Cloning and characterization of the promoters from Saccharomyces cerevisiae

Abstract

To provide regulatory elements for the regulation of metabolic fluxes in Saccharomyces cerevisiae cells and to control the efficient and precise expression of key genes at the transcriptional level in the cells, cloning and characterization studies of partial promoter elements of S. cerevisiae were carried out. The pRS41H-Promoter-EGFP expression vector was constructed using OE-PCR and enzymatic ligation using EGFP (enhanced green fluorescent protein) as the reporter gene. The relative strength and characterization of the S. cerevisiae promoters were characterized by measuring the fluorescence value of EGFP. The characterization results show that six new S. cerevisiae promoters are screened and cloned, namely PCBFI, PPAFI, PPOLI, PPOLII, PPOLIII and PRTFI, and their relative strengths are PPOLI>PPAFI>PPOLIII>PPOLII>PRTFI>PCBFI. Finally, the expression of promoter PPOLI in S. cerevisiae cells was investigated. It was found that PPOLI can continue to function in S. cerevisiae cells as they grow. This study provide a reference for the application of promoters in the regulation of metabolic flow homeostasis in chassis cells.

Keywords

• s. cerevisiae
• promoter
• enhanced green fluorescent protein(egfp)
• fluorescence intensity detection
• flow cytometry