Team:Slovenia/Protease signaling/Reporters

Project Achievements Medal requirements Idea

nbsp;Reporters

As our project was aimed develop novel orthogonal signaling pathways based on proteases, as well as at the development of the protein ER retention and release system, we tested and adapted several types of reporters, that will be useful for other iGEM teams.

To measure the activity of the proteases we used three types of reporters based on the firefly luciferase: the cleavable fLuc, the circularly permutated fLuc (cpLuc) and the cyclic fLuc (cycLuc). Additionally, we developed a split luciferase system that functions as an output of logic gates, which integrated the activity of orthogonal proteases.

Finally, to measure the ER protein retention and release, we used TagRFP and SEAP reporters.

Luciferase reporter of the proteolytic activity can be designed either to lead to the decrease of its activity due to proteolysis or to generate the activity by cleavage. Cleavable luciferase assay is expected to be relatively insensitive as it can only detect if a large fraction of the luciferase has been degraded, typically more than 20%, while an assay that leads to the activation of the luciferase might be able to detect much smaller fraction of the proteolytic cleavage.

Cleavable luciferase

Into the loop of the firefly luciferase (fLuc) we inserted amino acid sequence that is targeted by proteases. The substrate sequence thus divided the fLuc into two fragments (nLuc and cLuc), with a protease cleavage site between them (1A and B). The insertion site for the substrate sequence was based on the previously described split luciferase system Shekhawat2009, where we expected that this site would also be permissible to short linker insertion without significantly altering luciferase activity. Upon addition of an appropriate protease, the reporter would be cleaved at the substrate site and the two fragments would dissociate and in turn decrease the fLuc activity. In this system higher protease activity corresponds to a lower luciferase activity. The reporters were additionally equipped with a protein tag at the N-terminal and C-terminal end in order to allow immunostaining to detect protein cleavage by the western blot (1).

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