Figure 5

Assessment of C activity in CR.NsoJS138I. A. Schematic design of experiment. Top line indicates IPTG-inducible gene for T7 RNA polymerase in the host strain’s chromosome, middle indicates a plasmid (pJL200, called pNso for the figure) that carries the gene for CR.NsoJS138I linked to a T7 promoter, and the bottom indicates a plasmid (pJL300, called pBox-Lac for the figure) that carries the putative promoter and C box region from NsoJS138I linked to a promoterless gene for lacZ (β-galactosidase). B. Sequence of the putative promoter and C box region from NsoJS138I, showing the candidate C boxes (shaded) and promoter elements (−35 and −10 hexamers). This 161 nt sequence is what was included in pJL300 (pBoxLac). C. Timecourse of LacZ induction. Growing triplicate cultures of cells containing the indicated plasmids were treated at time = 0 with the inducer IPTG (which in these cells controls the gene for T7 RNA polymerase), and matched control cultures received no IPTG. LacZ activity was measured over time. The symbols indicate means of the triplicate cultures; standard errors are shown but mostly obscured by the symbols. D. Steady-state expression of lacZ. Triplicate cultures containing the plasmids indicated were grown for at least 10 generations in the presence or absence of IPTG, and LacZ activity was measured. In this case, activity is plotted vs. culture density, and so modified Miller units are used (in which the culture density term has been removed). Cultures approximating steady-state growth should give good linear fits, the slopes of which accurately measure relative expression levels. The symbols indicate means of the triplicate cultures; standard errors are shown but are in some cases obscured by the symbols.