylamide gel (BioRad.) and transferred to PVDF membranes using a Trans-blot semi-dry blotter (BioRad) according to the manufacturer's instructions. Membranes were blocked by Starting Block (TBS) blocking buffer (Thermo Scientific) and probed for one hour with mouse anti-FLAG antibody-horseradish peroxidase conjugate (Sigma) diluted 1:3000 in Starting Block buffer. After probing, membranes were washed four times with TBST, then developed with Supersignal West Dura chemiluminescent substrate (Thermo Scientific) and imaged using a CCD camera (Alpha Innotech). Results from a representative fusion construct LppOmpA-scFv5 are shown in
Nucleic acids encoding lectins such as those from H. pomatia, L. culinaris, T. vulgaris and C. ensiformis can be introduced into P. pastoris. Transforming DNA is shown graphically in
The transforming DNA was cloned into the pPIC9K vector (Invitrogen), thereby introducing the alpha-factor signal peptide at the 5′ end to facilitate secretion, and was introduced into GS115 competent cells (Invitrogen) according to the manufacturer's instructions. To determine whether the recombinant lectins are expressed, 1 L of each strain was grown centrifuged at 5000×g for 15 minutes. All subsequent manipulations were performed at 4° C. and in the presence of protease inhibitors. 1 mL Ni-NTA resin (Qiagen) was applied to the spent media for 1 hour. The resin was collected, washed with 30 column volumes of 1× Tris Buffered Saline (TBS) and eluted with 5 mL TBS and 400 mM imidazole pH 7.5. The eluate was then concentrated to a final concentration of 5 mg/mL using Amicon Ultra-15 Centrifugal Filter Units (Millipore). Representative lectins secreted by P. pastoris are labeled and shown in FIG. 10.
Nucleic acids encoding lectins from H. pomatia, L. culinaris, and T. vulgaris were fused to scaffold proteins such as the S. cerevisiae Pir1a, (SEQ ID NO: 6; SEQ ID NO: 7) Pir1b (SEQ ID NO: 8), the flocculin Flo1p, and the agglutinin protein Aga1p. All fusions, except those with Flo1p, are an N-terminal fusion placing the lectin 5′ of the scaffold. A metal affinity tag (MAT), Tobacco etch virus (TEV) protease cleavage site, and a Flag epitope were engineered in the linker between the two genes to facilitate characterization and visualization studies. Transforming DNA is shown graphically in
The transforming DNA was cloned into the pPIC9K vector (Invitrogen), thereby introducing the alpha-factor signal peptide at the 5′ end to facilitate secretion, and was introduced into GS1.15 competent cells (Invitrogen) according to the manufacturer's instructions. Colonies were screened for expression according to the manufacturer's instructions. Samples were then boiled and run on a 10% Bis-tris polyacryla.mide gel (BioRad) and transferred to PVDF membranes using a Trans-blot semi-dry blotter (BioRad) according to the manufacturer's instructions. Membranes were blocked by Starting Block (TBS) blocking buffer (Thermo Scientific) and probed for one hour with mouse anti-FLAG antibody-horseradish peroxidase conjugate (Sigma) diluted 1:3000 in Starting Block buffer. After probing, membranes were washed four times with TBST, then developed with Supersignal West Dura chemiluminescent substrate (Thermo Scientific) and imaged using a CCD camera (Alpha Innotech). Representative data showing Pir1b-H. pomatia lectin (1), Pir1b-L. culinaris lectin (2), and Pir1b-T. vulgaris lectin (3) from expression studies is shown in
