Heirarchical assembly methods for genome engineering
First Claim
1. A method of reducing or preventing translation of functional transposase in a cell, the method comprising:
- i) providing a plurality of cells comprising a plurality of polynucleotide constructs, wherein a portion of the plurality of polynucleotide constructs comprise sequence encoding a first selectable marker and a portion of the plurality of polynucleotide constructs comprise sequence encoding a second selectable marker;
ii) conducting pairwise conjugations by mixing pairs of cells, wherein each pair comprises a cell having at least one polynucleotide construct encoding said first selectable marker and a cell having at least one polynucleotide construct encoding said second selectable marker;
iii) selecting cells comprising at least portions of the polynucleotide constructs from both cells involved in the pairwise mixing that have been assembled in a desired manner by selecting cells comprising one of the first or second selectable markers; and
iv) reiteratively repeating said steps ii) and iii) to form a desired polynucleotide product;
wherein said plurality of polynucleotide constructs together comprise a modification in at least a substantial portion of open reading frames or regulatory regions of transposase genes, and wherein said modification causes a reduction in or prevents translation of functional transposase in a cell.
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Abstract
The present invention provides recombination based methods for assembling nucleic acids. In certain aspects the present invention provides hierarchical assembly methods for producing genome sized polynucleotide constructs. The methods may be used for assembling large polynucleotide constructs, for synthesizing synthetic genomes, or for introducing a plurality of nucleotide changes throughout the genome of an organism. In another aspect, the invention provides cells having increased genomic stability. For example, cells comprising alterations in at least a substantial portion of the transposons in the genome are provided.
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Citations
20 Claims
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1. A method of reducing or preventing translation of functional transposase in a cell, the method comprising:
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i) providing a plurality of cells comprising a plurality of polynucleotide constructs, wherein a portion of the plurality of polynucleotide constructs comprise sequence encoding a first selectable marker and a portion of the plurality of polynucleotide constructs comprise sequence encoding a second selectable marker;
ii) conducting pairwise conjugations by mixing pairs of cells, wherein each pair comprises a cell having at least one polynucleotide construct encoding said first selectable marker and a cell having at least one polynucleotide construct encoding said second selectable marker;
iii) selecting cells comprising at least portions of the polynucleotide constructs from both cells involved in the pairwise mixing that have been assembled in a desired manner by selecting cells comprising one of the first or second selectable markers; and
iv) reiteratively repeating said steps ii) and iii) to form a desired polynucleotide product;
wherein said plurality of polynucleotide constructs together comprise a modification in at least a substantial portion of open reading frames or regulatory regions of transposase genes, and wherein said modification causes a reduction in or prevents translation of functional transposase in a cell. - View Dependent Claims (2, 3)
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4. A method for introducing a plurality of predetermined nucleotide changes throughout a polynucleotide product, comprising:
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modifying one or more nucleotides on each of a plurality of polynucleotide segments from a genome to form a plurality of polynucleotide constructs; and
incorporating said plurality of polynucleotide constructs into said genome thereby introducing a plurality of nucleotide changes throughout said polynucleotide product. - View Dependent Claims (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
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18. A method of assembling a polynucleotide product comprising:
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(a) selecting a double stranded initiating polynucleotide construct;
(b) contacting said initiating polynucleotide construct with a next polynucleotide construct in the presence of a recombination system, wherein said next polynucleotide construct is double stranded and a terminal region of said next polynucleotide construct comprises substantial sequence homology with a terminal region of said initiating polynucleotide construct, and wherein said next polynucleotide construct is joined to said initiating polynucleotide construct by homologous recombination at the terminal regions having substantial sequence homology; and
(c) repeating (b) to sequentially add additional double stranded polynucleotide constructs to the extended initiating polynucleotide construct, whereby said polynucleotide product is synthesized. - View Dependent Claims (19, 20)
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Specification