Study Essential Gene Functions by Plasmid Shuffling.

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.