Microfluidic cell sorter-aided directed evolution of a protein-based calcium ion indicator with an inverted fluorescent response.
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 (Ca(2+)) indicators. The system was employed to pre-screen libraries of up to 10(6) variants of a yellow FP-based Ca(2+) 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 Ca(2+) indicators, exhibits bright fluorescence in the resting (Ca(2+) free) state, and gives a large response to intracellular Ca(2+) fluctuations in live cells.
Department of Chemistry, University of Alberta, 11227 Saskatchewan Drive, Edmonton, Alberta T6G 2G2, Canada. jed.harrison@ualberta.ca robert.e.campbell@ualberta.ca.
Yongxin
Y
Zhao
Ahmed S
AS
Abdelfattah
Yufeng
Y
Zhao
Araya
A
Ruangkittisakul
Klaus
K
Ballanyi
Robert E
RE
Campbell
D Jed
DJ
Harrison
D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000032:analysis*; D019020:Directed Molecular Evolution / Q000379:methods*; D005260:Female; D006367:HeLa Cells; D006624:Hippocampus / Q000737:chemistry; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D044085:Microfluidics / Q000295:instrumentation / Q000379:methods*; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley
D008164:Luminescent Proteins; D019151:Peptide Library; D002118:Calcium