EverGreen: A Calcium Sensing Fluorescent Protein with Ultra-Slow Response Properties

Abstract

Fluorescent calcium indicators are typically optimized for fast kinetics, but for some bio-engineering projects in synthetic biology slower indicators with long decay time constants may be more practical and easier to apply. We here present a green fluorescent protein calcium biosensor with unusually slow response kinetics. By molecular engineering we were able to extend a slow component of the decay time to 6.1 min. The fluorescence in EverGreen could thus be detected for minutes after the underlying calcium fluctuation had already dissipated. This may be useful when constraints in instrumentation and methodology do not allow close coupling of physiological activation of cells and read-out of signals, facilitating directed evolution projects run via plate readers or cell sorters.