Browsing by Author "Khalil, Ahmad S."

Now showing 1 - 4 of 4
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    Cooperative assembly confers regulatory specificity and long-term genetic circuit stability
    (Elsevier, 2023) Bragdon, Meghan D. J.; Patel, Nikit; Chuang, James; Levien, Ethan; Bashor, Caleb J.; Khalil, Ahmad S.
    A ubiquitous feature of eukaryotic transcriptional regulation is cooperative self-assembly between transcription factors (TFs) and DNA cis-regulatory motifs. It is thought that this strategy enables specific regulatory connections to be formed in gene networks between otherwise weakly interacting, low-specificity molecular components. Here, using synthetic gene circuits constructed in yeast, we find that high regulatory specificity can emerge from cooperative, multivalent interactions among artificial zinc-finger-based TFs. We show that circuits “wired” using the strategy of cooperative TF assembly are effectively insulated from aberrant misregulation of the host cell genome. As we demonstrate in experiments and mathematical models, this mechanism is sufficient to rescue circuit-driven fitness defects, resulting in genetic and functional stability of circuits in long-term continuous culture. Our naturally inspired approach offers a simple, generalizable means for building high-fidelity, evolutionarily robust gene circuits that can be scaled to a wide range of host organisms and applications.
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    Designing Automated, High-throughput, Continuous Cell Growth Experiments Using eVOLVER
    (JoVE, 2019) Heins, Zachary J.; Mancuso, Christopher P.; Kiriakov, Szilvia; Wong, Brandon G.; Bashor, Caleb J.; Khalil, Ahmad S.
    Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring fitness phenotypes and improving our understanding of how genotypes are shaped by selection. Extensive recent efforts to develop and apply niche continuous culture devices have revealed the benefits of conducting new forms of cell culture control. This includes defining custom selection pressures and increasing throughput for studies ranging from long-term experimental evolution to genome-wide library selections and synthetic gene circuit characterization. The eVOLVER platform was recently developed to meet this growing demand: a continuous culture platform with a high degree of scalability, flexibility, and automation. eVOLVER provides a single standardizing platform that can be (re)-configured and scaled with minimal effort to perform many different types of high-throughput or multi-dimensional growth selection experiments. Here, a protocol is presented to provide users of the eVOLVER framework a description for configuring the system to conduct a custom, large-scale continuous growth experiment. Specifically, the protocol guides users on how to program the system to multiplex two selection pressures - temperature and osmolarity - across many eVOLVER vials in order to quantify fitness landscapes of Saccharomyces cerevisiae mutants at fine resolution. We show how the device can be configured both programmatically, through its open-source web-based software, and physically, by arranging fluidic and hardware layouts. The process of physically setting up the device, programming the culture routine, monitoring and interacting with the experiment in real-time over the internet, sampling vials for subsequent offline analysis, and post experiment data analysis are detailed. This should serve as a starting point for researchers across diverse disciplines to apply eVOLVER in the design of their own complex and high-throughput cell growth experiments to study and manipulate biological systems.
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    Precise, automated control of conditions for high-throughput growth of yeast and bacteria with eVOLVER
    (Springer Nature, 2018) Wong, Brandon G.; Mancuso, Christopher P.; Kiriakov, Szilvia; Bashor, Caleb J.; Khalil, Ahmad S.
    Precise control over microbial cell growth conditions could enable detection of minute phenotypic changes, which would improve our understanding of how genotypes are shaped by adaptive selection. Although automated cell-culture systems such as bioreactors offer strict control over liquid culture conditions, they often do not scale to high-throughput or require cumbersome redesign to alter growth conditions. We report the design and validation of eVOLVER, a scalable do-it-yourself (DIY) framework, which can be configured to carry out high-throughput growth experiments in molecular evolution, systems biology, and microbiology. High-throughput evolution of yeast populations grown at different densities reveals that eVOLVER can be applied to characterize adaptive niches. Growth selection on a genome-wide yeast knockout library, using temperatures varied over different timescales, finds strains sensitive to temperature changes or frequency of temperature change. Inspired by large-scale integration of electronics and microfluidics, we also demonstrate millifluidic multiplexing modules that enable multiplexed media routing, cleaning, vial-to-vial transfers and automated yeast mating.
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    The sound of silence: Transgene silencing in mammalian cell engineering
    (Cell Press, 2022) Cabrera, Alan; Edelstein, Hailey I.; Glykofrydis, Fokion; Love, Kasey S.; Palacios, Sebastian; Tycko, Josh; Zhang, Meng; Lensch, Sarah; Shields, Cara E.; Livingston, Mark; Weiss, Ron; Zhao, Huimin; Haynes, Karmella A.; Morsut, Leonardo; Chen, Yvonne Y.; Khalil, Ahmad S.; Wong, Wilson W.; Collins, James J.; Rosser, Susan J.; Polizzi, Karen; Elowitz, Michael B.; Fussenegger, Martin; Hilton, Isaac B.; Leonard, Joshua N.; Bintu, Lacramioara; Galloway, Kate E.; Deans, Tara L.
    To elucidate principles operating in native biological systems and to develop novel biotechnologies, synthetic biology aims to build and integrate synthetic gene circuits within native transcriptional networks. The utility of synthetic gene circuits for cell engineering relies on the ability to control the expression of all constituent transgene components. Transgene silencing, defined as the loss of expression over time, persists as an obstacle for engineering primary cells and stem cells with transgenic cargos. In this review, we highlight the challenge that transgene silencing poses to the robust engineering of mammalian cells, outline potential molecular mechanisms of silencing, and present approaches for preventing transgene silencing. We conclude with a perspective identifying future research directions for improving the performance of synthetic gene circuits.