Fig. 2
From: Engineered ribosomes with tethered subunits for expanding biological function
Optimizing tether sequence improves performance. a Wild-type 23S rRNA helix 101 and 16S rRNA helix 44 are connected to create Ribo-T with 9A for 5′ tether, T1, and 8A for 3′ tether, T2. b Library 1: paired 5′ tether T1 poly A from 7–20 nucleotides, with 3′ tether T2 poly T from 7–20 nucleotides. Library 2: unpaired polyA on both T1 and T2, ranging in 7–20 nucleotides long. Library 3: randomized T1 (8N) and T2 (9N) keeping residues of opened H101 and h44 apex loops. Library 4: randomized apex-to-apex T1 (15N) and T2 (10N) of tether. c Selection scheme for improved tethers. Strains lacking genomic copies of rRNA operons (Δ7rrn) are transformed with plasmid-based Ribo-T tether libraries, and the wild-type pCSacB plasmid (wt) is removed. d Tether sequences and growth rates of analyzed colonies. Error bars = 1SD of noted independent colonies, n. The top 15 Ribo-T design winners (L4-1 through L4-13) were co-cultured and passaged for 3 days. Between each passage, the bulk culture populations were sequenced and analyzed. Source data for d can be found in the Source Data file