To determine whether the lectin-scaffold fusion is properly inserted in the plasma membrane, membrane isolations were performed. 25 mLs of a culture of at O.D.600=1 were centrifuged at 5000×g for 10 minutes. All subsequent manipulations were performed at 4° C. and in the presence of protease inhibitors. The pellet was resuspended in 3 mL 1× Tris Buffered Saline (TBS) and sonicated at 30% power using a macrotip for 3 cycles of 10 seconds. The solution was then centrifuged at 30,000×g for 30 minutes to remove the unlyrsed cells. 1 mL of the supernatant (mixture of membranes and cytosolic proteins) was carefully removed and centrifuged at 540,000×g in a TLA100.3 rotor (Beckman) for 20 minutes to separate the insoluble (membranes) from the soluble (cytosolic proteins). Samples were then boiled and run on a 10% Bis-tris polyacrylamide gel (BioRad) and transferred to PVDF membranes using a Trans-blot semi-thy blotter (BioRad) according to the manufacturer's instructions. Membranes were blocked by Starting Block (TBS) blocking buffer (Thermo Scientific) and probed for one hour with mouse anti-FLAG antibody-horseradish peroxidase conjugate (Sigma) diluted 1:3000 in Starting Block buffer. After probing, membranes were washed four times with TBST, then developed with Supersignal West Dura chemiluminescent substrate (Thermo Scientific) and imaged using a CCD camera (Alpha Innotech). Results from a representative fusion construct, Pir1a-T. vulgaris lectin fusion, is shown in
A nucleic acid encoding a lectin from L. culinaris, truncated L. culinaris, and E. cristagalli fused to Fas1 from C. reinhardtii was introduced into C. reinhardtii (SEQ ID NO. 9; SEQ ID NO. 10; SEQ ID NO. 11). Transforming DNA is shown graphically in
For these experiments, all transformations were carried out on C. reinhardtii strain 21gr. Cells were grown and transformed via electroporation. Cells were grown to mid-log phase (approximately 2-6×106 cells/ml). Tween-20 was added into cell cultures to a concentration of 0.05% before harvest to prevent cells from sticking to centrifugation tubes. Cells were spun down gently (between 2000 and 5000×g) for 5 min. The supernatant was removed and cells resuspended in TAP+40 mM sucrose media. 1 to 2 ug of transforming DNA was mixed with ˜1×108 cells on ice and transferred to electroporation cuvettes. Electroporation was performed with the capacitance set at 25 uF, the voltage at 800 V to deliver V/cm of 2000 and a time constant for 10-14 ms. Following electroporation, the cuvette was returned to room temperature for 5-20 min. Cells were transferred to 10 ml of TAP+40 mM sucrose and allowed to recover at room temperature for 12-16 hours with continuous shaking. Cells were then harvested by centrifugation at between 2000×g and 5000×g and resuspended in 0.5 ml TAP+40 mM sucrose medium. 0.25 ml of cells was plated on TAP+20 ug/ml. bleomycin. All transformations were carried out under bleomycin selection (20 μg/ml) in which resistance was conferred by the gene encoded by the segment in
Colonies growing in the presence of bleomycin were screened by dot blot. Briefly, colonies were lysed by BugBuster Protein Extraction Reagent (Novagen) and MAT-tagged proteins were separated using MagneHis Ni-particles (Promega), according to the manufacturer's instructions. After exposure to the proteins, the beads were washed three times by 150 ul of 1× Tris Buffered Saline with 0.05% Tween-20 (TBST) at room temperature. Proteins were released from beads by 150 μl 20 μM EDTA, 25 mM Tris-HCl pH 7.0, 400 mM NaCl, and the 150 μl eluates were dot blotted onto nitrocellulose membranes. Membranes were blocked by Starting Block (TBS) blocking buffer (Thermo Scientific) and probed for one hour with mouse anti-FLAG antibody-horseradish peroxidase conjugate (Sigma) diluted 1:3000 in Starting Block buffer. After probing, membranes were washed four times with TBST, then developed with Supersignal West Dura chemiluminescent substrate (Thermo Scientific) and imaged using a CCD camera (Alpha Innotech). Colonies showing positive results in the dot blot analysis were then screened by western blotting.
