Operator System -Development of Fluorescent Repressor(FROS) based on LexA for DNA labeling in single live cells

MSc. Graduate Seminar; Noa Eden Navon

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.