Publications
Proliferation history and transcription factor levels drive direct conversion
Wang NB, Lende-Dorn BA, Adewumi HO, Beitz AM, Han P, O’Shea TM, and Galloway, KE.
bioRxiv. 2023.
Highlights
- A minimal high-efficiency cocktail allows systematic interrogation of the reprogramming process
- Proliferation history provides a principal axis to distinguish transcription factors’ influence
- Encoding of the transcription factor cocktail impacts reprogramming efficiency and dynamics
- Levels of individual transcription factors differentially influence the rate of reprogramming
- Driving early hyperproliferation increases direct conversion of adult human fibroblasts
- Optimal cocktail allows neurotrophic factor-free reprogramming and in vivo grafting
#synbio #gene_circuits #reprogramming #proliferation
RNA-based controllers for engineering gene and cell therapies
Takahashi, K Galloway, KE. Current Opinion in Biotechnology. 2024.
Highlights
- RNA offers highly compact, modular, portable, and programmable gene regulation.
- Ligand-responsive riboswitches allow for user-guided control of transgenes.
- RNA-responsive RNA controllers detect and respond to transcriptional state.
#synbio #gene_circuits #RNA #cell_therapies
The sound of silence: Transgene silencing in mammalian cell engineering
Cabera, A* , Edelstein, HI*, Glykofrydis, F*, Love, KS*, Palacios, S* Tycko, J*, Zhang, M*, Lensch, S, Shields, CE, Livingston, M, Weiss, R, Zhao, H, Haynes, KA, Morsut, L, Chen, YY, Khalil, AS, Wong, WW, Collins, JJ, Rosser, SJ, Karen Polizzi, K, Elowitz, MB, Fussenegger, M, Hilton, IB, Leonard, JN, Bintu, L, Galloway, KE, Deans, TL.. Cell Systems. 2022.
Summary
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.
Supercoiling-mediated feedback rapidly couples and tunes transcription
Johnstone, CP and Galloway, KE. Cell Reports. 2022.
Highlights
- Supercoiling dynamics confer rapid, tunable coupling between adjacent genes
- Syntax—the relative order and orientation of genes—alters expression levels
- Supercoiling-dependent feedback tunes transcriptional variance and bursting
- Supercoiling coordinates the dynamics of transcriptional networks and gene circuits
Synthetic gene circuits as tools for drug discovery
Beitz, AM, Oakes, CG and Galloway, KE. Trends in Biotechnology. 2021.
Highlights
- Synthetic gene circuits enable real-time monitoring of diverse molecular events in live cells.
- Synthetic circuits provide internal controls to expedite hit validation and facilitate hit prioritization.
- Synthetic circuits enable on-line validation of induced pluripotent stem cell (iPSC)-derived cells.
- Synthetic circuits potentiate longitudinal tracking of neurodegenerative-associated phenotypes.
Engineering cellular symphonies out of transcriptional noise
Johnstone, CP and Galloway, KE. Nature Reviews Molecular Cell Biology. 2021.
Summary
Development unfolds through a series of orchestrated spatial and temporal gene-expression patterns. Despite relying on the noisy process of transcription, expression patterns remain robust to myriad disturbances. To achieve the goal of building complex tissues from the bottom up, synthetic biology must learn how to buffer and harness transcriptional noise.
Understanding and engineering chromatin as a dynamical system across length and time scales.
Johnstone, CP*, Wang, NB*, Sevier, SA, and Galloway, KE. Cell Systems. 2020.
Summary
Connecting the molecular structure and function of chromatin across length and timescales remains a grand challenge to understanding and engineering cellular behaviors. Across five orders of magnitude, dynamic processes constantly reshape chromatin structures, driving spaciotemporal patterns of gene expression and cell fate. Through the interplay of structure and function, the genome operates as a highly dynamic feedback control system. Recent experimental techniques have provided increasingly detailed data that revise and augment the relatively static, hierarchical view of genomic architecture with an understanding of how dynamic processes drive organization. Here, we review how novel technologies from sequencing, imaging, and synthetic biology refine our understanding of chromatin structure and function and enable chromatin engineering. Finally, we discuss opportunities to use these tools to enhance understanding of the dynamic interrelationship of chromatin structure and function.
Engineering cell fate: Applying synthetic biology to cellular reprogramming.
JWang, NB, Beitz, AM, Galloway, KE. Current Opinion in Systems Biology. 2020.
Highlights
- Roadblocks to cellular reprogramming can be overcome with synthetic biology tools.
