Title: Inducible cellular memory using switchable synthetic plasmid origins of replication
Abstract:
Control of plasmid replication rate is undertaken by many different cellular mechanisms, enabling compatible plasmids to be stably co-maintained in cells. This capability is essential for a multitude of engineering applications, including metabolic engineering, programmable genetic circuitry, and synthesis of biological products. In this work, we focus on RNA-based control mechanisms. We build upon previous work that modulated plasmid origins of replication [Liu25] via the pT181 transcriptional attenuator [Bra00], which was established and modified from a repurposed ColE1 origin antisense RNA that regulates plasmid replication initiation. We expand upon this technology by adding an inducible recombination system to this modified attenuator replication scheme to permanently change plasmid replication rate upon sensing of an induction signal. Reorienting relative promoter directionality between the transcriptional attenuator RNA and its target RNA from a convergent to a tandem-up orientation causes the plasmid to switch from a low-copy to a high-copy state [Liu25] and is directed by arabinose or
2,4-diacetylphloroglucinol (DAPG) induction of Bxb1 serine recombinase expression [Mer18]. This technology offers a method by which plasmid replication rate may be used as a biosensor in response to an outside chemical stimulus, causing a permanent switch in plasmid copy number that could function as a form of cellular memory in biosensing applications.