Patches of algae cells growing on TAP agar plates were lysed by resuspending cells in 50 μl of 1× SDS sample buffer with reducing agent (BioRad). Samples were then boiled and run on a 10% Bis-tris polyacrylamide gel (BioRad) and transferred to PVDF membranes using a Trans-blot semi-dry blotter (BioRad) according to the manufacturer's instructions. Membranes were blocked by Starting Block (TBS) blocking buffer (Thermo Scientific) and probed for one hour with mouse anti-FLAG antibody-horseradish peroxidase conjugate (Sigma) diluted 1:3000 in Starting Block buffer. After probing, membranes were washed four times with TBST, then developed with Supersignal West Dura chemiluminescent substrate (Thermo Scientific) and imaged using a CCD camera (Alpha Innotech). Results from three strains are shown in
To determine whether the lectin-Fas1 fusion was properly inserted in the plasma membrane in an orientation where the lectin is externally presented, membrane isolations were performed. 100 mL of a culture of a minimum 1×107 cells/mL were centrifuged at 5000×g for 10 minutes, All subsequent manipulations were performed at 4° C. and in the presence of protease inhibitors. The pellet was resuspended in 3 mL 1× Tris Buffered Saline (TBS) and sonicated at 30% power using a macrotip for 3 cycles of 10 seconds. The solution was then centrifitged at 30,000×g for 30 minutes to remove the unlysed cells. 1 mL of the supernatant (mixture of membranes and cytosolic proteins) was carefully removed and centrifuged at 540,000×g in a TLA100.3 rotor (Beckman) for 20 minutes to separate the insoluble (membranes) from the soluble (cytosolic proteins). Samples were then boiled and run on a 10% Bis-tris polyacrylamide gel (BioRad) and transferred to PVDF membranes using a Trans-blot semi-dry blotter (BioRad) according to the manufacturer's instructions. Membranes were blocked by Starting Block (TBS) blocking buffer (Thermo Scientific) and probed for one hour with mouse anti-FLAG antibody-horseradish peroxidase conjugate (Sigma) diluted 1:3000 in Starting Block buffer. After probing, membranes were washed four times with TBST, then developed with Supersignal West Dura chemiluminescent substrate (Thermo Scientific) and imaged using a CCD camera (Alpha Innotech). Results from several fusion constructs are shown in
A nucleic acid encoding a lectin from H. pomatia and L. culinaris used to GPI from C. reinhardtii was introduced into C. reinhardtii (SEQ ID NO. 12; SEQ ID NO. 13). Transforming DNA is shown graphically in
For these experiments, all transformations were carried out on C. reinhardtii strain 21gr. Cells were grown and transformed via electroporation. Cells were grown to mid-log phase (approximately 2-6×106 cells/ml). Tween-20 was added into cell cultures to a concentration of 0.05% before harvest to prevent cells from sticking to centrifugation tubes. Cells were spun down gently (between 2000 and 5000×g) for 5 min. The supernatant was removed and the cells resuspended TAP+40 mM sucrose media. 1 to 2 ug of transforming DNA was mixed with ˜1×108 cells on ice and transferred to electroporation cuvettes. Electroporation was performed with the capacitance set at 25 uF, the voltage at 800 V to deliver V/cm of 2000 and a time constant for 10-14 ms. Following electroporation, the cuvette was returned to room temperature for 5-20 min. Cells were transferred to 10 ml of TAP+40 mM sucrose and allowed to recover at room temperature for 12-16 hours with continuous shaking. Cells were then harvested by centrifugation at between 2000 g and 5000 g and resuspended in 0.5 ml TAP+40 mM sucrose medium, 0.25 ml of cells were plated on TAP+20 ug/ml bleomycin. All transformations were carried out under bleomycin selection (20 μg/ml) in which resistance was conferred by the gene encoded by the segment in
Colonies growing in the presence of bleomycin were screened by dot blot. Briefly, colonies were lysed by BugBuster Protein Extraction Reagent (Novagen) and MAT-tagged proteins were separated using MagneHis Ni-particles (Promega), according to manufacturer's instructions. After exposure to the proteins, the beads were washed three times by 150 μl of 1× Tris Buffered Saline with 005% Tween-20 (TEST) at room temperature. Proteins were released from beads by 150 μl of 10 μM EDTA, 25 mM Tris-HCl pH 7.0, 400 mM NaCl, and the 150 μl eluates were dot blotted onto nitrocellulose membranes. Membranes were blocked by Starting Block (TBS) blocking buffer (Thermo Scientific) and probed for one hour with mouse anti-FLAG antibody-horseradish peroxidase conjugate (Sigma) diluted 1:3000 in Starting Block buffer. After probing, membranes were washed four times with TBST, then developed with Supersignal West Dura chemiluminescent substrate (Thermo Scientific) and imaged using a CCD camera (Alpha Innotech). Colonies showing positive results in the dot blot analysis are then screened by western blotting.
