Study Essential Gene Functions by Plasmid Shuffling
Study Essential Gene Functions by Plasmid Shuffling
复制标题
通过质粒改组研究基本基因功能。
DOI：
10.1007/978-1-0716-0868-5_5
复制
发表时间：
2021-01-01
期刊：
影响因子：
--
通讯作者：
Xiao, Wei
中科院分区：
文献类型：
--
作者：
Fan, Li;Xiao, Wei
必需基因被定义为不能完全从基因组中移除的基因。研究一个重要的基因功能是有限的，因为它的删除菌株不能轻易地创建。在这里，我们描述了一种称为质粒洗牌的协议，可以方便地在酵母中用于研究基本基因功能。首先将基本基因克隆到以ycp为基础的质粒中，并将ura3作为可选择的标记物，然后将其转化到宿主细胞中。转化后的细胞可以用来删除基本基因的染色体拷贝。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，并且可以在体外改变基因序列。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。这些细胞生长在含有5-FOA的培养基中，这种培养基会选择3个细胞。抗5- foa细胞预计只携带含有突变基本基因的质粒，其功能可以评估。
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 withURA3as 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 selectsura3cells. The 5-FOA-resistant cells are expected to only carry the plasmid containing the mutated essential gene, whose functions can be assessed.