Abstract：
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.
摘要：
必需基因被定义为不能从基因组中完全去除的基因。对必需基因功能的研究是有限的，因为它的缺失菌株不容易产生。在这里，我们描述了一种称为质粒改组的协议，可以方便地用于酵母研究基本基因功能。首先将必需基因克隆到具有URA3作为选择标记的基于YCp的质粒中,然后转化到宿主细胞中。然后转化的细胞可用于删除必需基因的染色体拷贝。然后将该基因克隆到另一个具有不同选择标记的基于YCp的质粒中，基因序列可以在体外改变。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。这些细胞在含有选择ura3细胞的5-FOA的培养基中生长。预计5-FOA抗性细胞仅携带含有突变必需基因的质粒，可以评估其功能。