Patches of algae cells growing on TAP agar plates were lysed by resuspending cells in 50 μl of 1× SDS sample buffer with reducing agent (BioRad). Samples were then boiled and run on a 10% Bis-tris polyacrylamide gel (BioRad) and transferred to PVDF membranes using a Trans-blot semi-dry blotter (BioRad) according to the manufacturer's instructions. Membranes were blocked by Starting Block (TBS) blocking buffer (Thermo Scientific) and probed for one hour with mouse anti-FLAG antibody-horseradish peroxidase conjugate (Sigma) diluted 1:3000 in Starting Block buffer. After probing, membranes were washed four times with TBST, then developed with Supersignal test Dura chemiluminescent substitute (Thermo Scientific) and imaged using a CCD camera (Alpha Innotech). A small amount of intact fusion (Bleomycin resistance marker fused to GP1-lectin) is translated by the cells; as the resistance marker forms a dimer, these products migrate at a higher molecular weight.
To determine whether the lectin-GP1 fusion is properly inserted in the plasma membrane in an orientation where the lectin is externally presented, membrane isolations were performed. 100 mL of a culture of at minimum 1×107 cells/mL were centrifuged at 5000×g for 10 minutes. All subsequent manipulations were performed at 4° C. and in the presence of protease inhibitors. The pellet was resuspended in 3 mL 1× Tris Buffered Saline (TBS) 2M urea and sonicated at 30% power using a macrotip for 3 cycles of 10 seconds. The solution was then centrifuged at 30,000×g for 30 minutes to remove the unlysed cells. 1 mL of the supernatant (mixture of membranes and cytosolic proteins) was carefully removed and centrifuged at 540,000×g in a TLA100.3 rotor (Beckman) for 20 minutes to separate the insoluble (membranes) from the soluble (cytosolic proteins). Samples were then boiled and run on a 10% Bis-tris polyacrylamide gel (BioRad) in the presence of 2M urea and transferred to P′s7DF membranes using a Trans-blot semi-dry blotter (BioRad) according to the manufacturer's instructions. Membranes were blocked by Starting Block (TBS) blocking buffer (Thermo Scientific) and probed for one hour with mouse anti-FLAG antibody-horseradish peroxidase conjugate (Sigma) diluted 1:3000 in Starting Block buffer. After probing, membranes were washed four times with TBST, then developed with Supersignal West Dura chemiluminescent substrate (Thermo Scientific) and imaged using a CCD camera (Alpha Innotech). The results shown in
A nucleic acid encoding an antibody/single chain variable fragment (scFv5) fused to FaS1 was introduced into C. reinhardtii (SEQ ID NO 14). Transforming DNA is shown graphically in
For these experiments, all transformations were carried out on C. reinhardtii strain 21gr. Cells were grown and transformed via electroporation. Cells were grown to mid-log phase (approximately 2-6×106 cells/ml). Tween-20 was added into cell cultures to a concentration of 0.05% before harvest to prevent cells from sticking to centrifugation tubes. Cells were down gently (between 2000 and 5000×g) for 5 min. The supernatant was removed and cells resuspended in TAP+40 mM sucrose media. 1 to 2 ug of transforming DNA was mixed with ˜1×108 cells on ice and transferred to electroporation cuvettes. Electroporation was performed with the capacitance set at 25 uF, the voltage at 800 V to deliver V/cm of 2000 and a time constant for 10-14 ms. Following electroporation, the cuvette was returned to room temperature for 5-20 min. Cells were transferred to 10 ml of TAP+40 mM sucrose and allowed to recover at room temperature for 12-16 hours with continuous shaking. Cells were then harvested by centrifugation at between 2000 g and 5000 g and resuspended in 0.5 ml TAP+40 mM sucrose medium. 0,25 ml of cells were plated on TAP+20 ug/ml bleomycin. All transformations were carried out under bleomycin selection (20 μg/ml) in which resistance was conferred by the gene encoded by the segment in
