METHODS AND MATERIALS FOR TARGETED GENE DISRUPTION IN ACTINOMYCETE BACTERIA

摘要

A method is provided for generating a targeted nucleic acid disruption in an actinomycete, the method comprising:(1) providing a first nucleic acid construct comprising:(a) a nucleic acid sequence (the "marker sequence") encoding a marker;(b) a nucleic acid sequence (the "origin sequence") comprising an origin of transfer;(c) a pair of nucleic acid sequences (the "recombining sequences") which are capable of undergoing recombination with each other, thereby to eliminate nucleic acid lying between them and leave only a single recombining sequence; and(d) a pair of nucleic acid sequences (the "targeting sequences") capable of undergoing homologous recombination with a nucleic acid of interest of said actinomycete,wherein parts (a) and (b) lie between the pair (c) of recombining sequences and parts (a), (b) and (c) lie between the pair of targeting sequences;(2) recombining the first nucleic acid construct with a first plasmid bearing at least part of the actinomycete target nucleic acid of interest to form a "recombined plasmid";(3) transferring the recombined plasmid by conjugation from a first bacterial cell into an actinomycete bacterial cell; (4) integrating nucleic acid of the first nucleic acid construct from the transferred plasmid into the target actinomycete nucleic acid by homologous recombination; and(5) eliminating the origin sequence and marker sequence from the integrated nucleic acid, to leave an insertion in the actinomycete nucleic acid, the insertion having a single recombining sequence of the original pair of recombining sequences.Also provided are related materials, methods for producing them, and libraries of cells having such targeted nucleic acid disruptions.