Abstract

Proteins cannot adjust to environmental changes on a time scale faster than their half-life. When time-sensitive protein adaptations are required, cells must selectively and actively degrade proteins, which may be vital if cells need to respond more quickly than one generation. While the molecular mechanisms for targeted protein degradation are being elucidated, how the rates vary across individual cells has not been characterized. In this work, we use the “Mother machine,” a continuous-culture microfluidic device, to follow hundreds of cells for hundreds of cell divisions under constant growth conditions. We validate a tool to measure instantaneous proteolysis rates in single bacterial cells growing in the microfluidic device. We demonstrate that proteolysis mediated by the ssrA tag in Escherichia coli, one of the most common degradation signals in both natural and synthetic systems, follows the Michaelis-Menten kinetics with a very high affinity and a half-life of 30 seconds. We validate this tool further using an orthogonal method and obtain a saturation curve by accounting for the measured bleaching rate. We discuss the limitations of this dual reporter for in vivo measurement of protein degradation in single cells and suggest potential alternative methods.
We also design a dual reporter to quantify the rate of protein degradation in individual budding yeast. We discuss the difficulties encountered throughout constructing the dual reporter model for yeast in vivo measurements. We show preliminary results demonstrating that our reporter is being degraded and discuss further work on how this tool could be used to measure the proteolytic activity of humanized or mutant proteasomes.