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OPTOMIZING ELECTROPORATION EFFICIENCY IN SACCAROMYCES CEREVISAE SEC6-49 MUTANT CELLS TO ENABLE A HIGH SENSIVITY GENOMIC SUPPRESSOR SCREEN**

Abstract

The Exocyst is an evolutionarily conserved octameric complex essential for targeted vesicle delivery and tethering prior to membrane fusion at the plasma membrane. In Saccharomyces cerevisiae, the Sec6 subunit is thought to play a key role in proper Exocyst localization at sites of polarized secretion. The temperature-sensitive mutant sec6-49, which carries substitutions in key surface residues, exhibits severe growth defects at 37°C due to impaired membrane localization of Exocyst despite normal complex assembly. To identify genes capable of compensating for this defect, we initiated a multicopy genomic suppressor screen using a URA3-based genomic library. The power, sensitivity, and genomic coverage of this screen depends directly on achieving a high number of independent transformants, making electroporation efficiency a critical experimental bottleneck. Due to the temperature sensitivity of sec6-49 mutant phenotypes, the conventional lithium acetate-based plasmid DNA transformation was not successful in obtaining sufficient number of transformants. Instead, we decided to utilize electroporation for transformation, and it became necessary to develop a robust, reproducible library transformation protocol tailored specifically to sec6-49 mutant. We focused on optimizing three major variables that influence transformation success: electrical parameters such as applied voltage and DNA concentration, chemical pre-treatment that modifies cell wall permeability, and post-electroporation recovery conditions that support membrane repair and permit URA3 expression for transformant selection. Together, these optimization efforts are expected to increase transformant yield, expand genomic library coverage, and improve our ability to identify novel suppressors that restore Sec6-dependent Exocyst function, thereby advancing our understanding of conserved mechanisms of membrane trafficking.

Acknowledgements

GCSU Department of Biological and Enviornmental Sciences, Tri-Beta Research Grant

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