Fig. 1.

CFPS from extracts of a genomically recoded organism. a Schematic of the production and utilization of crude extract from genomically recoded organisms with plasmid overexpression of orthogonal translation components for cell-free protein synthesis (CFPS). CFPS reactions are supplemented with the necessary substrates (e.g., amino acids, NTPs, etc.) required for in vitro transcription and translation as well as purified orthogonal translation system (OTS) components to help increase the ncAA incorporation efficiency. aaRS, aminoacyl tRNA synthetase; ncAA, non-canonical amino acid; T7P, T7 RNA polymerase; UAG, amber codon. b Time course of superfolder green fluorescent protein (sfGFP) synthesis catalyzed by extracts derived from a genomically recoded organism, C321.∆A, and a commercial strain, BL21 Star (DE3). Three independent batch CFPS reactions (n = 3) were performed at 30 °C for each time point over 24 h. Error bar = 1 SD