Evaluation of Degron Motifs in Escherichia coli Using a Fluorescent Reporter

Fluorescent reporters provide a useful tool for studying degron motifs. Fusing a degron of interest to a fluorescent protein allows to accurately track protein levels overtime to characterize the degradation kinetics of studied degrons. Here, we describe a rapid and simple method to study degron peptides in Escherichia coli using plasmid-encoded eGFP-degron fusion constructs. The described methods provide an accessible workflow to evaluate degrons. We provide protocols for generation of pBAD plasmids encoding the studied constructs and two different methods for evaluating degrons: an end-point fluorescence measurement on agar plates and a kinetic measurement in liquid cultures in a 96-well format for high-throughput degron studies.