Inducible plasmid copy number control for synthetic biology in commonly used E. coli strains.

The ability to externally control gene expression has been paradigm shifting for all areas of biological research, especially for synthetic biology. Such control typically occurs at the transcriptional and translational level, while technologies enabling control at the DNA copy level are limited by either (i) relying on a handful of plasmids with fixed and arbitrary copy numbers; or (ii) require multiple plasmids for replication control; or (iii) are restricted to specialized strains. To overcome these limitations, we present TULIP (TUnable Ligand Inducible Plasmid): a self-contained plasmid with inducible copy number control, designed for portability across various Escherichia coli strains commonly used for cloning, protein expression, and metabolic engineering. Using TULIP, we demonstrate through multiple application examples that flexible plasmid copy number control accelerates the design and optimization of gene circuits, enables efficient probing of metabolic burden, and facilitates the prototyping and recycling of modules in different genetic contexts.

Stability and Robustness of Unbalanced Genetic Toggle Switches in the Presence of Scarce Resources.

While the vision of synthetic biology is to create complex genetic systems in a rational fashion, system-level behaviors are often perplexing due to the context-dependent dynamics of modules. One major source of context-dependence emerges due to the limited availability of shared resources, coupling the behavior of disconnected components. Motivated by the ubiquitous role of toggle switches in genetic circuits ranging from controlling cell fate differentiation to optimizing cellular performance, here we reveal how their fundamental dynamic properties are affected by competition for scarce resources. Combining a mechanistic model with nullcline-based stability analysis and potential landscape-based robustness analysis, we uncover not only the detrimental impacts of resource competition, but also how the unbalancedness of the switch further exacerbates them. While in general both of these factors undermine the performance of the switch (by pushing the dynamics toward monostability and increased sensitivity to noise), we also demonstrate that some of the unwanted effects can be alleviated by strategically optimized resource competition. Our results provide explicit guidelines for the context-aware rational design of toggle switches to mitigate our reliance on lengthy and expensive trial-and-error processes, and can be seamlessly integrated into the computer-aided synthesis of complex genetic systems.