A Modular Scaffold for Controlling Transcriptional Activation via Homomeric Protein–Protein Interactions

Protein–protein interactions (PPIs) have been extensively utilized in synthetic biology to construct artificial gene networks. However, synthetic regulation of gene expression by PPIs in E. coli has largely relied upon repressors, and existing PPI-controlled transcriptional activators have generally been employed with heterodimeric interactions. Here we report a highly modular, PPI-dependent transcriptional activator, cCadC, that was designed to be compatible with homomeric interactions. We describe the process of engineering cCadC from a transmembrane protein into a soluble cytosolic regulator. We then show that gene transcription by cCadC can be controlled by homomeric PPIs and furthermore discriminates between dimeric and higher-order interactions. Finally, we demonstrate that cCadC activity can be placed under small molecule regulation using chemically induced dimerization or ligand dependent stabilization. This work should greatly expand the scope of PPIs that can be employed in artificial gene circuits in E. coli and complements the existing repertoire of tools for transcriptional regulation in synthetic biology.