Protocols
For successful execution of all experiments, described in the previous pages, we used the following protocols:
Inovation in measurement
Sensor development and characterization
While calcium influx could be detected by exogenous fluorescent dyes such as Fura Red and Fluo-4, our project required a sensitive, genetically encoded calcium sensor that would couple a change in the calcium concentration to a biologically relevant output, such as the luciferase reporter or reconstitution of a split protease for the initiation of the signaling pathway. An ideal calcium sensor should be inactive at intracellular concentration of calcium and have a high response to calcium concentrations above physiological levels and should be detected by a quick and easy readout. For our intended application, the calcium sensor should also have the potential to act as the reconstitution mechanism for split proteins so that a conformational rearrangement in the presence of calcium would bring the two split protein fragments together and reconstitute the protein’s activity.
A wide palette of genetically encoded calcium sensors have been used for mapping intracellular calcium concentration
We replaced the FRET pair of CaMeleon2.12 by a split firefly luciferase (fLuc) as it provides a distinct signal even at small amounts and has a high
signal-to-noise ratio. The new luciferase based calcium sensor was named fLuc2.12. The fLuc2.12 was tested on HEK293 cells, but we found that the sensor
was active already in resting cells (1). We hypothesized the activation was a consequence of a close proximity of calmodulin and M13 in the
fusion even in the absence of calcium binding. In order to resolve this problem we set out to test a similar sensor based on two separate molecules.
Two-molecule-based CaM sensors have not been widely used, but lower leakage in comparison to a single molecule sensor has been reported by Miyawaki
et al.