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 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. Synthetic genomes or synthetic cells may be used for a variety of purposes, including the generation of synthetic fuels, such as hydrogen or ethanol.

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 invention provides a tamper resistant assembly that that securely contains a valuable material. The assembly has a container for holding the valuable material, an optional carrier that contains the container, one or more cover components that enclose the valuable material in the container, and one or more labels having a plurality of devices that reveal tampering by distortion of at least one of the plurality of devices. The label(s) are positioned so that dislodging a cover component causes a detectable distortion in at least one of the plurality of devices, thereby revealing tampering with the assembly. In one embodiment the label can be affixed partially to a surface of a cover component and partially to a surface of the container or the optional carrier. Also disclosed are methods of detecting tampering and method of manufacturing a temper-resistant assembly.

Abstract: The invention provides compositions and methods for assembling a DNA molecule having a desired sequence. The methods involve contacting a DNA polymerase, dNTPs, and a plurality of pairs of oligonucleotides. The oligonucleotides of a pair have a portion of the desired sequence, and an internal sequence that overlaps and is complementary to an internal sequence of the other oligonucleotide of the pair, and, when arranged in order, they have at least a portion of the desired sequence. The oligonucleotides also have a 3? or a 5? primer binding sequence having a binding site for a primer. The oligonucleotides that correspond to the end oligonucleotides of the desired sequence also have a universal 3? flanking sequence and a universal 5? flanking sequence, respectively.

Abstract: Methods for generating synthetic genomes, for example synthetic genomes having desired properties or viable genomes of reduced size, are disclosed. Also disclosed are synthetic genomes produced by the methods disclosed herein and synthetic cells containing the synthetic genomes disclosed herein.

Abstract: The present invention provides materials and methods useful for error correction of nucleic acid molecules. In one embodiment of the invention, a first plurality of double-stranded nucleic acid molecules having a nucleotide mismatch are fragmented by exposure to a molecule having unidirectional mismatch endonuclease activity. The nucleic acid molecules are cut at the mismatch site or near the mismatch site, leaving a double-stranded nucleic acid molecule having a mismatch at the end or near end of the molecule. The nucleic acid molecule is then exposed to a molecule having unidirectional exonuclease activity to remove the mismatched nucleotide. The missing nucleotides can then be filled in by the action of, e.g., a molecule having DNA polymerase activity. The result is double-stranded nucleic acid molecules with a decreased frequency of nucleotide mismatches.

Abstract: The invention provides a tamper resistant assembly that that securely contains a valuable material. The assembly has a container for holding the valuable material, an optional carrier that contains the container, one or more cover components that enclose the valuable material in the container, and one or more labels having a plurality of devices that reveal tampering by distortion of at least one of the plurality of devices. The label(s) are positioned so that dislodging a cover component causes a detectable distortion in at least one of the plurality of devices, thereby revealing tampering with the assembly. In one embodiment the label can be affixed partially to a surface of a cover component and partially to a surface of the container or the optional carrier. Also disclosed are methods of detecting tampering and method of manufacturing a temper-resistant assembly.

Abstract: The present invention provides materials and methods useful for error correction of nucleic acid molecules. In one embodiment of the invention, a first plurality of double-stranded nucleic acid molecules having a nucleotide mismatch are fragmented by exposure to a molecule having unidirectional mismatch endonuclease activity. The nucleic acid molecules are cut at the mismatch site or near the mismatch site, leaving a double-stranded nucleic acid molecule having a mismatch at the end or near end of the molecule. The nucleic acid molecule is then exposed to a molecule having unidirectional exonuclease activity to remove the mismatched nucleotide. The missing nucleotides can then be filled in by the action of, e.g., a molecule having DNA polymerase activity. The result is double-stranded nucleic acid molecules with a decreased frequency of nucleotide mismatches.

Abstract: The invention provides a tamper resistant assembly that that securely contains a valuable material. The assembly has a container for holding the valuable material, an optional carrier that contains the container, one or more cover components that enclose the valuable material in the container, and one or more labels having a plurality of devices that reveal tampering by distortion of at least one of the plurality of devices. The label(s) are positioned so that dislodging a cover component causes a detectable distortion in at least one of the plurality of devices, thereby revealing tampering with the assembly. In one embodiment the label can be affixed partially to a surface of a cover component and partially to a surface of the container or the optional carrier. Also disclosed are methods of detecting tampering and method of manufacturing a temper-resistant assembly.

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: 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: 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: 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: The invention provides a tamper resistant assembly that that securely contains a valuable material. The assembly has a container for holding the valuable material, an optional carrier that contains the container, one or more cover components that enclose the valuable material in the container, and one or more labels having a plurality of devices that reveal tampering by distortion of at least one of the plurality of devices. The label(s) are positioned so that dislodging a cover component causes a detectable distortion in at least one of the plurality of devices, thereby revealing tampering with the assembly. In one embodiment the label can be affixed partially to a surface of a cover component and partially to a surface of the container or the optional carrier. Also disclosed are methods of detecting tampering and method of manufacturing a temper-resistant assembly.

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 discloses methods for assembling a nucleic acid molecule from a set of overlapping oligonucleotides. The method involves contacting a set of overlapping oligonucleotides with a DNA polymerase, a mixture of dNTPs, and a crowding agent to form an assembly mixture. In one embodiment the crowding agent is polyethylene glycol (PEG). The presence of the crowding agent facilitates the nucleic acid assembly process of the invention. The assembly mixture is then subjected to multiple cycles, each cycle comprising an annealing phase, an extension phase, and a denaturation phase, and the desired nucleic acid molecule is thereby assembled. In some embodiments one or more of the phases are time varied.

Abstract: Improved methods for the production of reassortant influenza viruses are provided.