Syllabus
List of tasks:
• The use of various variants of light microscopy (phase contrast, Nomarski contrast, fluorescence microscopy) and staining methods for studying the subcellular structures of yeast cells.
• Identification of unknown yeast strain using classical microbiological methods to describe basic properties of yeast strains (e.g. ability to ferment sugars, osmotolerance, crossover, sporulation, acidification test).
Preparation of a genetically modified strain of S. cerevisiae. Comparison of the efficiency of genomic modification using a cassette for homologous recombination and plasmid transformation. Preparation of a GFP expressing strain localized to different cell compartments.
• Study of multicellular structures formed by yeast cells. Demonstration of preparation and microscopic analysis of thin sections from yeast colonies including observation of different expression of fluorescently labeled marker genes in fluorescence microscope and fluorescent magnifying glass.
Analysis of differential expression of marker genes in cell populations taken from a deficient yeast colony by flow cytometry.
• Determination of resistance to thermal shock in different populations from differentiated colonies. Evaluation by flow cytometer.
• High-throughput screening of a whole-genome collection of deletion mutants (i.e. about 4600 yeast strains) for osmoresistance and respiratory deficiency.
• Mutagenicity testing of chemicals used e.g. in households with the genetically modified strain of S. cerevisiae D7 (different phenotypic manifestations when different repair mechanisms are activated).
• Detection of Cu2+ ions in aqueous solution using genetically modified yeast strain cells immobilized in alginate.
• Analysis of the volatile substances produced by yeasts of different genera by gas chromatography and mass spectrometry GCxGC-MS
Annotation
The six-day course is complemental to lectures on Yeast Biology - MB140P24, Cell Cycles and Signals
- MB140P29 and Methods in molecular and cell biology - MB140S79.
During the course, students will be introduced to modern molecular biological and microscopic methods enabling study of cell organization and gene manipulation of yeast cells as well as classical microbiological methods used for determination of physiological characteristics or taxonomic classification of yeast. They will learn various yeast species from different parts of heterogeneous group of yeasts, especially Saccharomyces cerevisiae as one of the most important model organisms of contemporary experimental biology. Students will create genetically modified strain of S. cerevisiae expressing green fluorescent protein (GFP) localized to different cell compartments, which will allow observation of individual cell organelles using fluorescence microscope. In practice, students will also learn about flow cytometer and use it to evaluate different expression of marker gene in different cell subpopulations.
Part of the course is devoted to differentiated multicellular formations, i.e. yeast colonies and biofilms. Students will try to collect individual subpopulations from differentiated colonies and subsequently test resistance of these subpopulations to thermal shock as one of the parameters of physiological diversity of cellular subpopulations. Sensitive cell counts will be determined by fluorescence staining and subsequent flow cytometer evaluation. Students will test the conditions for induction of pseudohyphal growth and create different types of yeast biofilms. The technique of preparation of thin sections from yeast colonies to study the localization of individual subpopulations of differentiated cells labeled with marker proteins fused to GFP within colonies using a fluorescence microscope and a fluorescent magnifier will be demonstrated.
The last part of the course focuses on the possibilities of applied utilization of yeast strains. Using a patented method, students detect Cu2+ ions in various samples using an immobilized, specially genetically modified yeast strain. They will then use another strain to test the mutagenicity of household chemicals.
Students work in groups of two. The course is conducted in Czech. One day of the six-day course takes place at the Biotechnology and Biomedical Center of the Academy of Sciences and Charles University in Vestec (BIOCEV).