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. 2019 Nov 4;4(4):189–196. doi: 10.1016/j.synbio.2019.10.002

Fig. 4.

Fig. 4

Cell-free production of double mutant GFP protein (Y35UAAD193UAG), using extracts mixtures of different ratios. Fluorescence intensity of double mutant production was compared to WT protein or double mutant without any Uaas (negative control). a) mixtures of OTS pairs and mixtures of OTS single components extract comparison for double mutant GFP production. A two-extracts mixture composed of AzF-OTS for UAA suppression and Pyl-OTS for UAG suppression, while a four-extracts mixture composed of the following extracts: 1) AzF synthetase 2) AzF tRNA for UAA suppression 3) PrK synthetase 4) PrK tRNA for UAG suppression. b) OTS pairs extract mixture, between AzF-OTS for UAA suppression and increasing quantities of Pyl-OTS for UAG suppression, at different ratios. c) Linear regression of extract dilution for double mutant GFP expression measured by GFP fluorescence. d) Lyophilized extracts and mixing ratios of AzF-OTS for UAA supression and Pyl-OTS for UAG supression: 1:1, black; 1:2, blue Extracts were rehydrated with varying volumes of water (t-test, ns represents p-val>0.05, * represents p-val = 0.02).