Methods and compositions for cellular and metabolic engineering
First Claim
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1. A method of recombining one or more nucleic acids, the method comprising:
- introducing one or more sets of nucleic acids into a plurality of cells, thereby providing a plurality of modified cells, each of the plurality of modified cells comprising at least one member of the one or more sets of nucleic acids;
transferring at least a first member of the one or more sets of nucleic acids from a first of the plurality of modified cells into at least a second of the plurality of modified cells;
permitting recombination to occur between the at least one member of the one or more sets of nucleic acids and a nucleic acid present in the second of the plurality of modified cells to produce a recombinant nucleic acid;
introducing the recombinant nucleic acid into a third cell and permitting recombination between the recombinant nucleic acid and a third member present in a third cell of the plurality of modified cells, or between the recombinant nucleic acid and the first member or the second member, thereby producing a further recombined nucleic acid; and
, screening the further recombined nucleic acid for one or more properties or one or more encoded activities, thereby providing a selected recombinant nucleic acid.
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Abstract
The present invention is generally directed to the evolution of new metabolic pathways and the enhancement of bioprocessing through a process herein termed recursive sequence recombination. Recursive sequence recombination entails performing iterative cycles of recombination and screening or selection to “evolve” individual genes, whole plasmids or viruses, multigene clusters, or even whole genomes. Such techniques do not require the extensive analysis and computation required by conventional methods for metabolic engineering.
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Citations
134 Claims
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1. A method of recombining one or more nucleic acids, the method comprising:
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introducing one or more sets of nucleic acids into a plurality of cells, thereby providing a plurality of modified cells, each of the plurality of modified cells comprising at least one member of the one or more sets of nucleic acids;
transferring at least a first member of the one or more sets of nucleic acids from a first of the plurality of modified cells into at least a second of the plurality of modified cells;
permitting recombination to occur between the at least one member of the one or more sets of nucleic acids and a nucleic acid present in the second of the plurality of modified cells to produce a recombinant nucleic acid;
introducing the recombinant nucleic acid into a third cell and permitting recombination between the recombinant nucleic acid and a third member present in a third cell of the plurality of modified cells, or between the recombinant nucleic acid and the first member or the second member, thereby producing a further recombined nucleic acid; and
,screening the further recombined nucleic acid for one or more properties or one or more encoded activities, thereby providing a selected recombinant nucleic acid. - View Dependent Claims (2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134)
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3. A method of recombining one or more nucleic acids, the method comprising:
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introducing one or more sets of nucleic acids into a plurality of cells, thereby providing a plurality of modified cells, each of the plurality of modified cells comprising at least one member of the one or more sets of nucleic acids;
transferring at least a first member of the one or more sets of nucleic acids from a first of the plurality of modified cells into at least a second of the plurality of modified cells;
permitting recombination to occur between the first member and a second member present in the second of the plurality of modified cells, thereby producing a recombinant nucleic acid;
screening the recombinant nucleic acid for one or more properties or one or more encoded activities; and
,further recombining the selected recombinant nucleic acid with one or more additional nucleic acid, or with the first or second nucleic acid, thereby producing a further recombined selected nucleic acid.
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Specification