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. 2003 Oct 14;100(22):12672–12677. doi: 10.1073/pnas.2135496100

Fig. 1.

Fig. 1.

One-stage network. (A) A reporter gene (luc or egfp, promoter P) is expressed in the coupled transcription/translation T7/SP6 RNA polymerase wheat germ extract. Kinetics of expression of EGFP in LLM extract (0.1 nM SP6-egfp, filled circles) and SLM extract (0.5 nM T7-egfp, filled squares) with 20 nM T7 RNA polymerase. Three phases are observed: a lag phase of 10 min, an accumulation of synthesized EGFP, and a slowing down to a plateau. The initial lag phase is for transcription and translation. In the SLM extract, expression stops after 3 h, whereas, in the LLM extract, EGFP keeps accumulating beyond 6 h. (B) ATP concentration measurement in the SLM extract. With 20 nM T7 RNA polymerase and 0.5 nM T7-eGFP plasmid (filled circles) and without expression (open circles). The kinetics of expression of EGFP in SLM extract from A has been superimposed (gray filled squares). (C) Luc production in LLM extract measured after 6 h as a function of T7-luc plasmid concentration (filled circles) and SP6-Luc (open squares) with 20 nM RNA polymerase. (Inset) endogenous expression from the extract without addition of T7 or SP6 RNA polymerase as a function of the T7-luc (filled circles) and SP6-luc (open squares) plasmids. (D) Luc production in LLM extract as a function of T7 or SP6 RNA polymerase at 0.1 nM plasmid concentration, T7-luc (filled circles), and SP6-luc (open squares). (Inset) Luc production in LLM extract as a function of neutral plasmid pBR322 concentration, 20 nM RNA polymerase, 0.1 nM T7-luc plasmid (filled circles), and 0.1 nM SP6-luc (open squares).