Figure 3.

Engineering of translation using tetA^OPT. (a) Illustration of the workflow for modulating translation initiation rates by using tetA^OPT. In the first step, tetA^OPT is integrated upstream of the start codon of the gene of interest (goi) by selection on tetracycline. In the next step, tetA^OPT is removed using a degenerated oligonucleotide harboring homology (gray triangles; recombination sites) to the flanking regions of tetA^OPT and selection on NiCl₂. The degenerated oligonucleotide introduces a random sequence in the TIR. (b) Illustration of the TIR randomization of the native E. coli gene lacZ. (c) Relative beta-galactosidase activity of the TIR library variants (gray) normalized to the native TIR (dark blue). A negative control was included (tetA^OPT inserted upstream of lacZ; red). In c, bar graphs of the libraries shown are based on single data points.