RESUMEN

Genetic circuit engineering has emerged as a powerful methodology to program the behavior of mammalian cells to respond to internal and external cues. This approach is now used to develop new therapeutics and improve production processes. However, genetic interaction networks are complex and hard to engineer rationally. Moreover, a design may fail, and it may not be possible to identify the root cause of its breakdown. Introducing designated regulatory circuitry in the form of integral feedback can introduce performance guarantees by ensuring robust and precise operation.

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RESUMEN

Recent progress in protein engineering has established optogenetics as one of the leading external non-invasive stimulation strategies, with many optogenetic tools being designed for in vivo operation. Characterization and optimization of these tools require a high-throughput and versatile light delivery system targeting micro-titer culture volumes. Here, we present a universal light illumination platform - Diya, compatible with a wide range of cell culture plates and dishes. Diya hosts specially designed features ensuring active thermal management, homogeneous illumination, and minimal light bleedthrough. It offers light induction programming via a user-friendly custom-designed GUI. Through extensive characterization experiments with multiple optogenetic tools in diverse model organisms (bacteria, yeast, and human cell lines), we show that Diya maintains viable conditions for cell cultures undergoing light induction. Finally, we demonstrate an optogenetic strategy for in vivo biomolecular controller operation. With a custom-designed antithetic integral feedback circuit, we exhibit robust perfect adaptation and light-controlled set-point variation using Diya.

RESUMEN

The processes that keep a cell alive are constantly challenged by unpredictable changes in its environment. Cells manage to counteract these changes by employing sophisticated regulatory strategies that maintain a steady internal milieu. Recently, the antithetic integral feedback motif has been demonstrated to be a minimal and universal biological regulatory strategy that can guarantee robust perfect adaptation for noisy gene regulatory networks in Escherichia coli. Here, we present a realization of the antithetic integral feedback motif in a synthetic gene circuit in mammalian cells. We show that the motif robustly maintains the expression of a synthetic transcription factor at tunable levels even when it is perturbed by increased degradation or its interaction network structure is perturbed by a negative feedback loop with an RNA-binding protein. We further demonstrate an improved regulatory strategy by augmenting the antithetic integral motif with additional negative feedback to realize antithetic proportional-integral control. We show that this motif produces robust perfect adaptation while also reducing the variance of the regulated synthetic transcription factor. We demonstrate that the integral and proportional-integral feedback motifs can mitigate the impact of gene expression burden, and we computationally explore their use in cell therapy. We believe that the engineering of precise and robust perfect adaptation will enable substantial advances in industrial biotechnology and cell-based therapeutics.

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Despite recent advances in circuit engineering, the design of genetic networks in mammalian cells is still painstakingly slow and fraught with inexplicable failures. Here, we demonstrate that transiently expressed genes in mammalian cells compete for limited transcriptional and translational resources. This competition results in the coupling of otherwise independent exogenous and endogenous genes, creating a divergence between intended and actual function. Guided by a resource-aware mathematical model, we identify and engineer natural and synthetic miRNA-based incoherent feedforward loop (iFFL) circuits that mitigate gene expression burden. The implementation of these circuits features the use of endogenous miRNAs as elementary components of the engineered iFFL device, a versatile hybrid design that allows burden mitigation to be achieved across different cell-lines with minimal resource requirements. This study establishes the foundations for context-aware prediction and improvement of in vivo synthetic circuit performance, paving the way towards more rational synthetic construct design in mammalian cells.

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