No abstract

Synthetic hysteretic mammalian gene circuits generating sustained cellular responses to transient perturbations provide important tools to investigate complex cellular behaviors and reprogram cells for a variety of applications, ranging from protein production to cell fate decisions. The design rules of synthetic gene circuits with controlled hysteretic behaviors, however, remain uncharacterized. To identify the criteria for achieving predictable control of hysteresis, we built a genetic circuit for detection of proteasomal degradation (Hys-Deg). The Hys-Deg circuit is based on a tetracycline-controlled transactivator (tTA) variant engineered to interface with the ubiquitin proteasome system (UPS). The tTA variant activates its own expression, generating a positive feedback loop that is triggered by expression of another tTA gene that is constitutively regulated. Guided by predictive modeling, we characterized the hysteretic response of the Hys-Deg circuit. We demonstrated that control of the hysteretic response is achieved by modulating the ratio of expression of constitutive to inducible tTA. We also showed that the system can be finely tuned through dosage of the inducer tetracycline to calibrate the circuit for detection of the desired levels of UPS activation. This study establishes the design rules for building a hysteretic genetic circuit with an autoregulatory feedback loop and provides a synthetic memory module that could be easily integrated into regulatory gene networks to study and engineer complex cellular behaviors.

The production of high‐quality recombinant proteins is critical to maintaining a continuous supply of biopharmaceuticals, such as therapeutic antibodies. Engineering mammalian cell factories presents a number of limitations typically associated with the proteotoxic stress induced upon aberrant accumulation of off‐pathway protein folding intermediates, which eventually culminate in the induction of apoptosis. In this review, we will discuss advances in cell engineering and their applications at different hierarchical levels of control of the expression of recombinant proteins, from transcription and translational to posttranslational modifications and subcellular trafficking. We also highlight challenges and unique opportunities to apply modern synthetic biology tools to the design of programmable cell factories for improved biomanufacturing of therapeutic proteins.

Mammalian cells process information through coordinated spatiotemporal regulation of proteins. Engineering cellular networks thus relies on efficient tools for regulating protein levels in specific subcellular compartments. To address the need to manipulate the extent and dynamics of protein localization, we developed a platform technology for target-specific control of protein destination. This platform is based on bifunctional molecules comprising a target-specific nanobody and universal sequences determining target subcellular localization or degradation rate. We demonstrate that nanobody-mediated localization depends on the expression level of the target and the nanobody, and the extent of target subcellular localization can be regulated by combining multiple target-specific nanobodies with distinct localization or degradation sequences. We also show that this platform for nanobody-mediated target localization and degradation can be regulated transcriptionally and integrated within orthogonal genetic circuits to achieve the desired temporal control over spatial regulation of target proteins. The platform reported in this study provides an innovative tool to control protein subcellular localization which will be useful to investigate protein function and regulate large synthetic gene circuits.

The production of high-quality recombinant proteins is critical to maintaining a continuous supply of biopharmaceuticals, such as therapeutic antibodies. Engineering mammalian cell factories presents a number of limitations typically associated with proteotoxic stress induced upon aberrant accumulation of off-pathway protein folding intermediates, which eventually culminate with the induction of apoptosis. Recent progress in mammalian synthetic biology provides unique opportunities to endow cells with programmable, user-defined behaviors, thereby addressing some of the challenges of current methods. In this review, we will discuss advances in synthetic biology to design efficient strategies for biomanufacturing.