Abstract: The present invention relates, e.g., to a minimal set of protein-coding genes which provides the information required for replication of a free-living organism in a rich bacterial culture medium, wherein (1) the gene set does not comprise the 100 genes listed in Table 2; and/or wherein (2) the gene set comprises the 382 protein-coding genes listed in Table 3 and, optionally, one of more of: a set of three genes encoding ABC transporters for phosphate import (genes MG410, MG411 and MG412; or genes MG289, MG290 and MG291); the lipoprotein-encoding gene MG185 or MG260; and/or the glycerophosphoryl diester phosphodiesterase gene MG293 or MG385.

Abstract: The present invention relates to methods of joining two or more double-stranded (ds) or single-stranded (ss) DNA molecules of interest in vitro, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule of each pair share a region of sequence identity. The method allows the joining of a large number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes. It can be used, e.g., to join synthetically produced sub-fragments of a gene or genome of interest. Kits for performing the method are also disclosed. The methods of joining DNA molecules may be used to generate combinatorial libraries useful to generate, for example, optimal protein expression through codon optimization, gene optimization, and pathway optimization.

Abstract: The present invention relates to methods of joining two or more double-stranded (ds) or single-stranded (ss) DNA molecules of interest in vitro, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule of each pair share a region of sequence identity. The method allows the joining of a large number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes. It can be used, e.g., to join synthetically produced sub-fragments of a gene or genome of interest. Kits for performing the method are also disclosed. The methods of joining DNA molecules may be used to generate combinatorial libraries useful to generate, for example, optimal protein expression through codon optimization, gene optimization, and pathway optimization.

Abstract: The present invention relates, e.g., to a method for amplifying a small number of copies (e.g. a single copy) of a single-stranded circular DNA molecule (e.g. having a size of about 5-6 kb) by an isothermal rolling circle mechanism, using random or partially random primers and a F29-type DNA polymerase. The method, which can also be used for amplifying DNAs by non-rolling types of multiple displacement amplification, comprises incubating the reaction components in a small volume, e.g. about 10 ?l or less, such as about 0.6 ?l or less. The degree of amplification can be about 109 fold, or higher. A method for cell-free cloning of DNA, using the rolling circle amplification method of the invention, is described.

Abstract: The present invention relates to methods of joining two or more double-stranded (ds) or single-stranded (ss) DNA molecules of interest in vitro, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule of each pair share a region of sequence identity. The method allows the joining of a large number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes. It can be used, e.g., to join synthetically produced sub-fragments of a gene or genome of interest. Kits for performing the method are also disclosed. The methods of joining DNA molecules may be used to generate combinatorial libraries useful to generate, for example, optimal protein expression through codon optimization, gene optimization, and pathway optimization.

Abstract: Methods and apparatus are disclosed herein for encoding human readable text conveying a non-genetic message into nucleic acid sequences with a substantially reduced probability of biological impact and decoding such text from nucleic acid sequences. In one embodiment, each symbol of a symbol set of human readable symbols uniquely maps to a respective codon identifier. Mapping may ensure that each symbol will not map to a codon identifier that generates an amino acid residue which has a single-letter abbreviation that is the equivalent to the respective symbol. Synthetic nucleic acid sequences comprising such human readable text, and recombinant or synthetic cells comprising such sequences are provided, as well as methods of identifying cells, organisms, or samples containing such sequences.

Abstract: Compositions and methods are disclosed herein for cloning a synthetic or a semi-synthetic donor genome in a heterologous host cell. In one embodiment, the donor genome can be further modified within a host cell. Modified or unmodified genomes can be further isolated from the host cell and transferred to a recipient cell. Methods disclosed herein can be used to alter donor genomes from intractable donor cells in more tractable host cells.

Abstract: Compositions and methods are disclosed herein for cloning a donor genome in a heterologous host cell. In one embodiment, the donor genome can be further modified within a host cell. Modified or unmodified genomes can be further isolated from the host cell and transferred to a recipient cell. Methods disclosed herein can be used to alter donor genomes from intractable donor cells in more tractable host cells.

Abstract: The presently disclosed invention relates to methods of installing a genome isolated from one species (the donor) into suitably prepared cells of a second species (the recipient). Introduction of the donor genetic material into the recipient host cell effectively converts the recipient host cell into a new cell that, as a result of the operation of the donated genetic material, is functionally classified as belonging to the genus and species of the donor genetic material.

