Fig. 2.

Toxicity of GFP variants is independent of translation. (A and B) Fluorescence (A) and growth rate (B) of BL21 cells expressing GFP variants with functional and nonfunctional ribosome-binding sites (RBS). (C and D) Fluorescence (C) and growth rate (D) of cells expressing full-length GFP variants, truncated variants, and variants containing internal stop codons or transcription terminators. Inset in C shows location of toxic sequence element in GFP_170, which was calculated based on an analysis of growth rates of 36 shuffled constructs. The y axis shows the statistical significance of the association of particular positions with slow growth. Variants derived from nontoxic GFP_012 are shown in blue and variants derived from toxic GFP_170 are shown in magenta. Full-length constructs, truncated constructs, and constructs with internal stop codons have similar growth rates, suggesting that the element of toxicity resides within the truncated fragment and that the mechanism of toxicity is independent of translation. FL, full-length construct; TT, T7 transcription terminator. All data are averages of nine replicates, ±SEM.