Fig. 6: Applying f-MAGE to evolve antibiotic resistance and orthogonality in tethered ribosomes containing two introns.
From: Targeted editing and evolution of engineered ribosomes in vivo by filtered editing
a Introns were introduced at two sites in oRiboT: the engineered (CTt) intron at site 2 and the natural Tt intron at site 4. These orthogonal introns enable mutagenesis of two subunits of the ribosome at three sites: exit channel (site 2), PTC (site 2), and anti-SD (site 4). b, c Continuous in vivo editing with f-MAGE was used to introduce antibiotic resistance at site 2 with defined M4 mutation (Table 1) and orthogonality in the small subunit at site 4. b Post-MAGE cultures were induced for oGFP expression and flow cytometry was performed on cultures from f-MAGE cycles 0–6. Percentage of oGFP-positive cells was quantified for all cycles, and c normalized CFU counts in chloramphenicol (7.74 µM). Chloramphenicol-selected cells were induced for oGFP expression and flow cytometry was performed. Percentage of oGFP-positive cells was quantified for all cycles. Values and error bars represent the mean and standard deviation of n = 3 biologically independent replicates. d, e Continuous in vivo editing with f-MAGE was used to evolve chloramphenicol resistance at site 2 and orthogonality in the small subunit at site 4. d Post-MAGE cultures were induced for oGFP expression and flow cytometry was performed on cultures from f-MAGE cycles 0–6. Percentage of oGFP-positive cells was quantified for all cycles, and (e) normalized CFU counts in chloramphenicol (7.74 µM). Chloramphenicol-selected cells were induced for oGFP expression and flow cytometry was performed. Percentage of oGFP-positive cells was quantified for all cycles. Values and error bars represent the mean and standard deviation of n = 3 biologically independent replicates.
