CONTINUOUS DIRECTED EVOLUTION OF PROTEINS AND NUCLEIC ACIDS

摘要

The present invention discloses generalizable methods of evolving nucleic acids and proteins utilizing continuous directed evolution. The invention discloses methods of passing a nucleic acid from cell to cell in a desired function-dependent manner. The linkage of the desired function and passage of the nucleic acid from cell to cell allows for continuous selection and mutation of the nucleic acid.

主权项

1. An apparatus for continuous evolution of a gene of interest, the apparatus comprising
(a) a cellstat comprising
(i) a cell culture vessel comprising
a population of host cells wherein the host cells comprise phage genes required to package the selection phagemid into infectious phage particles,wherein at least one gene required to package the selection phagemid into infectious phage particles is expressed in response to expression of the gene to be evolved in the host cell,(ii) a population of phages comprising a gene of interest to be evolved;(iii) an inflow connected to a turbidostat;(iv) a drain to waste;(v) a pump effecting a flow of host cells through the cellstat, wherein the flow rate of the host cells through the cellstat permits replication of the phage, but not of the host cells, in the cellstat; and (b) a turbidostat comprising
(i) a population of uninfected host cells maintained at a substantially constant cell density;(ii) an outflow connected to the inflow of the cellstat.