Beyond the genetic information encoded in the linear DNA sequence, the spatial conformation of chromatin, namely the genome and its associate biomolecules, plays an important role in regulating a variety of processes in eukaryote cells. Over the years several approaches have been developed to reveal DNA dynamics that were hidden in the past. Among them are the Fluorescence Repressor-Operator System (FROS) which uses fluorescently labeled proteins that specifically bind to an operator array integrated at a desired locus within the genome. In this research, we have developed a new FROS based on the recognition between the bacterial LexA repressor, fused to iRFP720 fluorescent protein, to LexA operator. The new system enables DNA labeling with high signal-to-background, makes it suitable for super-resolution imaging techniques such as Point-Spread-Function (PSF) engineering. We used the new FROS in combination with the TetR/TetO system to simultaneously label two loci related to mating-type switching event in saccharomyces cerevisiae yeast cells.
