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.