Lack of an effective method for detection of Staphylococcus aureus is a major challenge for public health, as the bacterium is responsible for high rates of morbidity and
mortality worldwide. In this thesis, we aimed to introduce an innovative strategy that combines the advantages of phage particles and the GFP protein for a rapid and efficient
detection of S. aureus. By incorporating the gfp gene into the phage genome, GFP can be expressed during infection of the target bacteria, enabling detection by measuring
fluorescence. We attempted to construct a reporter phage using the CRISPR-Cas9 system, which would introduce a double-strand break in the phage genome and mediate recombination between the phage genome and donor DNA. This requires
electroporation of S. aureus with a plasmid containing the gfp gene, flanked by homologous regions and components of the CRISPR-Cas9 system. We tested various conditions for electroporation of S. aureus, but haven't been able to obtain transformants with electrocompetent cells prepared by standard protocol. Finally, we used commercial electrocompetent cells and managed to obtain two transformant colonies. In one of the transformants, the presence of the insert within the plasmid was also confirmed by the PCRKOL method. However, we obtained very low yields of isolated plasmid DNA from the transformed cells. This may be associated with poor cell lysis, low plasmid copy number, harvesting cells at an inappropriate stage of bacterial growth, plasmid instability or plasmid toxicity. Due to time constraints within the scope of the master's thesis, we were unable to proceed with further planned steps of the experiment.
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