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. 2014 May 3;42(10):6774–6785. doi: 10.1093/nar/gku307

Figure 6.

Figure 6.

Comparison of operon-based iSAT ribosomes, assembled 70S ribosomes, and purified 70S ribosomes for in vitro protein translation. (A) Diagram showing the difference between iSAT ribosomes, assembled ribosomes, and purified E. coli 70S ribosomes. iSAT ribosomes are made from rRNA transcribed in the iSAT reaction along with purified r-proteins. Assembled ribosomes are made from purified native rRNA and purified r-proteins. Purified E. coli 70S ribosomes are cell-derived and added intact to cell-free TX-TL reactions. (B) Comparison of luciferase production for operon-based iSAT ribosomes, in vitro assembled ribosomes, or purified 70S ribosomes in 15 μl cell-free reactions incubated for 4 h at 37°C. Each condition used a maximum of 100 nM ribosomes, as concentrations of iSAT and assembled ribosomes were limited by use of 100 nM TP70. For assembled and purified ribosomes, pT7rrnB was included at optimized conditions to maintain an equivalent transcriptional load on T7 RNA polymerase; pilot studies suggest that rRNA transcription had no effect on assembled or purified ribosomes. (C) Same comparison as (B) for sfGFP production, measured at 30 min intervals from 0 to 6 h while incubating at 37°C. Note that data for iSAT and assembled ribosomes result in overlapping data points. For panels (B) and (C), values show average reporter protein concentrations above background with error bars representing s.d. for three independent reactions.