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

Abstract

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 replications (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.

Phenotypic Patterning through Copy Number Adaptation to Environmental Gradients

“…Previous work on plasmid copy number regulation has successfully created small-molecule inducible copy number and mutated copy number variants. , Recently, single strains carrying coupled plasmid systems have been developed which are capable of copy number-mediated differentiation and thereby population adaptation. , The single strain contains two distinct plasmids with shared replication machinery, coupling their copy numbers and generating a new source of heterogeneity. The above work with colE1 plasmids has shown that due to shared replication inhibition, two distinct colE1 constructs will share a total copy number but have a flexible distribution of each plasmid.…”

“…These results align with the findings of Kumar et al and highlight that selective pressure from a single homogeneous inducer causes rapid lysis of subpopulations carrying high copy number of the induced plasmid and thereby leads to decline of this plasmid in the overall population. 14 A fourth strain with different origins of replication on each plasmid served as a comparison to traditional compatible plasmids. Without plasmid coupling the microfluidic experiments showed Strain 4 was unable to adapt to the environmental cues and large-scale cell death occurred across all cell traps (Figure S4).…”

“…Each plasmid in the coupled system has a unique environmental lysis inducer, either N-acylated-L-homoserine lactones (AHL) or arabinose, plus a unique reporter, either GFP or RFP, respectively. This coupled plasmid system with negative environmental feedback was kindly donated from Kumar et al 14 and served as a model system for testing bacterial selfadaptation to environmental conditions across spatial gradients. Figure 1b demonstrates the hypothesized spatial phenotypic plasticity with a coupled plasmid system compared to uniform expression expected from traditional compatible plasmids.…”

“…13,14 Specifically, Kumar et al showed that copy number flexibility in coupled plasmid systems allows populations to minimize encoded metabolic and other cellular burdens as a function of environment. 14 Bacterial cells housing plasmids sustain them at a specific copy number, steered by the autoregulation of the origin of replication. In synthetic biology, orthogonal origins of replication are typically employed when integrating multiple plasmids into a cell, a necessary approach for stable maintenance of the copy number for each construct.…”

“…Tang and Liu first showed the use of such systems to stably record environmental events by controlling output plasmid ratio . Later works employed dynamic copy number response of coupled plasmids for adaptation to specific environments, in one case directing reinforcement learning within a microbial population and in the other allowing burden reduction of synthetic constructs. , Specifically, Kumar et al showed that copy number flexibility in coupled plasmid systems allows populations to minimize encoded metabolic and other cellular burdens as a function of environment . Bacterial cells housing plasmids sustain them at a specific copy number, steered by the autoregulation of the origin of replication.…”