Fig. 4

Characterization of the set of constitutive promoters. a. The Hsmar1 gene is fused or not to 3x FLAG-tag on its C-terminus and cloned downstream of one of six different promoters (see text for more details) with an inactive or optimal RBS (defined in Fig. 2a). The construct is located between terminator sequences (T) upstream and downstream to avoid read-through transcription. To further control the number of copies, the plasmid backbone is a one-copy, pBACe3.6 (b), or a ~ 13-copy, pGMH491 (pIncQ, I), vector. b. Western blots using an antibody against the C-terminus of SETMAR, which corresponds to the domesticated Hsmar1, to compare the strongest promoters with an optimal RBS to the Ptac promoter induced with different concentration of IPTG. c. The promoter strength of each construct was determined by flow cytometry after cloning an EGFP gene in each vector (pRC1782–1807). The number EE to 6 corresponds to one of the six promoters. The single and ~ 13-copy vectors are annotated B or I, respectively. The vectors with an inactive or an optimal RBS are annotated – or ++, respectively. The fluorescence data were normalized to the strongest promoter, Ip6++. Average of the geometric mean ± standard deviation of two biological replicates, except for Bp-EE- where there is only replicate. Neg: negative control, Ip0 (empty vector). d. Plot of the relative mRNA production (as defined in [32]) versus the promoter strength determined by flow cytometry in Fig. 3c. The relative mRNA production of pEE was arbitrary defined as ten times less than p2