Colonies growing in the presence of bleomycin were screened by dot blot, Briefly, colonies were lysed by BugBuster Protein Extraction Reagent (Novagen) and MAT-tagged proteins were separated using MagneHis Ni-particles (Promega), according to manufacturer's instructions. After exposure to the proteins, the beads were washed three times by 150 μl of 1× Tris Buffered Saline with 0.05% Tween-20 (TBST) at room temperature. Proteins were released from beads by 150 μl 10 μM EDTA, 25 mM Tris-HCl pH 7.0, 400 mM NaCl, and the 150 μl eluates were dot blotted onto nitrocellulose membranes. Membranes were blocked by Starting Block (TBS) blocking buffer (Thermo Scientific) and probed for one hour with mouse anti-FLAG antibody-horseradish peroxidase conjugate (Sigma) diluted 1:3000 in Starting Block buffer. After probing, membranes were washed four times with TBST, then developed with Supersignal West Dura chemiluminescent substrate (Thermo Scientific) and imaged using a CCD camera (Alpha Innotech). Colonies showing positive results in the dot blot analysis were then screened by western blotting.
Patches of algae cells growing on TAP agar plates were lysed by resuspending cells in 50 μl of 1× SDS sample buffer with reducing agent (BioRad). Samples were then boiled and run on a 10% Bis-tris polyacrylamide gel (BioRad) and transferred to PVDF membranes using a Trans-blot semi-dry blotter (BioRad) according to manufacturers instructions. Membranes were blocked by Starting Block (TBS) blocking buffer (Thermo Scientific) and probed for one hour with mouse anti-FLAG antibody-horseradish peroxidase conjugate (Sigma) diluted 1:3000 in Starting Block buffer. After probing, membranes were washed four times with TBST, then developed with Supersignal West Dura chemiluminescent substrate (Thermo Scientific) and imaged using a CCD camera (Alpha Innotech). Results from 1 colony (and wild-type control) are shown in
To determine whether the seFv5-Fas1 fusion is properly inserted in the plasma membrane in an orientation where it is externally presented, membrane isolations were performed. 100 ml, of a culture of at minimum 1×107 cells/mL were centrifuged at 5000×g for 10 minutes. All subsequent manipulations were performed at 4° C. and in the presence of protease inhibitors. The pellet was resuspended in 3 mL 1× Tris Buffered Saline (TBS) and sonicated at 30% power using a macrotip for 3 cycles of 10 seconds. The solution was then centrifuged at 30,000×g for 30 minutes to remove the unlysed cells. 1 mL of the supernatant (mixture of membranes and cytosolic proteins) was carefully removed and centrifuged at 540,000×g in a TLA100.3 for 20 minutes to separate the insoluble (membranes) from the soluble (cytosolic proteins). Samples were then boiled and run on a 10% Bis-tris polyacrylamide gel (BioRad) and transferred to PVDF membranes using a Trans-blot semi-thy blotter (BioRad) according to the manufacturer's instructions. Membranes were blocked by Starting Block (TBS) blocking buffer (Thermo Scientific) and probed for one hour with mouse anti-FLAG antibody-horseradish peroxidase conjugate (Sigma) diluted 1:3000 in Starting Block buffer. After probing, membranes were washed four times with TBST, then developed with Supersignal West Dura chemiluminescent substrate (Thermo Scientific) and imaged using a CCD camera (Alpha Innotech). Results from a scFv-Fas1 membrane isolation are shown in
