Lezia, A. | bioRxiv Science

bioRxiv2023

Exploiting heterogeneity in coupled, two plasmid systems for dynamic population adaptation

In synthetic multi-plasmid systems, it is standard to use only plasmids with orthogonal replication mechanisms to avoid phenotypic heterogeneity and ensure plasmid stability. In nature, however, microbial populations actively exploit heterogeneity to survive in fluctuating environments. Here we show that the intentional use of distinct plasmids with identical origins of replication (oris) can help an engineered bacterial population adapt to its environment. We find that copy number coupling between distinct plasmids in such systems allows for copy number buffering of an essential, but high-burden construct through the action of a stably maintained, nonessential plasmid. Plasmid coupling also generates population state memory without additional layers of regulatory control. This work reimagines how we design synthetic populations to survive and adapt by strategically giving control back to the cells.

synthetic biology↗

bioRxiv2021

Multiplexed Screening of Population-level Synthetic Gene Oscillator Libraries

Directed evolution has become an invaluable tool in protein engineering and has also greatly influenced the construction of synthetic gene circuits. The ability to generate diversity at precise targets for directed evolution approaches has improved vastly, allowing researchers to create large, specific mutant libraries with relative ease. Screening approaches for large mutant libraries have similarly come a long way, especially when the desired behavior can easily be tested for with static, single time-point assays. For more complex gene circuits with dynamic phenotypes that change over time, directed evolution approaches to controlling and tuning circuit behavior have been hindered by the lack of sufficiently high-throughput screening methods to isolate variants with desired characteristics. Here we utilize directed mutagenesis and multiplexed microfluidics to develop a workflow for creating, screening and tuning dynamic gene circuits that operate at the population level. Specifically, we create a mutant library of an existing oscillator, the synchronized lysis circuit, and tune its dynamics while uncovering principles regarding its behavior. Lastly, we utilize this directed evolution workflow to construct a new synchronized genetic oscillator that exhibits robust dynamics over long time scales.

synthetic biology↗