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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1034—Isolating an individual clone by screening libraries
  • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease

Definitions

• the present invention relates generally to molecular biology, and more particularly to synthetic genomes.
• Embodiments and methods are provided for the design, synthesis, assembly and expression of synthetic genomes. Included are methods for rationally designing components of a genome; generating small nucleic acid fragments and assembling them into cassettes comprising portions of the genome; correcting errors in the sequences of the cassettes; cloning the cassettes (e.g., by in vitro methods such as rolling circle amplification); assembling the cassettes to form a synthetic genome (e.g., by methods of in vitro recombination); and transferring the synthetic genome into a biochemical system (e.g., by transplanting it into an intact cell, ghost cell devoid of functioning DNA, or other vesicle).
• a biochemical system e.g., by transplanting it into an intact cell, ghost cell devoid of functioning DNA, or other vesicle.
• the synthetic genome comprises sufficient information to achieve replication of a vesicle (e.g., a cell) in which it resides.
• a vesicle e.g., a cell
• the technology extends to useful end products that a synthetic genomic system can produce, such as energy sources (e.g., hydrogen or ethanol), and biomolecules such as therapeutics and industrial polymers.
• a synthetic genome comprising generating and assembling the nucleic acid components of the genome, wherein at least part of the genome is constructed from nucleic acid components that have been chemically synthesized, or from copies of chemically synthesized nucleic acid components.
• an entire synthetic genome is constructed from nucleic acid components that have been chemically synthesized, or from copies of chemically synthesized nucleic acid components.
• a synthetic genome may be a synthetic cellular genome (a genome which comprises all of the sequences required for replication of a vesicle (e.g., a cell or synthetic vesicle) in which it resides).
• Methods are provided for constructing a synthetic cell, comprising use of certain exemplary methods to construct a synthetic genome and introducing (transplanting) the synthetic genome into a vesicle (e.g., a cell or a synthetic membrane bound vesicle).
• Another method includes constructing a self-replicating synthetic cell, comprising use of exemplary methods to construct a synthetic cellular genome and introducing (transplanting) the synthetic cellular genome into a vesicle (e.g., a cell or a synthetic membrane bound vesicle), under conditions effective for the synthetic cell to replicate.
• Further methods include producing a product of interest, comprising culturing an exemplary synthetic cell under conditions effective to produce the product. When the product is produced from a synthetic cell comprising a synthetic cellular genome, the genome is contacted with the vesicle under conditions effective to replicate the synthetic cell and to produce the product.
• Other exemplary methods include making a synthetic cell, comprising removing part of or all of the resident (original) genome from a microorganism, such as a unicellular microorganism (e.g., a bacterium, fungus, etc.) and replacing it with a synthetic genome that is foreign to the organism (e.g., is from a different species of microorganism (e.g., bacterium)), which exhibits at least one property that is different from the resident genome.
• a synthetic cell produced by this method comprising removing part of or all of the resident (original) genome from a microorganism, such as a unicellular microorganism (e.g., a bacterium, fungus, etc.) and replacing it with a synthetic genome that is foreign to the organism (e.g., is from a different species of microorganism (e.g., bacterium)), which exhibits at least one property that is different from the resident genome.
• a synthetic cell produced by this method include
• One exemplary embodiment includes a synthetic genome that is capable of directing replication of a vesicle (e.g., cell) in which it resides, under particular environmental (e.g., nutritional or physical) conditions.
• the cellular genome is supplemented in the vesicle (e.g., cell) by small molecules, such as nutrients, ATP, lipids, sugars, phosphates etc, which serve as precursors for structural features or substrates for metabolic functions; and/or is supplemented with complex components, such as ribosomes, functional cell membranes, etc.
• These additional elements may complement or facilitate the ability of the genome to achieve (e.g., program) replication of the vesicle/cell.
• the sequences in the genome are capable of providing all of the machinery and components required to produce a cell and to allow the cell to replicate under particular energy or environmental (e.g., nutritional) conditions.
• inventions and methods include a "minimal genome" that may serve as a platform for introducing other sequences of interest, such as genes encoding biologic agents (e.g., therapeutic agents, drugs, vaccines or the like), or genes encoding products that, in the presence of suitable precursors, can produce useful compounds (e.g., biofuels, industrial organic chemicals, etc.).
