Method for the construction of randomized gene sequence libraries in cells

Abstract

An in vivo method for the construction of randomized gene libraries and/or domain replacement in gene libraries by homologous recombination using a Kluyveromyces lactis killer toxin, in particular the γ-subunit of the K. lactis killer toxin, as negative selection marker is described. The use of the γ-subunit of K. lactis as negative selectable marker increases the percentage of randomized clones.

1 . A DNA vector which comprises the following sequences: a first target sequence for homologous recombination, a TEF promoter from  Ashbya gossypii  driving transcription of a  K. lactis  killer toxin, a DNA sequence encoding at least a γ-subunit of a  K. lactis  killer toxin and a second target sequence for homologous recombination. 
     
     
         2 . A host cell comprising a vector of  claim 1 , preferably a yeast cell, more preferably a  Saccharomyces cerevisiae  cell. 
     
     
         3 . The host cell of  claim 2  which is a yeast cell. 
     
     
         4 . The host cell of  claim 3  which is a  Saccharomyces cerevisiae  cell.