Engineering Transcriptional Interference for Genetic Logic Gates

Transcriptional Interference (TI) is widespread in the genomes in all kingdoms of life and serves to regulate important cellular decisions. TI can occur through the collisions of RNA Polymerases (RNAPs) in tandem or in convergent orientation, and through the collision of RNAPs with protein roadblocks. Mathematical modeling and experiments have characterized several naturally occurring TI systems, but the design rules for constructing TI-based genetic devices are not yet well-defined. Here, we show that rationally controlling RNAP traffic on DNA through TI can lead to diverse gene expression profiles and facilitate the construction of TI-based logic gates. We demonstrate that tuning the dissociation constant of a protein roadblock enables optimized AND and OR logic gates, and that gate behavior can be predicted and validated through mathematical modeling. We then show that protein roadblocks coupled with RNAP collisions can produce NAND and NOR gates, and that the magnitude of gene repression from RNAP collisions can be tuned through inhibition of Rho-dependent transcription termination. These results expand the potential for TI as a novel tool for synthetic biology and offer insights into an important but relatively unexplored gene regulatory mechanism.