Back to results
US 10,179,911 B2Granted
Negative selection and stringency modulation in continuous evolution systems
AssigneePresident and Fellows of Harvard College
InventorsDavid R. Liu, Jacob Charles Carlson, Ahmed Hussein Badran, Kevin Michael Esvelt
Life of this patent
Estimating the term…
Abstract
Strategies, systems, methods, reagents, and kits for phage-assisted continuous evolution are provided herein. These include strategies, systems, methods, reagents, and kits allowing for stringency modulation to evolve weakly active or inactive biomolecule variants, negative selection of undesired properties, and/or positive selection of desired properties.
Claims21
A method for modulating the selection stringency during viral-assisted evolution of a gene product, the method comprising:
- (a) introducing host cells into a lagoon, wherein the host cell comprises a low selection stringency plasmid and a high selection stringency plasmid, wherein the low selection stringency plasmid comprises a viral gene required to package a selection viral vector into an infectious viral particle, wherein at least one gene required to package the selection viral vector into an infectious viral particle is expressed in response to a concentration of a small molecule, and wherein the high selection stringency plasmid comprises a second copy of the viral gene required to package the selection viral vector into the infectious viral particle, wherein at least one viral gene required to package the selection viral vector into an infectious viral particle is expressed in response to a desired activity property of a gene product encoded by the gene to be evolved or an evolution product thereof;
- (b) introducing a selection viral vector comprising a gene to be evolved into a flow of host cells through a lagoon, wherein the gene to be evolved produces an active gene product or a weakly active or inactive gene product, wherein the active gene product has an activity that drives the expression of the viral gene required to package the selection viral vector into infectious viral particles in the high selection stringency plasmid and wherein the weakly active or inactive gene product has a relatively lower activity than the activity of the active gene product;
- and
- (c) mutating the gene to be evolved within the flow of host cells, wherein the host cells are introduced through the lagoon at a flow rate that is faster than the replication rate of the host cells and slower than the replication rate of the virus thereby permitting replication of the selection viral vector in the lagoon.
1 more element
A method of using negative selection during viral-assisted evolution of a gene product, the method comprising:
- (a) introducing host cells into a lagoon, wherein the host cell comprises a negative selection gene and a dominant negative mutant gene of a phage gene that decreases or abolishes packaging of the selection viral vector into infectious viral particles, wherein the dominant negative gene is expressed in response to an undesired activity of the gene to be evolved or an evolution product thereof or in response to a concentration of a small molecule inducer;
- (b) introducing a mixture of selection viral vectors into a flow of host cells through a lagoon, wherein one type of selection viral vector comprises a negative selection gene comprising an undesired gene or gene encoding an undesired property of the gene to be evolved and wherein another type of selection viral vector comprises a positive selection gene comprising a desired gene or gene encoding a desired property of the gene to be evolved;
- and
- (c) mutating the gene to be evolved within the flow of host cells, wherein the host cells are introduced through the lagoon at a flow rate that is faster than the replication rate of the host cells and slower than the replication rate of the virus thereby permitting replication of the selection viral vector in the lagoon.
1 more element
Dependencies are inferred from the claim text.