Rosella is a pH-sensitive fluorescent biosensor that was recently deposited with Addgene by Dr. Mark Prescott. This system was developed for monitoring and analyzing autophagy of cytosol and organelles in yeast cells. Autophagy (Greek for “self-eating”) is induced by a lack of nutrients and targets cytosol and organelles to the vacuole/lysosome for degradation and recycling. The key to Rosella’s autophagy-sensing abilities is that its fluorescence emission spectra changes when it goes from a more neutral pH compartment, like the cytosol, to the higher pH of the vacuole. Read on to learn more about prior methods for studying autophagy and how Rosella improves upon them.
Scientists use deep mutational scanning to simultaneously test how multiple amino acid changes affect a protein of interest’s function. This technique relies on the generation of a plasmid library that expresses all desired variants of a protein. Applying a selective pressure winnows the pool down to plasmids expressing variants with optimal function. High-throughput DNA sequencing is then used to measure the frequency of each variant during the selection process. Each variant is assigned a functional score based on its library frequency before selection compared to its library frequency after selection. Key to this process is the ability to generate full libraries of mutant proteins. Researchers from the Whitehead lab developed One pot saturation mutagenesis as a quick and easy technique that can be used to generate complex libraries of mutant plasmids ready for deep mutational scanning.