In this example, a nucleic acid encoding a lectin from H. pomatia was introduced into C. reinhardtii (SEQ ID NO. 15). Transforming DNA is shown graphically in
For these experiments, all transformations were carried out on C. reinhardtii strain Nit I -305, deficient in functional nitrate reductase, from the Chlamydomonas center. Cells were grown and transformed via electroporation. Cells were grown to mid-log phase (approximately 2-6×106 cells/ml). Tween-20 was added into cell cultures to a concentration of 0.05% before harvest to prevent cells from sticking to centrifugation tubes. Cells were spun down gently (between 2000 and 5000×g) for 5 min. The supernatant was removed and cells resuspended in TAP+40 mM sucrose media. 1 to 2 ug of transforming DNA was mixed with ˜1×108 cells on ice and transferred to electroporation cuvettes. Electroporation was performed with the capacitance set at 25 uF, the voltage at 800 V to deliver V/cm of 2000 and a time constant for 10-14 ms. Following electroporation, the cuvette was returned to room temperature for 5-20 min. Cells were transferred to 10 ml of TAP+40 mM sucrose and allowed to recover at room temperature for 12-16 hours with continuous shaking. Cells were then harvested by centrifugation at between 2000 g and 5000 g and resuspended in 0.5 ml TAP+40 mM sucrose medium. 0.25 ml of cells were plated on TAP —NH4Cl, +7.4 mM KNO3. All transformations were carried out in the presence of 7.4 mM KNO3 in which the ability to utilize NO3 as the sole nitrogen source is conferred by nitrate reductase. Transformed strains are maintained in the presence of KNO3 to prevent loss of the exogenous DNA.
Colonies growing in TAP —NH4Cl, +7.4 mil KNO3 were screened by clot blot. Briefly, colonies were lysed by BugBuster Protein Extraction Reagent (Novagen) and MAT-tagged proteins were separated using MagneHis Ni-particles (Promega), according to manufacturer's instructions. After exposure to the proteins, the beads were washed three times by 150 μl of 1× Tris Buffered Saline with 0.05′% Tween-20 (TBST) at room temperature. Proteins were released from beads by 150 μl 20 μM EDTA, 25 mM Tris-HCl pH 7.0, 400 mM NaCl, and the 150 μl eluates were dot blotted onto nitrocellulose membranes. Membranes were blocked by Starting Block (TBS) blocking buffer (Thermo Scientific) and probed for one hour with mouse anti-FLAG antibody-horseradish peroxidase conjugate (Sigma) diluted 1:3000 in Starting Block buffer. After probing, membranes were washed four times with TBST, then developed with Supersignal West Dura chemiluminescent substrate (Thermo Scientific) and imaged using a CCD camera (Alpha Innotech). Colonies showing positive results in the dot blot analysis were then screened by western blotting.
Patches of algae cells growing on TAP agar plates were lysed by resuspending cells in 50 μl of 1× SDS sample buffer with reducing agent (BioRad). Samples were then boiled and run on a 10% Bis-tris polyacrylamide gel (BioRad) and transferred to PVDF membranes using a Trans-blot semi-dry blotter (BioRad) according to manufacturers instructions. Membranes were blocked by Starting Block (TBS) blocking buffer (Thermo Scientific) and probed for one hour with mouse anti-FLAG antibody-horseradish peroxidase conjugate (Sigma) diluted 1:3000 in Starting Block buffer. After probing, membranes were washed four times with TBST, then developed with Supersignal West Dura chemiluminescent substrate (Thermo Scientific) and imaged using a CCD camera (Alpha Innotech).
