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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session E

Poster U15 - Development of oscillatory biosensors for cellular events based on a bacterial reaction-diffusion system

Saturday, October 26, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  The E. Coli MinCDE system represents a model reaction-diffusion system that undergoes oscillatory behavior to orchestrate molecular transport and positioning involved in complicated tasks such as cell division and regulation of metabolism. The pole-to-pole oscillation is driven by the membrane-bound ATPase MinD and ATPase-activating protein MinE, where the recruitment of MinE stimulates MinD ATPase activity which in turn leads to detachment of MinDE from the membrane. Previous studies have illustrated the MinDE operated reaction-diffusion behaviors, i.e., protein oscillations, in mammalian cells, providing the feasibility of engineering the MinDE system as molecular reporters for mammalian cell events.

Materials and

Methods:  MinD and MinE were synthesized as gBlocks and were further engineered as molecular reporter plasmids using the NEBuilder HiFi DNA Assembly Master Mix or standard restriction digestion/ligation strategy. Transient transfection of HeLa and HEK293T cells was performed using Lipofectamine 3 000 (Invitrogen) reagent by following the manufacturer's instructions. After transfection for 16-24 hours, live-cell Images and movies were captured on a Nikon Ti2 Inverted microscope with a Yokogawa W‐1 dual spinning disk scanhead, Micro‐Scanner for photo‐stimulation, and stage top incubator.

Results, Conclusions, and Discussions: Herein, we present the design of a set of molecular reporters operated by MinDE oscillation (designated mRODEO). We demonstrated the feasibility of several common strategies, e.g., split/reassembly, insertion, allosteric modulation, payload co-trafficking, and sequestration, in engineering MinDE system as a molecular reporter. By integrating with different proteins/peptides of interest, mRODEO were able to transform different cellular events/signaling into MinDE oscillations. Collectively, mRODEO has successfully reported cellular calcium influx, protease activity, protein-protein interaction, and served as a drug/peptide screening platform.

Acknowledgements (Optional):