Optimization of eCFPS components.

Protein expression levels from the eCFPS system were measured using an Nanoluciferase (NLuc) reporter DNA. Green area in the graphs indicate the common concentration range used in published protocols for eCFPS. Error bars represent the standard error (SE) of at least three independent reactions. (A-E) Protein expression levels of the eCFPS system supplemented with different concentration of DTT (A), cAMP (B), PEG8000 (C), NH4+ (D), and folinic acid (E). (F-I) Protein expression levels of eCFPS with various concentrations of tRNA (F), amino acids (G), CTP (H) and UTP (I). (J) A summary of the supplement components before and after optimization.

Optimization of essential components for eCFPS system.

(A) Protein expression levels of the eCFPS system measured at varying concentrations of KGlu and MgGlu2. (B) Protein expression levels of the eCFPS system measured at varying concentrations of MgGlu2 and PEG8000. (C) Protein expression levels of the eCFPS system measured at varying concentrations of ATP and GTP. (D) Protein expression levels of the eCFPS system measured at varying concentrations of CrK and CrP. (E)Protein expression levels of the eCFPS system measured at varying pH and buffer concentrations. Data from all panels present mean ± SE, n = 3.

Characterization of the optimized eCFPS system.

(A) Kinetics of protein synthesis at 25°C, 30°C and 37°C over a 60-minute period. Data present mean ± SE, n= 3. (B) Protein expression levels of the eCFPS system measured at varying DNA concentrations for a reporter encoding a FLAG-tagged NLuc. The protein product was quantified via a luminescence assay and confirmed by western blotting. Data present mean ± SE, n = 3. (C) Comparison of protein expression levels from the initial and optimized eCFPS systems at various cell extract volume ratios. Data present mean ± SE, n = 4.

Benchmarking the optimized eCFPS system with different DNA templates.

(A) NLuc protein expression kinetics over time, comparing the PEP-based, initial CrP/CrK-based, and optimized CrP/CrK-based energy regeneration systems. Data present mean ± SE, n = 4. (B) sfGFP protein expression kinetics over time from the three energy regeneration systems. Data present mean ± SE, n = 3. (C-D) Western blot validation of protein expression for NLuc (C) and sfGFP (D) from the different eCFPS system shown in (A-B). Protein products were detected using an anti-FLAG antibody. The asterisk (*) indicates a non-specific band. (E) Western blot detection of His-FLAG-BsaI expressed by the optimized eCFPS system using an anti-FLAG antibody. (F) Agarose gel electrophoresis confirming the functional activity of eCFPS-synthesized BsaI via cleavage of a substrate plasmid. A 10-fold serial dilution of BsaI was with 1x representing 0.05 mg/mL. NC (negative control) indicates no plasmid in the eCFPS reaction. S, L, and O indicate the respective position of the supercoiled, linear, and open circular forms of the plasmid. (G) Western blot analysis of vimentin expressed by the optimized eCFPS system using an anti-vimentin antibody. (H) Negative-stain electron microscopy image showing that vimentin expressed via eCFPS can successfully self-assemble into filaments in vitro.

Preparation of eCFPS from cultured E. coli Cells.

(A) Flowchart of the eCFPS preparation procedures. (B) Comparison of reaction efficiency in eCFPS using lysate with bacteria cells harvested at different optical density. Data present mean ± SE, n = 3. (C) Sucrose gradient sedimentation analysis of different lysates used for eCFPS, revealing the presence of ribosome monomers. (D) Comparison of protein expression levels in eCFPS system using lysates prepared by runoff, dialysis, and rapid endogenous T7 RNA polymerase induction. Data present mean ± SE, n = 4. (E) Comparison of reaction efficiency in eCFPS using lysates after different numbers of freeze-thaw cycles. Data present mean ± SE, n = 3.