Extended Data Fig. 5: Optimal gene expression output may be obtained at intermediate TF concentrations.
From: Engineering AraC to make it responsive to light instead of arabinose
a, Schematic representation of the plasmid used in step 1. Regulatory sequences (O1 and O2 half-sites) upstream of the PBAD promoter have been deleted. Right, example of a scenario for which the highest mCherry levels are obtained at intermediate cTF levels (red line). b, In step 2, the range of transcriptional output (visualized through the fluorescent reporter expression) induced by different IPTG concentrations is mapped to the rates of constitutive promoters in a library (right), allowing for the identification of a constitutive promoter that matches the desired optimal BLADE expression level (schematically shown with a red line). A plasmid bearing the IPTG-inducible promoter as well as an extended library of constitutive promoters (http://parts.igem.org/Promoters/Catalog/Anderson) was used. To minimize the potential influence of individual promoters on mRNA transcription and translation initiation, we used a ribosome binding site (RBS) containing an insulating ribozyme (RiboJ59). Plotting the mCherry fluorescence levels obtained with the constitutive promoters and with the IPTG-inducible promoter at different IPTG concentrations in the same plot, it is possible to find the constitutive promoter that best matches the expression from the IPTG-inducible one at the desired IPTG concentration. This mapping has to be performed only once for a given strain background at specific conditions, which in our case as an example was done in E. coli BW25113ΔaraC attB::lacY. Values represent mean ± SD of more than three biological replicates acquired on different days.
