Continuous directed evolution

Patent metadata

Field	Value
Publication number	US-11214792-B2
Application number	US-201916410767-A
Country	US
Kind code	B2
Filing date	May 13, 2019
Priority date	Dec 22, 2010
Publication date	Jan 4, 2022
Grant date	Jan 4, 2022

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 vector system for phage-based continuous directed evolution comprising:
(a) a population of M13 phages comprising:
(i) a gene of interest to be evolved: and
(ii) all phage genes required to generate infectious phage particles, except a functional pIII gene;
wherein the M13 phages lack a functional pIII gene required for the generation of infectious phage, wherein the M13 phages allow for expression of the gene of interest in host cells, and wherein the host cells are suitable host cells for M13 phage infection, replication, and packaging; and

(b) an expression construct comprising a functional pIII gene required to generate infectious phage particles under the control of a conditional promoter, wherein the conditional promoter is activated by a function of a gene product encoded by the gene of interest, wherein the expression construct further encodes a pIII-neg protein under the control of a different promoter than the promoter controlling the functional pIII gene.

2. The vector system of claim 1 , wherein the M13 phage genome comprises a pI, pII, pIV, pV, pVI, pVII, pVIII, pIX, and a pX gene, but not a functional pIII gene.

3. The vector system of claim 1 , wherein the M13 phage genome comprises an F1 origin of replication.

4. The vector system of claim 1 , wherein the M13 phage genome comprises a 3′-fragment of a pIII gene.

5. The vector system of claim 1 , wherein the M13 phage genome comprises a multiple cloning site operably linked to a promoter.

6. The vector system of claim 1 further comprising a helper phage, and together the helper phage and the expression construct comprise all genes required for the generation of an infectious phage.

7. The vector system of claim 1 , wherein the conditional promoter comprises a cI binding site.

8. The vector system of claim 1 , wherein the promoter controlling expression of the pIII-neg protein comprises a cI binding site.

9. The vector system of claim 1 , wherein the vector system further comprises a mutagenesis plasmid comprising a gene expression cassette encoding a mutagenesis-promoting gene product.

10. The vector system of claim 9 , wherein the expression cassette comprises a nucleic acid encoding a gene involved in the SOS response.

11. The vector system of claim 10 , wherein the gene involved in the SOS response is UmuC, UmuD′, and/or RecA.

12. The vector system of claim 9 , 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.

13. The vector system of claim 1 further comprising a population of the host cells, wherein the host cells are E. coli cells.

14. The vector system of claim 10 , wherein the expression cassette comprises UmuC.

15. The vector system of claim 10 , wherein the expression cassette comprises UmuD′.

16. The vector system of claim 10 , wherein the expression cassette comprises UmuC and UmuD′.

17. The vector system of claim 16 , wherein the expression cassette further comprises dnaQ926.

18. The vector system of claim 17 , wherein the expression cassette further comprises recA730.

19. The vector system of claim 12 , wherein the inducer is arabinose.

20. The vector system of claim 12 , wherein the conditional promoter is a pBAD promoter.

Assignees

• Harvard College

Inventors

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

Classifications

• C12N15/85

  for animal cells · CPC title

• C12N15/86

  Viral vectors · 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

• C40B60/08

  Integrated apparatus specially adapted for both creating and screening libraries · CPC title

• C40B50/06

  Biochemical methods, e.g. using enzymes or whole viable microorganisms · CPC title