Fig. 3. Synthetic attC sites embedded into protein coding regions.

(A) In silico directed evolution approach that was used to generate synthetic attC sites with peptide linker properties (B-E) and to embed synthetic attC sites into lacZ (F-H). (B) Structures of the three synthetic attC sites encoding peptide linkers L1, L2, and L3, predicted using ViennaRNA 2.1.8. (C) Protein sequences of encoded peptide linkers. (D) Recombination frequencies of attC_aadA7 (positive control) and synthetic attC sites encoding peptide linkers. Values represent means of three independent experiments; error bars represent mean absolute error. Asterisk (*) indicates that the recombination frequency was below detection level, indicated by the bar height. (E) Results of a bacterial two-hybrid assay with the two domains of Bordetella pertussis adenylate cyclase fused either with a natural linker (p5, positive control), a natural linker with a frameshift mutation (p5_FS, negative control), or synthetic attC sites encoding peptide linkers. Values represent means of three independent experiments; error bars represent mean absolute error. (F) Predicted structures of the synthetic attC sites embedded into four regions of the lacZ gene encoding β-galactosidase, predicted using ViennaRNA 2.1.8. (G) Protein sequences of the four β-galactosidase target regions and sequences after attC site embedding. Blue, mutations that preserve the amino acid physicochemical properties; red, other nonsilent mutations. (H) Strains with the four synthetic attC sites embedded into lacZ and streaked on an LB agarose plate with X-gal and isopropyl-β-D-thiogalactopyranoside. Blue color indicates a functional β-galactosidase, as in lacZ_wt. White color indicates that an embedded attC site perturbed the function of the β-galactosidase, as in lacZ::attC_aadA7.