Construction of two-input logic gates using Transcriptional Interference

Transcriptional Interference (TI) has been shown to regulate gene expression at the DNA level via different molecular mechanisms. The obstacles present on the DNA that a transcribing RNA polymerase might encounter, e.g. a DNA bound protein or another RNA polymerase, can result in TI causing termination of transcription, thus reducing gene expression. However, the potential of TI as a new strategy to engineer complex gene expression modules has not been fully explored yet. Here we created a series of two-input devices using the presence of a roadblocking protein using both experimental and mathematical modeling approaches. We explore how multiple characteristics affect the response of genetic devices engineered to act like either AND, OR, or Single Input logic gates. We show that the dissociation constant of the roadblocking protein, inducer activation of promoter and operator sites, and distance between tandem promoters tune gate behavior. Our results demonstrate that controlling RNAP traffic using the TI mechanism of roadblock can generate genetic devices capable of more diverse gene expression patterns than traditional single input promoters. This work highlights the potential of rationally creating different types of genetic responses using the same transcription factors in subtly different genetic architectures.