A palette of bright and photostable monomeric fluorescent proteins for bacterial time-lapse imaging

We report the engineering of novel cyan, green, yellow, and red fluorescent proteins that exhibit improved photostability and aggregation properties while retaining high in vivo brightness. Using sitedirected mutagenesis in Escherichia coli , we first derived the superfolder green fluorescent protein (sfGFP) into mChartreuse, a brighter, more photostable, and monomeric fluorophore. This mChartreuse variant was further derived into cyan and yellow variants with enhanced photostability and dispersibility. We also report a mutation that eliminates residual oligomerization in red fluorescent proteins derived from Discosoma sp , such as mCherry or mApple. Incorporation of this mutation in mApple among other substitutions yielded mLychee, a bright and photostable monomeric red fluorescent protein. These novel fluorescent proteins outperform current standards and are, therefore, highly desirable for fluorescence time-lapse analysis in bacteria.