Figure 2

Construction of 18-members N₆-RedLibs and GLOS-RedLibs genomic lacZ RBS libraries. Randomized RBS sequences were created with N₆-degeneracy or according to the GLOS rules and subsequently reduced to 18-members libraries with RedLibs to uniformly span the TIR range. Frequencies of the 18 members of the N₆-RedLibs library in (a) the MMR⁻ Ec^- strain and (b) the MMR⁺ EcNR1 strain. N₆-RedLibs degenerate oligonucleotide sequence: DDKGAG. (d) Frequencies of the 18 members of the GLOS-RedLibs RBS library in the MMR⁺ EcNR1 strain. GLOS-RedLibs degenerate oligonucleotide sequence: BDGGGW. Each library was integrated in duplicate and individual clones were analyzed by Sanger sequencing (n = 96 for each library). Red: wild type lacZ RBS sequence. RBS sequences showing indels were excluded. (c and e) Distribution of sequences in the oligonucleotide pool after chemical synthesis for the oligonucleotides used for the N₆-RedLibs library (c) and GLOS-RedLibs library (e). Illumina-based oligonucleotide pool sequencing for the different oligonucleotide libraries. Assuming a uniform distribution, each sequence should be represented in the pool with 5.5%, which correspond to 0% discrepancy from uniform distribution. A discrepancy of 50% corresponds to 2.75% abundance in the oligonucleotide pool. Bars in black indicate oligonucleotides for which no corresponding strains could be recovered in the single library preparations.