Lakin, M. R. | bioRxiv Science

bioRxiv2025

Title: Engineering Plasmids with Synthetic Origins of Replication

Plasmids remain by far the most common medium for delivering engineered DNA to microorganisms. However, the reliance on natural plasmid replication mechanisms limits their tunability, compatibility, and modularity. Here we refactor the natural pMB1 origin and create plasmids with customizable copy numbers by tuning refactored components. We then create compatible origins that use synthetic RNA regulators to implement independent copy control. We further demonstrate that the synthetic origin of replication (SynORI) can be engineered modularly to respond to various signals, allowing for multiplexed copy-based reporting of environmental signals. Lastly, a library of 6 orthogonal SynORI plasmids is created and co-maintained in E. coli for a week. This work establishes the feasibility of creating plasmids with SynORI that can serve as a new biotechnology for synthetic biology.

synthetic biology↗

bioRxiv2017

Sequential Super-Resolution Imaging using DNA Strand Displacement

Sequential labeling and imaging in fluorescence microscopy allows the imaging of multiple structures in the same cell using a single fluorophore species. In super-resolution applications, the optimal dye suited to the method can be chosen, the optical setup can be simpler and there are no chromatic aberrations between images of different structures. We describe a method based on DNA strand displacement that can be used to quickly and easily perform the labeling and removal of the fluorophores during each sequence. Site-specific tags are conjugated with unique and orthogonal single stranded DNA. Labeling for a particular structure is achieved by hybridization of antibody-bound DNA with a complimentary dye-labeled strand. After imaging, the dye is removed using toehold-mediated strand displacement, in which an invader strand competes off the dye-labeled strand than can be subsequently washed away. Labeling and removal of each DNA-species requires only a few minutes. We demonstrate the concept using sequential dSTORM super-resolution for multiplex imaging of subcellular structures.

bioengineering↗