Browsing by Author "Bachhav, Bhagyashree"

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    A platform for post-translational spatiotemporal control of cellular proteins
    (Oxford University Press, 2021) Jayanthi, Brianna; Bachhav, Bhagyashree; Wan, Zengyi; Martinez Legaspi, Santiago; Segatori, Laura; Systems, Synthetic, and Physical Biology Program
    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 the 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.
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    Expanding the mammalian synthetic biology toolbox: Orthogonal tools and circuits for cellular reprogramming
    (2020-12-04) Bachhav, Bhagyashree; Segatori, Laura
    Cell engineering presents great potential for next-generation biotechnological and biomedical applications that employ engineered mammalian cells to sense intracellular or extracellular signals and respond in a user-defined manner. Programming mammalian cells requires tools that operate orthogonally to the cellular machinery for sensing and controlling the levels, localization, and function of endogenous proteins. This work describes my efforts to expand the mammalian synthetic biology toolbox and develop orthogonal tools and gene circuits to reprogram cells for a diverse range of applications. I first illustrate the development of a gene signal amplifier platform to monitor chromosomal gene expression that recapitulates gene expression dynamics from the native chromosomal context and generates a readily detectable signal output. The gene signal amplifier links transcriptional and post-translational regulation of a fluorescent output to the expression of a chromosomal target gene. By recapitulating the transcriptional and translational control mechanisms underlying the expression of a target gene with high sensitivity, this platform provides a novel technology for monitoring target gene expression with superior sensitivity and dynamic resolution. To illustrate the utility of such a reporter system, I used the gene signal amplifier platform to monitor the activation and dynamics of the unfolded protein response (UPR), a complex signal transduction pathway that remodels gene expression in response to proteotoxic stress in the endoplasmic reticulum (ER). The UPR manifests as a series of signaling cascades aimed at relieving proteotoxic stress and restoring homeostasis or executing apoptosis to eliminate irremediably damaged cells. To control cellular fate upon UPR induction, typically caused by overexpression of recombinant proteins, I designed a series of orthogonal genetic circuits that interface with the UPR, sense and integrate signals associated with activation of the UPR and, in response remodel the stress response pathway to enhance stress attenuation and delay apoptosis. Finally, I developed a nanobody-based platform for controlling the selective localization of a target protein to multiple cellular compartments and investigated different circuit architectures to obtain dynamic control over the localized target protein. Combining nanobody-mediated localization and degradation with orthogonal regulation, I generated a nanobody-based platform to control the relative concentration and localization of a target protein in cells. The tools described in this work are expected to have a significant impact on the design of strategies to reprogram mammalian cells for a diverse range of applications including the development of cell-based therapies and the production of biologics.