Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, SK, Canada.
College of Life Sciences, Capital Normal University, Beijing, China.
Methods Mol Biol. 2021;2196:53-62. doi: 10.1007/978-1-0716-0868-5_5.
An essential gene is defined as a gene that cannot be completely removed from the genome. Investigation of an essential gene function is limited because its deletion strain cannot be readily created. Here we describe a protocol called plasmid shuffling that can be conveniently employed in yeast to study essential gene functions. The essential gene is first cloned into a YCp-based plasmid with URA3 as a selectable marker and then transformed into host cells. The transformed cells can then be used to delete the chromosomal copy of the essential gene. The gene is then cloned into another YCp-based plasmid with a different selectable marker, and the gene sequence can be altered in vitro. Plasmids carrying the mutated gene sequences are transformed into the above cells, resulting in carrying two plasmids. These cells are grown in medium containing 5-FOA that selects ura3 cells. The 5-FOA-resistant cells are expected to only carry the plasmid containing the mutated essential gene, whose functions can be assessed.
必需基因被定义为不能从基因组中完全去除的基因。由于不能轻易地创建缺失该基因的菌株，对必需基因功能的研究受到限制。在这里，我们描述了一种称为质粒洗牌的方案，它可以方便地用于酵母中研究必需基因的功能。首先将必需基因克隆到带有 URA3 作为选择标记的 YCp 质粒中，然后转化到宿主细胞中。然后，可使用转化的细胞来删除必需基因的染色体拷贝。然后，将基因克隆到另一个带有不同选择标记的 YCp 质粒中，并在体外改变基因序列。将携带突变基因序列的质粒转化到上述细胞中，导致携带两个质粒。将这些细胞在含有 5-FOA 的培养基中生长，5-FOA 选择ura3 细胞。预计只有携带突变必需基因的质粒的细胞能够在培养基中存活，其功能可以进行评估。
