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Library of a Collection of Cells

a cell collection and gene technology, applied in the field of combinatorial gene expression libraries, can solve the problems of large chance, heterologous genes are lethal or sub-lethal to the cell,

Inactive Publication Date: 2012-04-19
EVOLVA SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0017]Each unique cell according to the invention may comprise a selection of expressible nucleotide sequences from just one expression state and can thus be assembled from one library representing this expression state or it may comprise cassettes from of a number of different expression states. The variation among and between cassettes in the cells may be such as to minimise the chance of cross over as the host cell undergoes cell division such as through minimising the level of repeat sequences occurring in concatemers, since it is not an object of this embodiment of the invention to obtain inter- or intrachromosomal recombination of the concatemers. Nor to obtain recombination with epitopes of the host cell.
[0031]In the evolution of novel biochemical pathways based on the insertion and expression of a high number of heterologous genes in a population of cells, it is highly likely that cells will be killed due to the formation of lethal gene products. If each cell comprises just one lethal gene, the co-expression of a number of heterologous genes will not lead to any novel biochemical pathways. By having a random combination of promoters and expressible nucleotide sequences, it may be possible to down regulate lethal or sub-lethal genes without affecting the expression of the other heterologous expression constructs.
[0033]According to a further aspect the invention relates to a library comprising at least one library or at least one sub-library as defined above. In the evolution of novel biochemical pathways, it may be preferable to use a number of libraries- or sub-libraries and to evolve these in parallel or mix the libraries in order to improve the chances of identifying a desired property.
[0040]The assembly of concatemers is facilitated by the common structure of the expression cassettes. When the rs1-rs2 restriction site produces sticky ends with a predetermined nucleotide sequence the assembly of the concatemers becomes especially easy to perform.
[0048]According to this method for producing a library of individual cells the source expressible nucleotide sequences are first ligated into a primary vector comprising a cloning site and a cloning cassette. This primary vector may be maintained in a cDNA library and reisolated for excision of the expression cassettes and insertion into a host cell. Through this process the expressible nucleotide sequences are given a common structure which makes it possible to clone the cassettes into a predetermined cloning site in a vector and to remove the cassettes selectively from the host cells later.

Problems solved by technology

One drawback of the libraries of the prior art is that evolution of the libraries may only be obtained through crossing and recombination between cells whereby homologous or homeologous genes are recombined thereby resulting in novel genes yielding gene products with slightly changed properties such as substrate specificity, solubility, cellular location etc.
When a large number of heterologous genes from a wide variety of distantly related species is assembled in one cell, chances are great that some of the heterologous genes are lethal or sub-lethal to the cell, or that several gene products will compete for the same substrates.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0313]In the examples 1-3 an AscI site was introduced into the EcoR1 site in pYAC4 (Sigma, Burke D T et al. 1987, Science vol 236, p 806), so that sticky ends match the Asc1 site(=RS2 in general formula of this patent) of the cassettes in pEVE vectors

Preparation of EVACS (Evolvable Artificial Chromosomes) Including Size Fractioning

[0314]Preparation of pYAG4-Asc Arms

1. inoculate 150 ml of LB (sigma) with a single colony of E. coli DH5α containing pYAC4-Asc

2. grow to OD600˜1, harvest cells and make plasmid preparation

3. digest 100 μg pYAC4-Asc w, BamH1 and Asc1

4. dephosphorylate fragments and heat inactivate phosphatase (20 min, 80 C)

5. purify fragments(e.g. Qiaquick Gel Extraction Kit)

6. run 1% agarose gel to estimate amount of fragment

Preparation of Expression Cassettes

[0315]1. take 100 μg of plasmid preparation from each of the following libraries

a) pMA-CAR

b) pCA-CAR

c) Phaffia cDNA library

d) Carrot-cDNA library

2. digest w. Srf1(10 units / prep, 37 C overnight)

3. dephosphorylate (10 u...

example 2

Preparation of EVACs (Evolvable Artificial Chromosomes) with Direct Transformation

[0335]Preparation of pYAC4-Asc Arms

1. inoculate 150 ml of LB with a single colony of DH5α containing pYAC4-Asc

2. grow to OD600˜1, harvest cells and make plasmid preparation

3. digest 100 μg pYAC4-Asc w. BamH1 and Asc1

4. dephosphorylate fragments and heat inactivate phosphatase(20 Min, 80 C)

5. purify fragments(e.g. Qiaquick Gel Extraction Kit).

1. run 1% agarose gel to estimate amount of fragment

Preparation of Expression Cassettes

[0336]1. take 100 μg of plasmid preparation from each of the following libraries

e) pMA-CAR

f) pCA-CAR

g) Phaffia cDNA library

h) Carrot cDNA library

2: digest w. Srf1 (10 units / prep, 37 C overnight)

3. dephosphorylate (10 units / prep, 37 C, 2 h)

4. heat inactivate 80 C, 20 min

5. concentrate and change buffer (precipitation or ultra filtration),

6. digest w. Asc1. (10 units / prep, 37 C, overnight)

7. adjust volume of preps to 100 μL

Preparation of EVACs

[0337]Different types of EVACs have bee...

example 3

Preparation of EVACs (EVolvable Artificial Chromosomes) (Small Scale Preparation)

Preparation of Expression Cassettes

[0355]1. inoculate 5 ml of LB-medium (Sigma) with library inoculum corresponding to a 10+fold representation of library. Grow overnight[0356]2. make plasmid miniprep from 1.5 ml of culture (E.g. Qiaprep spin miniprep kit)[0357]3. digest plasmid w. Sri 1[0358]4. dephosphorylate fragments and heat inactivate phosphatase (20 min, 80 C)[0359]5. digest w. Asc1[0360]6. run 1 / 10 of reaction in 1% agarose to estimate amount of fragment

Preparation of pYAC4-Asc Arms[0361]1. inoculate 150 ml of LB with a single colony of E. coli DH5α containing pYAC4-Asc[0362]2. grow to OD600˜1, harvest cells and make plasmid preparation[0363]3. digest 100 μg pYAC4-Asc w. BamH1 and Asc1[0364]4. dephosphorylate fragments and heat inactivate phosphatase (20 min, 80 C)[0365]5. purify fragments(E.g. Qiaquick Gel Extraction Kit)[0366]6. run 1% agarose gel to estimate amount of fragment

Preparation of E...

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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 prosecution 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.

Description

[0001]This application is a nonprovisional of U.S. provisional application Ser. No. 60 / 300,863 filed 27, Jun. 2001, which is hereby incorporated by reference in its entirety. The application claims priority from Danish patent application number PA 2001 00128 filed 25, Jan. 2001 and PA 2001 00679 filed on 1, May 2001, which are hereby incorporated by reference in their entirety. All patent and nonpatent references cited in the application, or in the present application, are also hereby incorporated by reference in their entirety.[0002]The present invention relates to a library of a collection of cells and a method for producing said library. The library is useful as a starting material for evolving cells or compositions having new properties.TECHNICAL FIELD[0003]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 comp...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C40B40/02C40B50/06C12N15/09C12N1/00C12N1/15C12N1/19C12N1/21C12N5/10C12N15/10C12N15/66
CPCC12N15/1034C12N15/66C12N15/1086
Inventor GOLDSMITH, NEILSORENSEN, ALEXANDRA M.P. SANTANANIELSEN, SOREN V.S.
Owner EVOLVA SA
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