Cutting Translation out of the Equation: Exploring the P4 CI RNA as the Basis for a Novel Family of Translation-Independent Genetic Parts

Synthetic biology (SynBio) aims to engineer living organisms for applications in industry, medicine, and environmental health. Central to SynBio is the concept of genetic circuits, DNA sequences fabricated from standardized biological parts to perform specific functions. Traditionally, these circuits rely on translation, the biochemical process of synthesizing proteins from mRNA. However, translation-independent logic presents a promising alternative paradigm for engineering genetic circuits that are less burdensome to their hosts, function with less latency, and have a number of other potential advantages. One relevant approach uses antisense RNA-mediated transcriptional attenuation, a molecular mechanism through which small antisense RNAs halt transcription by binding to attenuation sites on nascent RNA transcripts. Engineered orthogonal (mutually non-interfering) antisense-attenuator pairs have enabled RNA-only genetic circuits. However, RNA-based inhibitors of transcription remain scarce compared to their protein counterparts, in part because very few naturally existing antisense-attenuator pairings have been thoroughly investigated for this purpose. This project aims to expand translation-independent genetic parts by characterizing and working towards a genetic parts library derived from an alternative antisense RNA, designated CI, and derived from the bacteriophage satellite P4.