Abstract: The present invention relates to methods of joining two or more double-stranded (ds) or single-stranded (ss) DNA molecules of interest in vitro, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule of each pair share a region of sequence identity. The method allows the joining of a large number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes. It can be used, e.g., to join synthetically produced sub-fragments of a gene or genome of interest. Kits for performing the method are also disclosed. The methods of joining DNA molecules may be used to generate combinatorial libraries useful to generate, for example, optimal protein expression through codon optimization, gene optimization, and pathway optimization.

Abstract: A method to assemble any desired nucleic acid molecule by combining cassettes in vitro to form assemblies which are further combined in vivo, or by assembling large numbers of DNA fragments by recombination in a yeast culture to obtain desired DNA molecules of substantial size is described.

Abstract: The present invention relates, e.g., to a method for amplifying a small number of copies (e.g. a single copy) of a single-stranded circular DNA molecule (e.g. having a size of about 5-6 kb) by an isothermal rolling circle mechanism, using random or partially random primers and a F29-type DNA polymerase. The method, which can also be used for amplifying DNAs by non-rolling types of multiple displacement amplification, comprises incubating the reaction components in a small volume, e.g. about 10 ?l or less, such as about 0.6 ?l or less. The degree of amplification can be about 109 fold, or higher. A method for cell-free cloning of DNA, using the rolling circle amplification method of the invention, is described.

Abstract: A method is provided for introducing a genome into a cell or cell-like system. The introduced genome may occur in nature, be manmade with or without automation, or may be a hybrid of naturally occurring and manmade materials. The genome is obtained outside of a cell with minimal damage. Materials such as a proteins, RNAs, polycations, nucleoid condensation proteins, or gene translation systems may accompany the genome. The genome is installed into a naturally occurring cell or into a manmade cell-like system. A cell-like system or synthetic cell resulting from the practice of the provided method may be designed and used to yield gene-expression products, such as desired proteins. By enabling the synthesis of cells or cell-like systems comprising a wide variety of genomes, accompanying materials and membrane types, the provided method makes possible a broader field of experimentation and bioengineering than has been available using prior art methods.

Abstract: Methods are provided for constructing a synthetic genome, comprising generating and assembling nucleic acid cassettes comprising portions of the genome, wherein at least one of the nucleic acid cassettes is constructed from nucleic acid components that have been chemically synthesized, or from copies of the chemically synthesized nucleic acid components. In one embodiment, the entire synthetic genome is constructed from nucleic acid components that have been chemically synthesized, or from copies of the chemically synthesized nucleic acid components. Rational methods may be used to design the synthetic genome (e.g., to establish a minimal genome and/or to optimize the function of genes within a genome, such as by mutating or rearranging the order of the genes). Synthetic genomes of the invention may be introduced into vesicles (e.g., bacterial cells from which part or all of the resident genome has been removed, or synthetic vesicles) to generate synthetic cells.

Abstract: The present invention relates, e.g., to a minimal set of protein-coding genes which provides the information required for replication of a free-living organism in a rich bacterial culture medium, wherein (1) the gene set does not comprise the 101 genes listed in Table 2; and/or wherein (2) the gene set comprises the 381 protein-coding genes listed in Table 3 and, optionally, one of more of: a set of three genes encoding ABC transporters for phosphate import (genes MG410, MG411 and MG412; or genes MG289, MG290 and MG291); the lipoprotein-encoding gene MG185 or MG260; and/or the glycerophosphoryl diester phosphodiesterase gene MG293 or MG385.

Abstract: The present invention provides the nucleotide sequence of the entire genome of Mycoplasma genitalium, SEQ ID NO: 1. The present invention further provides the sequence information stored on computer readable media, and computer-based systems and methods which facilitate its use. In addition to the entire genomic sequence, the present invention identifies protein encoding fragments of the genome, and identifies, by position relative to two (2) genes known to flank the origin of replication, any regulatory elements which modulate the expression of the protein encoding fragments of the Mycoplasma genitalium genome.

Abstract: The present invention provides the nucleotide sequence of the entire genome of Mycoplasma genitalium, SEQ ID NO:1. The present invention further provides the sequence information stored on computer readable media, and computer-based systems and methods which facilitate its use. In addition to the entire genomic sequence, the present invention identifies protein encoding fragments of the genome, and identifies, by position relative to two (2) genes known to flank the origin of replication, any regulatory elements which modulate the expression of the protein encoding fragments of the Mycoplasma genitalium genome.