Predicting the quantitative regulatory function of transcription factors (TFs) based on factors such as binding sequence, binding location, and promoter type is not possible. The interconnected nature of gene networks and the difficulty in tuning individual TF concentrations make the isolated study of TF function challenging. Here, we present a library of Escherichia coli strains designed to allow for precise control of the concentration of individual TFs enabling the study of the role of TF concentration on physiology and regulation. We demonstrate the usefulness of this resource by measuring the regulatory function of the zinc-responsive TF, ZntR, and the paralogous TF pair, GalR/GalS. For ZntR, we find that zinc alters ZntR regulatory function in a way that enables activation of the regulated gene to be robust with respect to ZntR concentration. For GalR and GalS, we are able to demonstrate that these paralogous TFs have fundamentally distinct regulatory roles beyond differences in binding affinity.