Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Yijie Lai
Date created: 2016-10-03 11:00:00
Date modified: 2016-10-05 10:23:22
Nano-lantern(cAMP-1.6)
| Types | DnaRegion |
| Roles | Reporter engineered_region |
| Sequences | BBa_K1969000_sequence (Version 1) |
Description
Nano-lantern(cAMP-1.6) is a fusion protein designed by professor Nagai's group, It is a detector for changes in intracellular cAMP level.The reporter mainly contains three parts: the enhanced yellow fluorescent protein, Venus△C10, the mutated Renilla luciferase, RLuc8△N3, and the cAMP-recognition motif of human EPAC1 with a Q270E mutation(Saito, Chang et al. 2012). The yellow flourescent part is used to increase the emitted photon number of RLuc8△N3 by a Forster resonance energy transfer (FRET) mechanism, in which the excited energy of the luminescent substrate, coelenterazine, bound to RLuc is efficiently transferred intermolecularly to the acceptor Venus△C10. The cAMP-recognition motif of human EPAC1 is flanked by two split parts of RLuc8△N3 based on the complementation of split luciferase (CSL) mechanism, thus the conformational change in cAMP-recognition motif reconstitutes the catalytic activity of the split-Luc in a target-molecule-dependent fashion. Upon binding with cAMP, the fusion protein, Nano-lantern(cAMP-1.6), showed the signal increase of about 130%.
We aimed to use the reporter to detect the cAMP surge upon stimulation by ligands of G-protein coupled receptors, so cloned Nano-lantern(cAMP-1.6) downstream three kinds of promoters and navigated its expression, localization and function in yeast Saccharomyces cerevisiae.