Fraikin, N.; Couturier, A.; Lesterlin, C.

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 site-directed 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.