In order to characterize the inducibility of expression, 50 mL, of the Nit-2A-H. pomatia lectin strain was grown in TAP media under light. Once the density of the cell culture reached 1×106 cells/mL, the culture was divided into two 25 mL samples. Each was centrifuged at 5000×g for 10 minutes. The supernatant was removed and one sample was resuspended in 25 mL TAP. The other sample was resuspended in 25 mL. TAP —NH4Cl+7.4 mM KNO3. 20×106 cells were centrifuged and frozen at time points 0, 12 hrs, and 24 hrs. These samples were processed and MAT-tagged proteins were separated using MagneHis Ni-particles (Promega), according to manufacturer's instructions. After exposure to the proteins, the beads were washed three times by 150 μl of 1× Tris Buffered Saline with 0.05% Tween-20 (TBST) at room temperature. Proteins were released from beads by 150 μl 20 μM EDTA, 25 mM Tris-HCl pH 7.0, 400 mM NaCl. 50 uL of 4× SDS sample buffer with reducing agent (BioRad) was added. Samples were then boiled and run on a 10% Bis-tris polyacrylamide gel (BioRad) and transferred to PVDF membranes using a Trans-blot semi-dry blotter (BioRad) according to the manufacturer's instructions. Membranes were blocked by Starting Block (TBS) blocking buffer (Thermo Scientific) and probed for one hour with mouse anti-FLAG antibody-horseradish peroxidase conjugate (Sigma) diluted 1:3000 in Starting Block buffer. After probing, membranes were washed four times with TBST, then developed with Supersignal West Dura chemiluminescent substrate (Thermo Scientific) and imaged using a CCD camera (Alpha Innotech). The induction of H. pomantia lectin expression is very dramatic as shown in
In this example, the two Chlamydomonas reinhardtii strains 21gr and 6145c, representing the two mating types plus (MT+) and minus (MT−), respectively were grown in Tris-Acetate-Phosphate (TAP) media to 5×106 cells/mL. 1 L of each culture was centrifuged at 5000×g for 10 minutes. The supernatant was removed and the pellet was resuspended in 1 L TAP media without ammonium chloride (TAP −NH4Cl). The culture was centrifuged again to wash away residual ammonium chloride at 5000×g for 10 minutes. The supernatant was removed and the pellet was resuspended in 1 L TAP —NH4Cl. The culture was centrifuged again at 5000×g for 10 minutes. The pellet was resuspended in 1 L TAP —NH4Cl and allowed to shake for 12-16 hours under light. The cultures were combined in a 1:1 ratio and allowed to shake for 12 hours under light before observing the sedimentation. Sedimentation or settling of 7.5 mL, of the culture was observed in 13×100 mm borosilicate disposable culture tubes over time. Results are shown in
A variation of the method involves minimizing the amount of nitrogen in the media so that the culture will naturally begin starvation as it approaches saturation. Strains 21gr and 6145c were grown in TAP until approximately 2-6×106 cells/mL. Approximately 1×108 cells were centrifuged and resuspend in 1 mL of TAP —NH4Cl. These were diluted 10,000 fold in TAP —NH4Cl+5 mM NaNO3. Saturation was reached after three days of growth under light. Sedimentation of 7.5 mL of the culture was observed in a 13×100 mm borosilicate disposable culture tubes over time. Results are shown in
H. pomatia
T. vulgaris
H. pomatia
L. culinaris
L. culinaris
H. pomatia
L. culinaris
T. vulgaris
L. culinaris
L. culinaris
E. cristagalli
H. pomatia
L. culinaris
H. pomatia
This application claims the benefit of U.S. Provisional Application Number 61/076,430, tiled Jun. 27, 2008, the entire contents of which are incorporated herein by reference for all purposes.
| Number | Date | Country | |
|---|---|---|---|
| 61076430 | Jun 2008 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13001027 | Mar 2011 | US |
| Child | 14604445 | US |
