Characterization of a Fluorescent Protein Degron System for Mammalian Systems

Synthetic gene circuits require the ability to dynamically react to stimuli and change their expression in order to improve potential applicability. Thus, a method to track changes in gene expression in real time is required. However, the persistence of the fluorescent proteins commonly used as reporters complicates the understanding of these gene circuits by masking transient behaviors such as transcriptional bursting. Here we describe the development of a series of degron-tagged fluorescent proteins to allow the detection and live tracking of transient signals. These constructs consist of an mRuby2 control linked via a T2A peptide to either an eGFP or mGL reporter, themselves with one of 4 different N-degron tags reported in literature. Using flow cytometry snapshots of protein levels at 15-minute intervals following cycloheximide addition, the constructs containing eGFP-MODC had a half-life of 50 minutes, those with mGL-MODC a half-life of 130 minutes, and those with mGL-MODCmut a half-life of 50 minutes. This was further validated by timelapse microscopy using our CellBaum tracking pipeline, which takes a series of microscopy images and identifies, follows, and records features such as size and brightness of individual cells over time. While similarly designed constructs have proved effective in yeast, there are few implemented for use in mammalian systems, letting this degron system offer a new tool for the interrogation of gene dynamics in mammalian cells.