New families of single integration vectors and gene tagging plasmids for genetic manipulations in budding yeast

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• 作者：Victoria Wosika ; Eric Durandau ; Clémence Varidel…
• 关键词：Plasmid integration ; Gene tagging ; Fluorescent protein ; Transformation ; Molecular biology ; Genetic modification ; Yeast expression
• 刊名：Molecular Genetics and Genomics
• 出版年：2016
• 出版时间：December 2016
• 年：2016
• 卷：291
• 期：6
• 页码：2231-2240
• 全文大小：651 KB
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Life Sciences
  Cell Biology
  Biochemistry
  Microbial Genetics and Genomics
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1617-4623
• 卷排序：291

文摘

The tractability of the budding yeast genome has provided many insights into the fundamental mechanisms regulating cellular life. With the advent of synthetic biology and single-cell measurements, novel tools are required to manipulate the yeast genome in a more controlled manner. We present, here, a new family of yeast shuttle vectors called single integration vectors (pSIV). Upon transformation in yeast, these plasmids replace the entire deficient auxotrophy marker locus by a cassette containing an exogenous marker. As shown using flow cytometry, this complete replacement results in a unique integration of the desired DNA fragment at the marker locus. In addition, a second transcriptional unit can be inserted to achieve the simultaneous integration of two constructs. The selection marker cassettes, present in the pSIV, were also used to generate a complete set of gene tagging plasmids (pGT) encompassing a large palette of fluorescent proteins, from a cyan fluorescent protein to a near-infrared tandem dimer red fluorescent protein. These tagging cassettes are orthogonal to each other thanks to the use of different TEF promoter and terminator couples, thereby avoiding marker cassette switching and favoring integration in the desired locus. In summary, we have created two sets of robust molecular tools for the precise genetic manipulation of the budding yeast.