• the other sequences of interest result in the production of products in sufficient quantities to be commercially valuable.
• a synthetic version of the Mycoplasma genitalium genome having 482 protein- coding genes and 43 RNA genes comprising a 580-kilobase circular chromosome is assembled from gene cassettes.
• Each cassette may be made from chemically synthesized oligonucleotides.
• Several versions of each cassette may be made such that combinatorial assembly into a complete chromosome results in millions of different genomes. These genomes may be tested for functionality by "genome transplantation," replacement of a cell's resident chromosome by the synthetic genome.
• synthetic cells may be assembled from various subcellular components. Additionally, a genome in a cell-free environment may be established, comprising the necessary transcriptional and translation "machinery" to express genes.
• FIG. 1 shows an illustration of suitable oligonucleotides for preparing M. genitalium.
• Example I illustrates the use of mycoplasma comparative genomics to identify genes that may be involved in a minimal gene set for mycoplasma.
• Example II shows the design of 682 48-mer oligonucleotides, and the assembly of those oligonucleotides into three overlapping segments (cassettes).
• Example III shows the synthesis of 5 kb cassettes of an essential region of the M. genitalium genome.
• Cellular genome or a “synthetic cellular genome” is a genome that comprises sequences which encode and may express nucleic acids and proteins required for some or all of the processes of transcription, translation, energy production, transport, production of cell membranes and components of the cell cytoplasm, DNA replication, cell division, and the like.
• a "cellular genome” differs from a viral genome or the genome of an organelle, at least in that a cellular genome contains the information for replication of a cell, whereas viral and organelle genomes contain the information to replicate themselves (sometimes with the contribution of cellular factors), but they lack the information to replicate the cell in which they reside.
• Form gene or genome is a gene or genome derived from a source other than the resident (original) organism, e.g., from a different species of the? organism.
• Gene may include viral genomes, the genomes of organelles (e.g., mitochondria or chloroplasts), and genomes of self- replicating organisms, such as bacteria, yeast, archebacteria, or eukaryotes.
• a genome may also be an entirely new construct for an organism that does not fall into any known Linnean category.
• the genes are from a microorganism, e.g., a unicellular microorganism, such as a bacterium.
• the genes may be in the order found in the microorganism, or they may be shuffled; and mutant versions of some of the genes may also be included.
• “Membrane-bound vesicle,” refers to a vesicle in which a lipid-based protective material encapsulates an aqueous solution.
• “Minimal genome,” with respect to a cell, as used herein, refers to a genome consisting of or consisting essentially of a minimal set of genetic sequences that are sufficient to allow for cell survival under specified environmental (e.g., nutritional) conditions.
• a “minimal genome,” with respect to an organelle, as used herein, refers to a genome consisting of or consisting essentially of a minimal set of genetic sequences that are sufficient to allow the organelle to function.
• a minimal genome must contain sufficient information to allow the cell or organelle to carry out essential biological processes, such as, for example, transcription, translation, use of an energy source, transport of salts, nutrients and the like into and out of the organelle or cell, etc.
• a "minimal replicating genome,” with respect to either a cell or an organelle, contains, in addition, genetic sequences sufficient to allow for self replication of the cell or organelle,
• a “minimal replicating synthetic genome” is a single polynucleotide or group of polynucleotides that is at least partially synthetic and that contains the minimal set of genetic sequences for a cell or organelle to survive and replicate under specific environmental conditions.
• Nucleic acid and “Polynucleotide” are used interchangeably herein. They include both DNA and RNA. Other types of nucleic acids, such as PNA, LNA, modified DNA or RNA, etc. are also included, provided that they can participate in the synthetic operations described herein and exhibit the desired properties and functions. A skilled worker will recognize which forms of nucleic acid are applicable for any particular embodiment or method described herein.
• Synthetic genome includes a single polynucleotide or group of polynucleotides that contain the information for a functioning organelle or organism to survive and, optionally, replicate itself where particular environmental (e.g., nutritional or physical) conditions are met. All or at least part of the genome (e.g., a cassette) is constructed from components that have been chemically synthesized, or from copies of chemically synthesized nucleic acid components. The copies may be produced by any of a variety of methods, including cloning and amplification by in vivo or in vitro methods. In one embodiment, an entire genome is constructed from nucleic acid that has been chemically synthesized, or from copies of chemically synthesized nucleic acid components.
