Patent ID: 7833703

Claim:
A method for the construction of randomized gene libraries in yeast cells sensitive to Kluyveromyces lactis γ toxin and which are capable of homologous recombination comprising the following steps: introducing into said yeast cells a) a target vector comprising a first DNA sequence coding for at least a γ-subunit of a Kluyveromyces lactis killer toxin as negative selection marker, wherein said Kluyveromyces lactis killer toxin lacks the signal peptide and wherein said DNA sequence is flanked at its 5′ end by a first target sequence and at its 3′ end by a second target sequence and; b) a donor DNA sequence which is flanked at its 5′ end by a DNA sequence which is homologous to said first target sequence and flanked at its 3′ end by a DNA sequence which is homologous to said second target sequence; and cultivation of said yeast cells under conditions allowing the selection of cells in which said DNA sequence in the target vector encoding at least a γ-subunit of a Kluyveromyces lactis killer toxin has been replaced by said donor sequence by means of homologous recombination thereby abolishing expression of said γ-subunit of a K. lactis killer toxin.