Microfluidic cell sorter-aided directed evolution of a protein-based calcium ion indicator with an inverted fluorescent response

Abstract

We demonstrate a simple, low cost and disposable microfluidic fluorescence activated cell sorting system (μFACS) for directed evolution of fluorescent proteins (FP) and FP-based calcium ion (Ca2+) indicators. The system was employed to pre-screen libraries of up to 106 variants of a yellow FP-based Ca2+ indicator (Y-GECO) with throughput up to 300 cells per s. Compared to traditional manual screening of FP libraries, this system accelerated the discovery of improved variants and saved considerable time and effort during the directed evolution of Y-GECO. Y-GECO1, the final product of the μFACS-aided directed evolution, has a unique fluorescence hue that places it in the middle of the spectral gap that separates the currently available green and orange FP-based Ca2+ indicators, exhibits bright fluorescence in the resting (Ca2+ free) state, and gives a large response to intracellular Ca2+ fluctuations in live cells. This journal is © the Partner Organisations 2014.