Library of a collection of cells

• US 7,838,287 B2
• Filed: 01/25/2002
• Issued: 11/23/2010
• Est. Priority Date: 01/25/2001
• Status: Expired due to Fees

• Current Assignee: Evolva Holding SA
• Original Assignee: Evolva Holding SA
• Inventors: Goldsmith, Neil, Nielsen, Soren V. S., Sorensen, Alexandra M. P. Santana
• Primary Examiner(s): SAJJADI, FEREYDOUN GHOTB
• Application Number:
  US10/466,960
• Publication Number:
  US 20040241672A1
• Time in Patent Office: 3,224 Days
• Field of Search: 435/320.1, 435/91.2, 536/23.1
• US Class Current: 435/320.1
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/1086 Preparation or screening of expression libraries, e.g. reporter assays
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease

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.

Claims

1. A library comprising a collection of individual cells, the cells being denoted

cell₁, cell₂, . . . , wherein i≧

2,

each cell comprising

a concatemer of individual oligonucleotide cassettes, said concatemer comprising

a nucleotide sequence of the following formula;

[rs₂-SP--PR-X-TR-SP-rs₁]_n

wherein rs₁ and rs₂ together comprise a rare cutting restriction site, denoted rs₁-rs₂, 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≧

2

and wherein at least one concatemer of cell₁ is different from a concatemer of cell₂.

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.

11. The expression library of claim 1, wherein the rs₁ and rs₂ together comprise a rare cutting restriction site, denoted rs₁-rs₂, comprising a recognition sequence of 8 to 38 bases.

12. The expression library of claim 1, wherein the rs₁ and rs₂ together comprise a rare cutting restriction site, denoted rs₁-rs₂, comprising a recognition sequence of 10 to 38 bases.

13. The expression library of claim 1, wherein the rs₁ and rs₂ together comprise a rare cutting restriction site, denoted rs₁-rs₂, which is cleavable by a restriction enzyme to leave a unique palindromic overhang.

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

[rs₂--SP-PR-X-TR-SP-rs₁],

wherein rs₁ and rs₂ together comprise a rare cutting restriction site, denoted rs₁-rs₂, 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.