Ubiquitin SynBioCopyright: © Marco Trujillo
One of the ultimate aims is to engineer systems allowing Targeted Protein Degradation (TPD). However, the study of ubiquitinated proteins faces many challenges such as the low stoichiometry of ubiquitin, rapid cleavage of ubiquitin moieties and the degradation of modified proteins. By reconstituting the ubiquitination cascade in an orthogonal system such as E. coli, we are able to circumvents some of these limitations. For this purpose, we developed a synthetic biology toolbox that is based on golden gate cloning (Kowarschik et al. 2018). Expression operons containing all components of the ubiquitination cascade, enable the easy analysis E3 activity or substrate ubiquitination, which can be expensive and time-consuming (Winkler et al. 2017; Turek et al. 2018; Kowarschik et al. 2018; Yu et al. 2023). Furthermore, UbiGate is also amenable for easy up-scaling, rendering ubiquitinated products accessible to further down-stream analysis such as mass-spectrometry or NMR.