Claims for Patent: 7,838,287
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Summary for Patent: 7,838,287
Title: | Library of a collection of cells |
Abstract: | The present invention relates to combinatorial gene expression libraries and methods for making these. Such libraries are useful in discovery of novel and/or enhanced metabolic pathways leading to the production of novel compounds for e.g drug discovery and/or to the production of known compounds in novel quantities or in novel compartments of the cells. The expression libraries in particular are composed of host cells capable of co-ordinated and controllable expression of large numbers of heterologous genes in the host cells. |
Inventor(s): | Goldsmith; Neil (Oxford, GB), Sorensen; Alexandra M. P. Santana (Holte, DK), Nielsen; Soren V. S. (Allerod, DK) |
Assignee: | Evolva SA (CH) |
Application Number: | 10/466,960 |
Patent Claims: | 1. A library comprising a collection of individual cells, the cells being denoted cell.sub.1, cell.sub.2, . . . , wherein i.gtoreq.2, each cell comprising a
concatemer of individual oligonucleotide cassettes, said concatemer comprising a nucleotide sequence of the following formula: [rs.sub.2-SP--PR-X-TR-SP-rs.sub.1].sub.n wherein rs.sub.1 and rs.sub.2 together comprise a rare cutting restriction site,
denoted rs.sub.1-rs.sub.2, comprising a recognition sequence of 7 to 38 bases where substantially all restriction sites are complete restriction sites recognized by the same enzyme, SP denotes a spacer of 2 to 2500 bases, X denotes an expressible
nucleotide sequence, PR denotes a promoter capable of regulating the expression of X in the cell, TR denotes a terminator, and n.gtoreq.2 and wherein at least one concatemer of cell.sub.1 is different from a concatemer of cell.sub.2.
2. The library according to claim 1, wherein said cells are yeast cells, said yeast being selected from the group consisting of budding yeast, Phaffia rhodozyma, Saccharomyces boulardii, Yarrowia lipolytica, Schwanniomyces castellii, Rhodotorula glutinis, Lipomyces lipofer, Cryptococcos curvatus, Candida spp., Rhodotorula spp., Saccharomyces spp., Aureobasidium pullulans, Torulopsis spp., Saccharomyces cerevisiae, Eremothecium ashbyii, Pichia spp., Kluyveromyces spp., Hansenula spp., Kloeckera spp., and Pachysolen spp., and mutants thereof. 3. The library according to claim 1, wherein at least one cassette in one cell comprises an intron between the promoter and the expressible nucleotide sequence. 4. The library according to claim 1, wherein at least two expressible nucleotide sequences come from different expression states or tissues. 5. The library according to claim 1, wherein at least one concatemer is integrated into an artificial or host chromosome in the host cell. 6. The library according to claim 1, wherein the PR is an externally controllable promoter. 7. A method of producing a library comprising a collection of individual cells, comprising the steps: i) providing a population of nucleotide cassettes having the general formula [rs.sub.2--SP-PR-X-TR-SP-rs.sub.1], wherein rs.sub.1 and rs.sub.2 together comprise a rare cutting restriction site, denoted rs.sub.1-rs.sub.2, comprising a recognition sequence of 7 to 38 bases where substantially all restriction sites are complete restriction sites recognized by the same enzyme, SP denotes a spacer of 2 to 2500 bases, X denotes an expressible nucleotide sequence, PR denotes a promoter capable of regulating the expression of X in the cell, and TR denotes a terminator, ii) assembling random subsets of the cassettes into concatemers comprising at least two cassettes, iii) ligating the concatemers into vectors, iv) introducing the vectors into host cells, and v) mixing at least two cells so that at least one concatemer of a first cell comprises a random sub-set of cassettes being different from a random sub-set of cassettes of a concatemer of a second cell. 8. The method according to claim 7, whereby the vectors comprise an artificial chromosome. 9. An expression library obtainable by the method of claim 7. 10. An expression library obtainable by the method of claim 8. 11. The expression library of claim 1, wherein the rs.sub.1 and rs.sub.2 together comprise a rare cutting restriction site, denoted rs.sub.1-rs.sub.2, comprising a recognition sequence of 8 to 38 bases. 12. The expression library of claim 1, wherein the rs.sub.1 and rs.sub.2 together comprise a rare cutting restriction site, denoted rs.sub.1-rs.sub.2, comprising a recognition sequence of 10 to 38 bases. 13. The expression library of claim 1, wherein the rs.sub.1 and rs.sub.2 together comprise a rare cutting restriction site, denoted rs.sub.1-rs.sub.2, which is cleavable by a restriction enzyme to leave a unique palindromic overhang. |
Details for Patent 7,838,287
Applicant | Tradename | Biologic Ingredient | Dosage Form | BLA | Approval Date | Patent No. | Expiredate |
---|---|---|---|---|---|---|---|
Merck Sharp & Dohme Corp. | INTRON A | interferon alfa-2b | For Injection | 103132 | 06/04/1986 | ⤷ Try a Trial | 2021-01-25 |
Merck Sharp & Dohme Corp. | INTRON A | interferon alfa-2b | For Injection | 103132 | ⤷ Try a Trial | 2021-01-25 | |
Merck Sharp & Dohme Corp. | INTRON A | interferon alfa-2b | Injection | 103132 | ⤷ Try a Trial | 2021-01-25 | |
>Applicant | >Tradename | >Biologic Ingredient | >Dosage Form | >BLA | >Approval Date | >Patent No. | >Expiredate |
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