Fig. 2.
The construction of an orthogonal AND function. (A) The logic of orthogonal gene expression (Left). A molecular strategy (Right) for the realization of orthogonal gene expression. Plac, lac promoter; T7 RNAP, the gene encoding T7 RNAP; PT7, T7 promoter; gfp, gene encoding the GFP; Ptrc, a hybrid of the tryptophan and lactose promoters; O-rDNA, encodes an orthogonal ribosomal RNA. (B) Sequences selected after 3 rounds of FACS sorting for O-ribosome-dependent translation of a T7 transcript. T7S2 was represented 3 times and T71503 was represented twice among the best clones for GFP expression. (C) Orthogonal transcription–translation shows AND logic. The expression construct pT7 O-rbs GST-GFP contains the regulatory sequences from T71504 (in B) upstream of a genetic fusion between GST and sfGFP. (Top) Expressed and purified GST-GFP. (Middle) A Northern blot to detect sfGFP mRNA. (Bottom) A loading control for total RNA.