Engineering repressors with coevolutionary cues facilitates toggle switches with a master reset

Abstract

Engineering allosteric transcriptional repressors containing an environmental sensing module (ESM) and a DNA recognition module (DRM) has the potential to unlock a combinatorial set of rationally designed biological responses. We demonstrated that constructing hybrid repressors by fusing distinct ESMs and DRMs provides a means to flexibly rewire genetic networks for complex signal processing. We have used coevolutionary traits among LacI homologs to develop a model for predicting compatibility between ESMs and DRMs. Our predictions accurately agree with the performance of 40 engineered repressors. We have harnessed this framework to develop a system of multiple toggle switches with a master OFF signal that produces a unique behavior: each engineered biological activity is switched to a stable ON state by different chemicals and returned to OFF in response to a common signal. One promising application of this design is to develop living diagnostics for monitoring multiple parameters in complex physiological environments and it represents one of many circuit topologies that can be explored with modular repressors designed with coevolutionary information.

Epistatic contributions promote the unification of incompatible models of neutral molecular evolution

“…For example, by assuming that the distribution of possible ancestors remains consistent over evolutionary time, pools of ancestral sequences can be constructed such that their Hamiltonians match the extant distribution of family members. Lastly, our model can direct protein-design approaches that utilize the Hamiltonian and conditional probability to design and optimize novel protein functions into existing protein scaffolds (77).…”

Synthetic Biology for Terraformation Lessons from Mars, Earth, and the Microbiome

“…Presently, we can successfully engineer cells to sense exogenous inputs [46,47], and control gene expression [48][49][50][51] and communication [52][53][54]. Synthetic interactions within a microbial consortia are typically engineered using the natural quorum sensing system for bacteria [55,56], or different pheromones in fungi [57,58]; but adhesion proteins or ion channels can be also used [59,60].…”

Engineering DNA recognition and allosteric response properties of TetR family proteins by using a module-swapping strategy

“…In previous studies, we established a module-swapping strategy to develop hybrid regulators with LacI family proteins, in which the resulting regulators possess desirable and predictable combinations of DNA-binding and ligand-binding properties originated from different native regulators (18,19). Based on this discovery, we constructed a set of hybrid regulators that enable flexible connections between small molecule sensing and promoter control.…”

“…Based on this discovery, we constructed a set of hybrid regulators that enable flexible connections between small molecule sensing and promoter control. These engineered parts were harnessed to establish two novel circuit designs with potential biotechnological applications, including a Passcode kill switch (18), and a system of multiple toggle switches with a master OFF signal (19). These studies show that transcriptional regulators are promising for creating robust, modular parts that facilitate cell engineering.…”