Construction of a histone-reporter gene fusion for proliferation tracking in Saccharomyces cerevisiae.

• Grant number: 24/07852-7
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: September 01, 2024
• End date: August 31, 2025
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Fellipe da Silveira Bezerra de Mello
• Grantee: Osmar Nucessor de Macedo Neto
• Host Institution: Instituto de Biologia (IB). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil

Abstract

Directed evolution techniques, protein design, and mutagenesis experiments are widely employed in biotechnological studies for the manipulation and generation of gene variant libraries. However, current screening tools for analyzing potential phenotype gains with these libraries still have deficiencies that make analyses with high-throughput rates hard to be made. Although high-throughput screening systems demonstrate efficiency on scales of up to 103-4 variants, they have technical limitations associated with the high cost of specialized equipment and trained personnel. In this regard, this project aims to develop a high-throughput selection tool based on a genetic proliferation marker in Saccharomyces cerevisiae, where the choice of the variant of interest is tightly coupled with the reproductive capacity of a transformed strain in selective medium. For this purpose, a fluorescent reporter gene fused to histone H2A (HTA2) will be employed - a protein with high intracellular abundance, long half-life, and a pattern of equal segregation among daughter cells. Thus, a population of yeast transformed with a variant library can be monitored for the emission of its fluorescence, and the strength of the emitted signal correlated to the influence of the variant on the phenotype. In this way, the selection throughput is done at the scale of a single cells which, coupled with flow cytometry, would allow exponentially higher throughput rates facilitating the manipulation and analysis of genetic libraries.