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Preparation of organelle markers in the yeast Saccharomyces cerevisiae
Dekha, Daniella ; Vopálenská, Irena (advisor) ; Papoušková, Klára (referee)
The aim of the work was to prepare a set of strains with fluorescently labelled organelles. The organism used is a haploid strain with a high frequency of homologous recombination Saccharomyces cerevisiae BY4742. The yeast S. cerevisiae is a unicellular organism and is one of the most studied experimental living systems. Its generation time tends to be 1.5-2 hours, which allows rapid monitoring of culture development on both solid and liquid media. For each organelle, specific proteins were selected, namely proteins localized on the vacuole membrane (Vph1, Vtc3), peroxisomes (Pex3), nucleus (Nvj1), endoplasmic reticulum (Sec63), mitochondria (Cox4) and in the vacuole lumen (Prc1). Selected proteins were labeled by attaching fluorescent proteins (yEGFP, yomCherry or yomRuby2) to their C-termini. The prepared strains were analyzed in liquid and solid media. Growth curves were constructed, fluorescence at different growth stages was compared and the effect of two different carbon sources on the expression of selected genes was determined. A total of 12 strains with fluorescent organelle markers for the vacuolar membrane, peroxisomes, nucleus, endoplasmic reticulum, mitochondria, and vacuolar lumen and 5 strains with a pair of fluorescent markers for vacuolar membrane and selected other organelle...
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