DNA Double-Strand Break-Induced Gene Amplification in Yeast

Tomas Strucko, Michael Lisby, Uffe Hasbro Mortensen*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingBook chapterResearchpeer-review

Abstract

Precise control of the gene copy number in the model yeast Saccharomyces cerevisiae may facilitate elucidation of enzyme functions or, in cell factory design, can be used to optimize production of proteins and metabolites. Currently, available methods can provide high gene-expression levels but fail to achieve accurate gene dosage. Moreover, strains generated using these methods often suffer from genetic instability resulting in loss of gene copies during prolonged cultivation. Here we present a method, CASCADE, which enables construction of strains with defined gene copy number. With our present system, gene(s) of interest can be amplified up to nine copies, but the upper copy limit of the system can be expanded. Importantly, the resulting strains can be stably propagated in selection-free media.
Original languageEnglish
Title of host publicationHomologous Recombination: Methods and Protocols
EditorsAndres Aguilera, Aura Carreira
Number of pages14
Volume2153
PublisherSpringer
Publication date2021
Pages239-252
Chapter17
ISBN (Print)978-1-0716-0643-8
ISBN (Electronic)978-1-0716-0644-5
DOIs
Publication statusPublished - 2021
SeriesMethods in Molecular Biology
ISSN1064-3745

Keywords

  • CEN.PK
  • DNA double-strand break
  • Gene amplification
  • Gene targeting
  • Homology-directed recombination
  • I-SceI nuclease
  • Metabolic engineering
  • Saccharomyces cerevisiae

Cite this

Strucko, T., Lisby, M., & Mortensen, U. H. (2021). DNA Double-Strand Break-Induced Gene Amplification in Yeast. In A. Aguilera, & A. Carreira (Eds.), Homologous Recombination: Methods and Protocols (Vol. 2153, pp. 239-252). Springer. Methods in Molecular Biology https://doi.org/10.1007/978-1-0716-0644-5_17