Tool generation to characterize DTR1, a member of the poorly characterized DHA1 transporter family of proteins in yeast

• Autor(a) principal: Sousa, Ana Rita Lopes Nogal Lemos de
• Data de Publicação: 2021
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.1/16851
• topic: Mfs-mdr; DTR1; Saccharomyces cerevisiae; GFP; Pgal1; Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias

Nitrogen is necessary for the synthesis of a large number of compounds, such as amino acids, which are very important for several yeast dynamics, and for industrial purposes. This project aims at the identification and characterization of new transporters involved in the excretion of amino acids through the study of a specific gene of the MFS superfamily of transporters in eukaryotes, the DTR1 gene. In the MDR-MFS (MultiDrug Resistance-Major Facilitator Superfamily) family of proteins, up to 100 are unknown, 24 proteins are Multidrug Resistance, and may be involved in the excretion of amino acids. DTR1 is a multi-drug resistance protein with a physiological role assigned to the yeast cell where the layers of chitosan and dityrosine on the external spore wall provide greater resistance to environmental stresses. In the present study, techniques such as E. coli and S. cerevisiae transformation, PCR, RT-PCR, qPCR analysis were applied. The characterization of the DTR1 gene was carried out through its location and expression using various tools, such as the Green Fluorescent Protein by observing when it is expressed in the control of its own promoter, and a fusion with the GAL1 promoter, verifying overexpression of the protein or whether the expression is normally done. The construction of the PGAL1-DTR1-GFP cassette did not occur as expected, therefore was not possible to observe the overexpression of the DTR1 gene and its location. As for characterization on a plasmidic level, plasmids containing the GAP1 promoter were used and the promoter induced, this was possible to observe the expression analysis of the GAP1-DTR1 construction with a change of medium. Additionally, a study of the influence of glycerol and temperature, on S. cerevisiae culture was carried out where it was observed that high temperature and presence of glycerol might be stress conditions enough to DTR1 to be expressed.