Name: Christopher M. Grilly
Email: cgrilly @ ucsd.edu
Grad Year: 2007
Synthetic biology has benefited enormously from the development of fluorescent reporter proteins for monitoring the behavior of gene regulatory networks in living cells. These reporters must strike a balance between sufficient fluorescence for signal detection and temporal resolution for tracking dynamical responses. We have used components of the Escherichia coli degradation machine to construct a modular system in Saccharomyces cerevisiae that allows for tunable degradation of an ssrA-tagged protein. Using a microfluidic platform tailored for single-cell fluorescence measurements in yeast, we monitor GFP-ssrA decay after induction of a promoter driving expression of genes coding for the essential proteases of the degradation machine. We observe a GFP decay time that ranges from approximately 75 down to 15 minutes, depending on the level of promoter induction. We use computational modeling to illustrate how the underlying set of enzymatic reactions leads to GFP decay curves that are in excellent agreement with the observations. Importantly, the exogenous proteases do not have any apparent deleterious cellular effects over a wide range of degradation rates, implying that the tunable degradation module is robust. In addition to providing a reporter with tunable dynamical resolution, our findings set the stage for explorations of the effect of protein degradation on gene regulatory and signalling pathways.
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