Continuous directed evolution

Patent metadata

Field	Value
Publication number	US-10336997-B2
Application number	US-201615188627-A
Country	US
Kind code	B2
Filing date	Jun 21, 2016
Priority date	Dec 22, 2010
Publication date	Jul 2, 2019
Grant date	Jul 2, 2019

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. An apparatus for continuous evolution of a gene of interest, the apparatus comprising
(a) a lagoon comprising
a cell culture vessel comprising 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, and a population of bacterial host cells, 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 functional pIII gene;
(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;
(4) 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;
an inflow connected to a turbidostat;
an outflow;
a controller controlling inflow and outflow rates; and,
(b) a turbidostat comprising
a cell culture vessel comprising a population of fresh host cells;
an outflow connected to the inflow of the lagoon;
an inflow connected to a vessel comprising liquid media
a turbidity meter measuring the turbidity of the culture of fresh host cells in the turbidostat; and,
a controller controlling the inflow of sterile liquid media and the outflow into the waste vessel based on the turbidity of the culture liquid.

2. The apparatus of claim 1 , wherein the bacterial host cells are E. coli cells.

3. The apparatus of claim 1 , wherein the bacterial host cells express pIII under the control of a conditional promoter, the activity of which depends on an activity of a product encoded by the gene of interest.

4. The apparatus of claim 1 , wherein the fresh host cells are not infected by the M13 phage.

5. The apparatus of claim 1 , wherein the rate of inflow of fresh host cells and the rate of outflow are substantially the same.

6. The apparatus of claim 1 , wherein the inflow and outflow rates are controlled to maintain a substantially constant number of host cells in the lagoon.

7. The apparatus of claim 1 , wherein the inflow and outflow rates are controlled to maintain a substantially constant frequency of fresh host cells in the lagoon.

8. The apparatus of claim 1 , wherein the lagoon comprises an inflow connected to a vessel comprising a mutagen, and wherein the inflow of mutagen is controlled to maintain a concentration of the mutagen in the lagoon that is sufficient to induce mutations in the bacterial host cells.

9. The apparatus of claim 1 , wherein the lagoon comprises an inflow connected to a vessel comprising an inducer.

10. The apparatus of claim 1 , wherein the inducer induces expression of mutagenesis-promoting genes in the bacterial host cells.

11. The apparatus of claim 1 , wherein the bacterial host cells comprise a mutagenesis plasmid comprising an expression cassette encoding a mutagenesis-promoting gene product.

12. The apparatus of claim 11 , wherein the expression cassette encoding a mutagenesis-promoting gene product comprises a nucleic acid encoding a gene involved in the SOS stress response.

13. The apparatus of claim 12 , wherein the gene involved in the SOS stress response is UmuC, UmuD′, or RecA.

14. The apparatus 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.

15. The apparatus of claim 1 , wherein the turbidostat comprises a vessel comprising a mutagen, a vessel comprising an inducer, or a vessel comprising a mutagen and a vessel comprising an inducer.

16. The apparatus 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.

17. The apparatus of claim 1 , wherein the bacterial host cells comprise an accessory plasmid.

18. The apparatus of claim 17 , wherein the accessory plasmid comprises an expression construct encoding the pIII protein.

19. The apparatus of claim 17 , wherein the bacterial host cells comprise the accessory plasmid and together the phage genome and the accessory plasmid comprise all genes required for the generation of infectious phage.

20. The apparatus of claim 1 , wherein the pIII-neg expression construct comprises a conditional promoter regulating the expression of the pIII-neg gene, wherein the activity of the conditional promoter depends on an undesired function of a gene product encoded by the gene of interest.

Assignees

• Harvard College

Inventors

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

Classifications

• C07K14/005

  from viruses · CPC title

• C12N15/85

  for animal cells · CPC title

• C12N15/86

  Viral vectors · CPC title

• C40B60/08

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

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title