• Such a genome is sometimes referred to herein as a "completely synthetic" genome.
• one or more portions of the genome may be assembled from naturally occurring nucleic acid, nucleic acid that has been cloned, or the like.
• Such a genome is sometimes referred to herein as a "partially synthetic" genome.
• Synthetic genomes offer numerous advantages over traditional recombinant DNA technology. For example, the selection and construction of synthetic genome sequences allow for easier manipulation of sequences than with classical recombination techniques, and permits the construction of novel organisms and biological systems. Furthermore, various embodiments and methods are amenable to automation and adaptation to high throughput methods, allowing for the production of synthetic genomes and synthetic cells by computer-mediated and robotic methods that do not require human intervention.
• the inventive technology opens the door to an integrated process of synthetic genome design, construction, introduction into a biological system, biological production of useful products, and recursive improvement to the design.
• a gene set is identified that constitutes a minimal genome, e.g., of a bacterium, such as Mycoplasma genitalia (M. genitalium), M. capricolum (e.g., subspecies cap ⁇ colum), E. coli, B. subtilis, or others.
• M. genitalium M. genitalium
• M. capricolum e.g., subspecies cap ⁇ colum
• E. coli e.g., B. subtilis, or others.
• One or more conventional or novel methods, or combinations thereof, may be used to accomplish this end.
• One method includes using random saturation global transposon mutagenesis to knock out the function of each gene in a microbial genome (e.g., a bacterial genome) individually, and to determine on this basis putative genes that may be eliminated without destroying cell viability. See, e.g., Smith et al. (1999) Proc Natl Acad Sci USA 87, 826-830.
• Another method is to use comparative genomics of a variety of related genomes (e.g., analyzing the sequences of orthologous organisms, metagenomics, etc.) to predict common genes which are basic to the function of a microorganism of interest (e.g., a bacterium), e.g., to identify genes common to all members of a taxon.
• Existing databases may be used, or new databases may be generated by sequencing additional organisms, using conventional methods.
• the identification of genes in a minimal genome is facilitated by isolating and expanding clones of individual cells, using a method for disrupting cell aggregates.
• Example I herein illustrates the use of mycoplasma comparative genomics to identify genes that may be involved in a minimal gene set for mycoplasma.
• a candidate minimal genome may be constructed as described herein.
• a set of overlapping nucleic acid cassettes are constructed, each generally having about 5 kb, which comprise subsets of the genes; and the cassettes are then assembled to form the genome.
• the function/activity of the genome may be further studied by introducing the assembled genome into a suitable biological system and monitoring one or more functions/activities encoded by the genome.
• the synthetic genome may be further manipulated, for example, by modifying (e.g., deleting, altering individual nucleotides, etc.) portions of genes or deleting entire genes within one or more of the cassettes; by replacing genes or cassettes by other genes or cassettes, such as functionally related genes or groups of genes; by rearranging the order of the genes or cassettes (e.g., by combinatorial assembly); etc.
• the consequences of such manipulations may be examined by re-introducing the synthetic genes into a suitable biological system. Factors that may be considered include, e.g., growth rate, nutritional requirements and other metabolic factors. In this manner, one may further refine which genes are required for a minimal genome.
• Another aspect of rational design according to further methods involves the determination of which sites within a synthetic genome may withstand insertions, such as unique identifiers (e.g., watermarks), expressible sequences of interest, etc., without disrupting gene function.
• sites within a genome that can withstand such disruption lie at the junctions between genes, in non-coding regions, or the like.
• Another aspect of rational design according to even further methods includes the selection of suitable regulatory control elements.
• regulatory control elements include promoters, terminators, signals for the modulation of gene expression (e.g., repressors, stimulatory factors, etc.), signals involved in translation, signals involved in modification of nucleic acids (e.g., by methylation), etc.
• further regulatory control elements include signals involved in splicing, post-translational modification, etc.
• a further design procedure that may be applied is the design of suitable cassettes to be combined to form a synthetic genome.
• cassettes are selected which lie adjacent to one another in that
• each synthetic about 5 kb piece is a cassette comprising one or more complete genes.
• cassettes are designed to be interchangeable, e.g., the cassettes are bounded by unique sequences such as restriction enzyme or adaptor sites, which allow the cassettes to be excised from the genome.
