Team:William and Mary/Degradation Rates
Overview
While Cameron and Collins had previously demonstrated the functionality of protein degradation tags (pdt) and the Mesoplasma florum Lon (mf-Lon) protease in E. coli, they did their work exclusively using genomically integrated constructs. Since the majority of iGEM teams work mainly with plasmid constructs, we first wanted to confirm and characterize the parts using iGEM backbones. To do this we assembled constitutive and ATC inducible constructs carrying the red fluorescent protein mScarlet-I, tagged with each of our six different pdts, or left untagged as a control. Further, to ensure that our project will work with a variety of different proteins, we made identical constructs encoding for superfolder GFP (sfGFP) and performed preliminary characterization (Figure 2). This page shows the degradation rates calculated from our pTet mScarlet-I series of parts (Bba_ K2333427-K2333433), at the end of the same 4 hour time courses detailed in our speed control section. Unfortunately, due to time constraints, we were unable to successfully measure one of our constructs (pdt D), via time course. However, we had performed functional test previously with the missing construct, and have used the tag successfully on different constructs, such as our J23100 mScarlet-I (K2333417) and copper sensing (K2333440) parts. Degradation rate for other constructs is located on their registry parts pages .
Figure 1: Schematic of a generic reporter construct used to test degradation rates. Analogous constructs were built with sfGFP reporters.
Figure 2: Degradation rates measured in the above constructs. Each data point represents the population geometric mean of at least 10,000 cells of a distinct biological replicate. Degradation was calculated relative to the geometric mean fluorescence of the untagged control at the final point of a 200 minute time course.