Figure 3. Performance of rationally reduced libraries for two fluorescent proteins.

(a) A vector design (plasmid pMJ1) was chosen in which the genes for sfGFP and mCherry are placed under control of opposing constitutive promoters separated by a spacer to allow independent randomization of both RBSs. (b) The performance of irrationally randomized RBSs (N₆ × N₆ and N₈ × N₈) as well as of three libraries optimized by RedLibs (sizes 4 × 4, 12 × 12 and 24 × 24) were compared by random in silico picking (252 picks). (c) The behaviour of the rationally reduced libraries was analysed in vivo by measuring the cell-specific green and red fluorescence of 252 independent cultures of E. coli for each library (N₆, N₈, 4, 12 and 24).