• the cassettes may be: removed, manipulated (e.g., mutated) and returned to the original location in the genome; substituted by other cassettes, such as cassettes having functionally related genes; re-assorted (rearranged) with other cassettes, for example in a combinatorial fashion; etc.
• Mutations or other changes may be introduced, for example, by inserting mutated nucleic acid from a natural source; by site-directed mutagenesis, either in vivo or in vitro; by synthesizing nucleic acids to contain a desired variation, etc,
• genes of interest which directly or indirectly lead to the production of desired products (e.g., therapeutic agents, biofuels, etc.) may be present in a synthetic genome.
• desired products e.g., therapeutic agents, biofuels, etc.
• the genes may be manipulated and the effects of the manipulations evaluated by introducing the modified synthetic genome into a biological system.
• Features may be altered including, e.g., coding or regulatory sequences, codon usage, adaptations for the use of a particular growth medium, etc.
• factors that may be evaluated are, e.g., the amount of desired end product produced, tolerance to end product, robustness, etc. Additional rounds of such manipulations and assessments may be performed to further the optimization.
• a cassette of interest is generally subdivided into smaller portions from which it may be assembled.
• the smaller portions are oligonucleotides of about between about 30 nt and about 1 kb, e.g., about 50 nt (e.g., between about 45 and about 55).
• the oligonucleotides are designed so that they overlap adjacent oligonucleotides, to facilitate their assembly into cassettes.
• the entire sequence may be divided into a list of overlapping 48-mers with 24 nucleotide overlaps between adjacent top and bottom oligonucleotides.
• An illustration of suitable oligonucleotides for preparing M. genitalium is shown in Figure 1.
• the oligonucleotides may be synthesized using conventional methods and apparatus, or they may be obtained from well-known commercial suppliers.
• PCA polymerase cycle assembly
• the cassettes are cloned and amplified by conventional cell-based methods.
• the cassettes are cloned in vitro.
• One such in vitro method which is discussed in co-pending U.S. Provisional Patent Application Serial Nos. 60/675,850; 60/722,070; and 60/725,300, uses rolling circle amplification, under conditions in which background synthesis is significantly reduced.
• Cassettes which may be generated according to various exemplary methods may be of any suitable size.
• cassettes may range from about 1 kb to about 20 kb in length.
• a convenient size is about 4 to about 7 kb, e.g., about 4.5 to about 6.5 kb, preferably about 5 kb.
• each cassette overlaps the cassettes on either side, e.g., by at least about 50, 80, 100, 150, 200, 250 or 1300 nt. Larger constructs (up to the size of, e.g., a minimal genome) comprising groups of such cassettes are also included, and may be used in a modular fashion according to various exemplary embodiments and methods.
• cassettes may be assembled in vitro, using methods of recombination involving "chew-back" and repair steps, which employ either 3' or 5' exonuclease activities, in a single step or in multiple steps.
• the cassette:3 may be assembled with an in vitro recombination system that includes enzymes from the Dienocuccus radiodurans homologous recombination system. Methods of in vivo assembly may also be used.
• Example II describes the generation of a synthetic mouse mitochondrial genome of 16.3 kb by the assembly of three cassettes.
• Example II shows the design of 682 48-mer oligonucleotides, and the assembly of those oligonucleotides into three overlapping segments (cassettes). The oligonucleotides are then assembled into cassettes, by such methods as the method described in Smith et al. (2003), supra, modified in order to reduce heat damage to the synthetic DNA.
• a cassette once a cassette is assembled, its sequence may be verified. It is usually desirable to remove errors which have arisen during the preparation of the cassettes, e.g., during the synthesis or assembly of the nucleic acid components.
• error correction methods which may be used are; (1) methods to modify, tag and/or separate mismatched nucleotides so that amplification errors may be prevented; (2) methods of global error correction, using enzymes to recognize and cleave mismatches in DNA, having known or unknown sequences, to produce fragments from which the errors may be removed and the remaining error- free pieces reassembled; (3) methods of site-directed mutagenesis; and (4) methods to identify errors, select portions from independent synthetic copies which are error-free, and assemble the error-free portions, e.g., by overlap extension PCR (OE-PCR).
• OE-PCR overlap extension PCR
• identifying features such as a unique sequence (e.g., encoding a particular symbol or name, or, e.g., spelling with the alphabet letter designations for the amino acids) or an identifiable mutation which does not disrupt function are introduced into the synthetic genome.
