Difference between revisions of "Team:Slovenia/Protease signaling/Reporters"
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| + | <h1 class = "ui centered dividing header"><span class="section">nbsp;</span>Reporters</h1> | ||
| + | <div class = "ui segment"> | ||
| + | <p>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. | ||
| + | </p> | ||
| + | <p>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. | ||
| + | </p> | ||
| + | <p>Finally, to measure the ER protein retention and release, we used TagRFP and SEAP reporters.</p> | ||
| + | <p>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. | ||
| + | </p> | ||
| + | |||
| + | </div> | ||
| + | </div> | ||
| + | |||
</div> | </div> | ||
Revision as of 22:52, 15 October 2016
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.
