Advertisement

Applied Microbiology and Biotechnology

, Volume 48, Issue 3, pp 339–345 | Cite as

Improved efficiency and stability of multiple cloned gene insertions at the δ sequences of Saccharomyces cerevisiae

  • F. W. F. Lee
  • N. A. Da Silva
ORIGINAL PAPER

Abstract

Two δ-integration vectors were evaluated for the insertion of an inducible expression cassette (the yeast CUP1 promoter fused to the Escherichia coli lacZ structural gene, CUP1p-lacZ) and a bacterial neomycin-resistance gene (neo) into the genome of Saccharomyces cerevisiae via homologous recombination. Cells containing integrations were selected by resistance to the aminoglycoside G418. The first vector was a traditional construct containing only one δ sequence; with this vector, the transformation efficiency and the number of integrations per cell were quite low. The second carried two δ sequences flanking the desired insert, and the unneeded bacterial sequences were removed by restriction-enzyme digestion immediately before transformation. When this double δ vector was employed, the integrated copy number was more than doubled relative to the single δ system and final β-galactosidase levels exceeded those obtained with the 2μ-based plasmid. Furthermore, the integrations appeared more stable in long-term sequential culture (both with and without induction of the lacZ gene) than those obtained via the single δ vector.

Keywords

Escherichia Coli Recombination Saccharomyces Cerevisiae Saccharomyces Homologous Recombination 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Copyright information

© Springer-Verlag Berlin Heidelberg 1997

Authors and Affiliations

  • F. W. F. Lee
    • 1
  • N. A. Da Silva
    • 1
  1. 1.Department of Chemical and Biochemical Engineering, University of California, Irvine, CA 92697-2575, USA Fax: (714) 824 2541US

Personalised recommendations