Wosika Victoria, Durandau Eric, Varidel Clémence, Aymoz Delphine, Schmitt Marta, Pelet Serge
Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland.
Mol Genet Genomics. 2016 Dec;291(6):2231-2240. doi: 10.1007/s00438-016-1249-1. Epub 2016 Sep 16.
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.
出芽酵母基因组的易处理性为深入了解调节细胞生命的基本机制提供了诸多见解。随着合成生物学和单细胞测量技术的出现，需要新的工具来更可控地操纵酵母基因组。在此，我们展示了一类名为单整合载体（pSIV）的新型酵母穿梭载体。在酵母中转化后，这些质粒会被一个含有外源标记的盒式结构取代整个缺陷型营养缺陷标记基因座。如通过流式细胞术所示，这种完全取代导致所需DNA片段在标记基因座处进行独特整合。此外，可以插入第二个转录单元以实现两个构建体的同时整合。pSIV中存在的选择标记盒式结构也被用于生成一套完整的基因标签质粒（pGT），涵盖从青色荧光蛋白到近红外串联二聚体红色荧光蛋白等多种荧光蛋白。由于使用了不同的TEF启动子和终止子对，这些标签盒式结构相互正交，从而避免了标记盒式结构的切换，并有利于在所需基因座处进行整合。总之，我们创建了两组强大的分子工具，用于对出芽酵母进行精确的基因操作。
