E2-Orange and E2-Red/Green do not form higher-order aggregates and are noncytotoxic to bacteria. (A) To assay higher-order aggregation of oligomeric FPs, the percent fluorescence in the pellet fraction of a lysate from E. coli cells expressing E2-Orange, E2-Red/Green, or KO* was measured for eight independent replicates. Error bars represent s.e.m. (B) To measure bacterial cytotoxicity of FPs, E. coli DH10B cells harboring the pREP4 repressor plasmid were transformed with pQE-60NA encoding DsRed-Express2, E2-Red/Green, E2-Orange, mOrange2, KO, or KO*. Equal volumes of transformation mixtures were plated onto adjacent sectors of plates under either repressing (no IPTG) or derepressing (1 mM IPTG) conditions. Large colonies under derepressing conditions (right panel) indicate low cytotoxicity. (C) Quantitation of FP expression under derepressing conditions. Cells were grown to an OD600 of ~ 0.6 and then treated with 1 mM IPTG for 4 h. Whole-cell lysates were separated using SDS-PAGE followed by staining with Coomassie Blue. Control cells were transformed with empty pQE-60NA.