Figure 3
Genomic integration of a chromosomal 18-members GLOS-RedLibs ribA RBS library into an MMR+ strain. (a) Schema of the reduced ribA RBS library construction. Upper: wild type ribA RBS sequence; lower: GLOS-RedLibs degenerate ribA RBS sequence. (b) Library clones were analyzed by Sanger sequencing (n = 88). The 18 possible RBS sequence variations (grey) as well as the wild type RBS sequence (red) are listed in ascending order of their predicted TIR. RBS sequences showing indels were excluded from analysis. Therefore, only 86 of 88 sequenced clones are displayed. (c) Riboflavin production of all isolated RBS library member strains (n = 88, grey, single measurements) compared to wild type (red, in triplicate, mean ± standard deviation). (d-e) Analysis of the separately constructed ribA library members CATAGA, GAGAGA, GAGGGA not found in the sequenced library clones in b (d) Riboflavin production of the strains compared to wild type (wt, red), values in triplicate are mean ± standard deviation. (e) Growth curves of the corresponding strains in LB medium at 30 °C (n = 3). (f) Distribution of sequences in the oligonucleotide pool after chemical synthesis. Illumina-based oligonucleotide pool sequencing for the oligonucleotide used to construct this library. Bars in black indicate oligonucleotides for which no corresponding strains could be recovered during library-based genome editing.
