Synthetic biology provides an ideal approach to build functional biological devices by assembling biological parts. Using synthetic biology, efficient control of gene regulation may be achieved to a degree that is not possible using natural genetic structures.1 However, previous studies on promoter engineering have focused on natural transcription factors (TFs),2 including the lac repressor, which produces a switch-like “all-or-none” response.3 In this project, we worked to develop a new system for transcriptional control based on tunable synthetic TFs, which are designed to yield programmable linear responses in gene expression. To accomplish this, we used zinc finger proteins (ZFPs) as regulators of engineered promoters assayed by green fluorescent protein (GFP) as a fluorescent transcriptional reporter probe. In particular, we designed strong-binding three finger ZFPs as proof-of-principle regulatory elements, with the intention of moving to weaker binding two finger ZFPs and the addition of the accessory binding module PAR (part of the protein Adr1).4 To generate engineered promoters, we integrated ZFP binding sites into known promoters of varying strength. To analyze the engineered activity of each promoter, we cultured E. coli cells transformed with plasmids containing sequences for both ZFP production and our engineered promoters and measured the resulting fluorescence intensity. In this way, we constructed a novel method for tuning gene expression as well as testing the DNA binding affinity of synthetic TFs. We anticipate that this general approach could be used in the future for designing and characterizing synthetic TFs for gene therapy and gene regulation applications
