Fig. 3: Generation of fully autonomous AcK-synthesizing E. coli and mammalian cells.
From: Engineering unnatural cells with a 21st amino acid as a living epigenetic sensor
a Comparison of the production of sfGFP-Y151AcK, sfGFP-Y151AcK-Y182AcK, and sfGFP-N39AcK-Y151AcK-Y182AcK from cells supplemented with AcK versus those producing biosynthesized AcK. b SDS-PAGE analysis of sfGFPs expressed in the presence (+) or absence (−) of exogenous 20 mM AcK (fed), or with ( + bio) or without (-bio) RDW23166.1 induction. Bio: biosynthesis; Fed: feeding. c ESI-MS analysis of sfGFP-AcK proteins expressed in E. coli, with either 20 mM AcK supplementation or AcK biosynthesis, as well as sfGFP-2AcK and sfGFP-3AcK proteins expressed via AcK biosynthesis. d A schematic representation of the genetic circuits used to generate completely autonomous mammalian cells producing AcK-containing proteins. e The confocal images of HEK293T cells expressing MmAcKRS-3, MmPyltRNACUA, and EGFP with an amber codon at the Tyr39 position, with or without feeding of 1 mM or 5 mM AcK, and the biosynthesis of AcK. Scale bar = 100 μm. Three times of the experiment were repeated with the same results. f Intracellular concentrations of AcK from exogenous AcK feeding or AcK biosynthesis. g The fluorescence signal of EGFP-Y39TAG without AcK, with 1 or 5 mM AcK supplementation, or with AcK biosynthesis. a, f, g Data are plotted as the mean ± standard deviation from n = 3 independent biological replicates. Statistical analysis was performed using two-tailed unpaired t-test. **P < 0.01; ****P < 0.0001. Exact p-values: P = 0.0014 for the TAG group, P < 0.0001 for the 2TAG group, and P < 0.0001 for the 3TAG group in (a); P < 0.0001 in (f); P = 0.0061 in (g). arb. units, arbitrary units. Source data are provided as a Source Data file.
