|
|
The monitoring of intracellular ion concentrations in microbial cells
Vodáková, Adéla ; Plášek, Jaromír (advisor) ; Krůšek, Jan (referee)
The Master Thesis focuses on monitoring of intracellular ion concentrations in bacteria Escherichia coli and yeast Saccharomyces cerevisiae using genetically encoded fluorescent probes with green fluorescent protein (GFP). Aquired knowledge about this protein and its spectral characteristics is summarized in the introduction. For experimental study a pH-sensitive sensor which displays a ratio change of two excitation fluorescence peaks - pHluorin - was chosen. This probe was tested in bacteria and yeast cells. The experiments concentrated on the ability of the cell to maintain a constant cytosolic pH under various conditions like different pH values of the suspension, addition of glucose or KCl to the suspension. Another topic discussed in the thesis is the elimination of the cell autofluorescence from the GFP signal. For this purpose the synchronous fluorescence scan technique was succesfully used. I have found out that by using this method the measurements of cytosolic pH values are even more accurate thanks to the improved signal to noise ratio.
|
|
|
Response of plasma membrane potential and intracellular pH of yeast to changes in the concentration of extracellular K+
Babuka, David ; Plášek, Jaromír (advisor) ; Heřman, Petr (referee)
The bachelor thesis is focused on studying yeast cells and their response to various external conditions. Main focus was on the study of intracellular pH and membrane potential change under the condition of varying extracellular concentration of K+ ions. In particular we studied to what extent are the yeast cells able to compensate these changes. The ability of yeasts to resist the changes of external pH of the cell medium was studied in an experiment complementary to the measurements of intracellular pH. To measure the intracellular pH a genetically encoded fluorescent probe ratiometric pHluorin was used and to measure the changes of membrane potential a fluorescent probe diS-C3(3) was used. Also we successfully applied a method of synchronously scanned fluorescence to supress the cell autofluorescence.
|
|
|
Response of plasma membrane potential and intracellular pH of yeast to changes in the concentration of extracellular K+
Babuka, David ; Plášek, Jaromír (advisor) ; Heřman, Petr (referee)
The bachelor thesis is focused on studying yeast cells and their response to various external conditions. Main focus was on the study of intracellular pH and membrane potential change under the condition of varying extracellular concentration of K+ ions. In particular we studied to what extent are the yeast cells able to compensate these changes. The ability of yeasts to resist the changes of external pH of the cell medium was studied in an experiment complementary to the measurements of intracellular pH. To measure the intracellular pH a genetically encoded fluorescent probe ratiometric pHluorin was used and to measure the changes of membrane potential a fluorescent probe diS-C3(3) was used. Also we successfully applied a method of synchronously scanned fluorescence to supress the cell autofluorescence.
|
|
|
The monitoring of intracellular ion concentrations in microbial cells
Vodáková, Adéla ; Plášek, Jaromír (advisor) ; Krůšek, Jan (referee)
The Master Thesis focuses on monitoring of intracellular ion concentrations in bacteria Escherichia coli and yeast Saccharomyces cerevisiae using genetically encoded fluorescent probes with green fluorescent protein (GFP). Aquired knowledge about this protein and its spectral characteristics is summarized in the introduction. For experimental study a pH-sensitive sensor which displays a ratio change of two excitation fluorescence peaks - pHluorin - was chosen. This probe was tested in bacteria and yeast cells. The experiments concentrated on the ability of the cell to maintain a constant cytosolic pH under various conditions like different pH values of the suspension, addition of glucose or KCl to the suspension. Another topic discussed in the thesis is the elimination of the cell autofluorescence from the GFP signal. For this purpose the synchronous fluorescence scan technique was succesfully used. I have found out that by using this method the measurements of cytosolic pH values are even more accurate thanks to the improved signal to noise ratio.
|
guest ::