• Such sequences may serve not only to show that the genome has, in fact, been artificially synthesized and to enable branding and tracing, but also to distinguish the synthetic genome from naturally occurring genomes.
• genes or cassettes contain selectable markers, such as drug resistance markers, which aid in selecting nucleic acids that comprises the genes or cassettes. The presence of such selectable markers may also distinguish the synthetic genomes from naturally occurring nucleic acids.
• selectable markers such as drug resistance markers, which aid in selecting nucleic acids that comprises the genes or cassettes. The presence of such selectable markers may also distinguish the synthetic genomes from naturally occurring nucleic acids.
• a synthetic genome which is identical to a naturally occurring genome, but which contains one or more identifying markers as above, is sometimes referred to herein as being “substantially identical" to the naturally occurring genome.
• a synthetic genome according to one embodiment may be present in any environment that allows for it to function.
• a synthetic genome may be present in (e.g., introduced into) any of the biological systems described herein, or others.
• the functions and activities of a synthetic genome, and the consequences of modifying elements of the genome, can be studied in a suitable biological system.
• a suitable biological system allows proteins of interest (e.g., therapeutic agents) to be produced.
• suitable substrates are provided, downstream, non-proteinaceous products, such as energy sources (e.g., hydrogen or ethanol) may also be produced, e.g., in commercially useful amounts.
• a synthetic genome is contacted with a solution comprising a conventional coupled transcription/translation system.
• the nucleic acid may be able to replicate itself, or it may be necessary to replenish the nucleic acid, e.g., periodically.
• a synthetic genome is introduced into a vesicle such that the genome is encapsulated by a protective lipid-based material.
• the synthetic genome is introduced into a vesicle by contacting the synthetic genome, optionally in the presence of desirable cytoplasmic elements such complex organelles (e.g., ribosomes) and/or small molecules, with a lipid composition or with a combination of lipids and other components of functional cell membranes, under conditions in which the lipid components encapsulate the synthetic genome and other optional components to form a synthetic cell.
• a synthetic genome is contacted with a coupled transcription/translation system and is then packaged into a lipid-based vesicle.
• the internal components are encapsulated spontaneously by the lipid materials.
• Exemplary embodiments also include a synthetic genome introduced into a recipient cell, such as a bacterial cell, from which some or all of the resident (original) genome has been removed.
• a synthetic genome may be introduced into a recipient cell which contains some or all of its resident genome.
• the resident (original) and the synthetic genome will segregate, and a progeny cell will form that contains cytoplasmic and other epigenetic elements from the cell, but that contains, as the sole genomic material, the synthetic genome (e.g., a copy of a synthetic genome).
• a cell is a synthetic cell according to various embodiments and methods, and differs from the recipient cell in certain characteristics, e.g., nucleotide sequence, nucleotide source, or non-nucleotide biochemical components.
• a variety of in vitro methods may be used to introduce a genome (synthetic, natural, or a combination thereof) into a cell. These methods include, e.g., electroporation, lipofection, the use of gene guns, etc.
• a genome such as a synthetic genome
• a genome is immobilized in agar; and the agar plug is laid on a liposome, which is then inserted into a host cell.
• a genome is treated to fold and compress before it is introduced into a cell.
• Methods for inserting or introducing large nucleic acid molecules, such as bacterial genomes, into a cell are sometimes referred to herein as chromosome transfer, transport, or transplantation.
• a synthetic cell may comprise elements from a host cell into which it has been introduced, e.g., a portion of the host genome, cytoplasm, ribosomes, membrane, etc.
• the components of a synthetic cell are derived entirely from products encoded by the genes of the synthetic genome and by products generated by those genes.
• nutritive, metabolic and other substances as well as physical conditions such as light and heat may be provided externally to facilitate the growth, replication and expression of a synthetic cell.
• Various exemplary methods may be readily adapted to computer-mediated and/or automated (e.g., robotic) formats.
• Many synthetic genomes (including a variety of combinatorial variants of a synthetic genome of interest) may be prepared and/or analyzed simultaneously, using high throughput methods.
• Automated systems for performing various methods as described herein are included.
• An automated system permits design of a desired genome from genetic components by selection using a bioinformatics computer system, assembly and construction of numerous genomes and synthetic cells, and automatic analysis of their characteristics, feeding back to suggested design modifications.

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