Continuous directed evolution

Patent metadata

Field	Value
Publication number	US-9394537-B2
Application number	US-201113996208-A
Country	US
Kind code	B2
Filing date	Dec 22, 2011
Priority date	Dec 22, 2010
Publication date	Jul 19, 2016
Grant date	Jul 19, 2016

Abstract

The invention provides systems, methods, reagents, apparatuses, vectors, and host cells for the continuous evolution of nucleic acids. For example, a lagoon is provided in which a population of viral vectors comprising a gene of interest replicates in a stream of host cells, wherein the viral vectors lack a gene encoding a protein required for the generation of infectious viral particles, and wherein that gene is expressed in the host cells under the control of a conditional promoter, the activity of which depends on a function of the gene of interest to be evolved. Some aspects of this invention provide evolved products obtained from continuous evolution procedures described herein. Kits containing materials for continuous evolution are also provided.

First claim

What is claimed is:

1. A method of phage-assisted continuous evolution, comprising
(a) contacting a population of bacterial host cells in a culture medium with a population of M13 phages comprising a gene of interest to be evolved and lacking a functional pIII gene required for the generation of infectious phage particles, wherein
(1) the M13 phages allow for expression of the gene of interest in the host cells,
(2) the host cells are suitable host cells for M13 phage infection, replication, and packaging, wherein the M13 phage comprises all phage genes required for the generation of phage particles, except a full length pIII gene; and
(3) the host cells comprise an expression construct encoding the pIII protein, wherein expression of the pIII gene is dependent on a function of a gene product of the gene of interest;
(b) incubating the population of host cells under conditions allowing for the mutation of the gene of interest, the production of infectious M13 phage, and the infection of host cells with M13 phage, wherein infected cells are removed from the population of host cells, and wherein the population of host cells is replenished with fresh host cells that are not infected by M13 phage, wherein the function of the gene of interest is a desired function, wherein desired functional library members induce production of pIII and release M13 progeny into the culture medium capable of infecting new host cells, wherein undesired non-functional library members do not produce pIII and release only non-infectious M13 progeny into the culture medium;
(c) isolating a mutated M13 phage replication product encoding an evolved protein from the population of host cells, wherein the host cells comprise an expression construct encoding a dominant-negative pIII protein (pIII-neg), wherein the pIII-neg protein is driven by a promoter the activity of which depends on an undesired function of a gene product encoded by the gene of interest.

2. The method of claim 1 , wherein the phage genes required for the generation of phage particles are pI, pII, pIV, pV, pVI, pVII, pVIII, pIX, and/or pX.

3. The method of claim 1 , wherein the host cells comprise an accessory plasmid.

4. The method of claim 3 , wherein the accessory plasmid comprises an expression construct encoding the pIII protein.

5. The method of claim 3 , wherein the cell comprises the accessory plasmid and together, the phage genome and the accessory plasmid comprise all genes required for the generation of an infectious phage.

6. The method of claim 3 , wherein the cell comprises a helper phage and an accessory plasmid and together, the helper plasmid and the accessory plasmid comprise all genes required for the generation of an infectious phage in the presence of a phage genome.

7. The method of claim 1 , wherein the pIII expression construct comprises a conditional promoter regulating the expression of the pIII gene, wherein the activity of the conditional promoter depends on a function of a gene product encoded by the gene of interest.

8. The method of claim 7 , wherein the function of the gene of interest is a binding activity or an enzymatic activity.

9. The method of claim 8 , wherein the enzymatic activity is a polymerase activity, a recombinase activity, a phospho-transferase activity, a kinase activity, a phosphatase activity, or a protease activity.

10. The method of claim 9 , wherein the expression of the pIII protein is driven by a promoter comprising a cI binding site, and wherein the cell expresses a cI protein comprising a desired target protease cleavage site.

11. The method of claim 9 , wherein the expression of the pIII-neg protein is driven by a promoter comprising a cI-neg binding site, and wherein the cell expresses a cI-neg protein comprising an undesired target protease cleavage site.

12. The method of claim 1 , wherein the host cells further comprise a mutagenesis plasmid.

13. The method of claim 11 , wherein the mutagenesis plasmids comprises a gene expression cassette encoding a mutagenesis-promoting gene product.

14. The method of claim 12 , wherein the expression cassette comprises a nucleic acid encoding a gene involved in the SOS stress response.

15. The method of claim 11 , wherein the expression cassette encoding a mutagenesis-promoting gene product comprises a conditional promoter, the activity of which depends on the presence of an inducer.

16. The method of claim 12 , further comprising contacting the host cells with an inducer that induces the expression of the mutagenesis-promoting genes in the host cells.

17. The method of claim 1 , wherein the host cells are E. coli cells.

18. The method of claim 1 , wherein the cells are exposed to a mutagen.

19. The method of claim 1 , wherein the expression construct encoding the pIII protein and the expression construct encoding the pIII-neg protein are situated on the same plasmid.

20. The method of claim 14 , wherein the gene involved in the SOS stress response is UmuC, UmuD′, and/or RecA.

Assignees

• Liu David R
• Esvelt Kevin Michael
• Carlson Jacob Charles
• Harvard College

Inventors

• Liu David R
• Esvelt Kevin Michael
• Carlson Jacob Charles

Classifications

• C12N15/86

  Viral vectors · CPC title

• C12N2795/14152

  relating to complementing cells and packaging systems for producing virus or viral particles · CPC title

• C12N7/00

  Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) · CPC title

• C12N15/1058 Primary

  Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms · CPC title

• C12N15/85

  for animal cells · CPC title