- Highly interconnected aspects of latent donor cell identity affect reprogramming.
- Recent systems-level studies of reprogramming identify key drivers of reprogramming.
- Advances in synthetic biology offer new tools for coordinating reprogramming processes.
Mitigating antagonism between transcription and proliferation mediated near-deterministic reprogramming
Highlights
- Chemical, genetic cocktail reduces genomic stress induced by TF reprogramming
- DDRR cocktail expands population of hypertranscribing, hyperproliferating cells (HHCs)
- Supported by topoisomerases, HHCs reprogram at near-deterministic rates
- Topoisomerase expression reduces negative DNA supercoiling and R-loop formation
Comparative genomic analysis of embryonic, lineage-converted, and stem cell-derived motor neurons.
Abstract
Advances in stem cell science allow the production of different cell types in vitro either through the recapitulation of developmental processes, often termed ‘directed differentiation’, or the forced expression of lineage-specific transcription factors. Although cells produced by both approaches are increasingly used in translational applications, their quantitative similarity to their primary counterparts remains largely unresolved. To investigate the similarity between in vitro-derived and primary cell types, we harvested and purified mouse spinal motor neurons and compared them with motor neurons produced by transcription factor-mediated lineage conversion of fibroblasts or directed differentiation of pluripotent stem cells. To enable unbiased analysis of these motor neuron types and their cells of origin, we then subjected them to whole transcriptome and DNA methylome analysis by RNA sequencing (RNA-seq) and reduced representation bisulfite sequencing (RRBS). Despite major differences in methodology, lineage conversion and directed differentiation both produce cells that closely approximate the primary motor neuron state. However, we identify differences in Fas signaling, the Hox code and synaptic gene expression between lineage-converted and directed differentiation motor neurons that affect their utility in translational studies.
Modeling neurodegenerative diseases and neurodevelopmental disorders with reprogrammed cells
Summary
Induced pluripotent stem cells and reprogrammed cells offer the opportunity to generate disease-relevant human cells from readily-available patient cells for studying disease and identifying therapeutics.
Feedback loops in biological networks
Abstract
We introduce fundamental concepts for the design of dynamics and feedback in molecular networks modeled with ordinary differential equations. We use several examples, focusing in particular on the mitogen-activated protein kinase (MAPK) pathway, to illustrate the concept that feedback loops are fundamental in determining the overall dynamic behavior of a system. Often, these loops have a structural function and unequivocally define the system behavior. We conclude with numerical simulations highlighting the potential for bistability and oscillations of the MAPK pathway re-engineered through synthetic promoters and RNA transducers to include positive and negative feedback loops.
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Dynamically reshaping signaling networks to program cell fate via genetic controllers
Galloway, KE, Franco, E, and Smolke, CD. Science. 2013.
Abstract
Engineering of cell fate through synthetic gene circuits requires methods to precisely implement control around native decision-making pathways and offers the potential to direct cell processes. We demonstrate a class of genetic control systems, molecular network diverters, that interface with a native signaling pathway to route cells to divergent fates in response to environmental signals without modification of native genetic material. A method for identifying control points within natural networks is described that enables the construction of synthetic control systems that activate or attenuate native pathways to direct cell fate. We integrate opposing genetic programs by developing network architectures for reduced antagonism and demonstrate rational tuning of performance. Extension of these control strategies to mammalian systems should facilitate the engineering of complex cellular signaling systems.
Synthetic biology: Advancing biological frontiers by building synthetic systems.
Chen, YY*, Galloway, KE*, and Smolke, CD. Genome Biology. 2012.
Abstract
Synthetic biology is an emerging field of interdisciplinary research that seeks to transform our ability to probe, manipulate, and interface with living systems by combining the knowledge and techniques of biology, chemistry, computer science, and engineering. Its main aim is to increase the ease and efficiency with which biological systems can be designed, constructed, and characterized. Core efforts in the field have focused on the development of tools to support this goal, including new approaches to biological design and fabrication. Although the first generation of synthetic systems demonstrated genetic circuits that encode dynamic behavior, cellular computational operations, and biological communication channels, more recent research has focused on implementing synthetic biological devices and systems in diverse applications, including disease therapy, environmental remediation, and biosynthesis of commodity chemicals. As the field matures, synthetic biology is advancing biological frontiers by expanding biomanufacturing capabilities, developing next-generation therapeutic approaches, and providing new insights into natural biological systems. Here, we review the theoretical foundations, diverse tool kits, and engineered systems that have emerged from synthetic biology and discuss current as well as potential future applications, which include in-depth studies of basic biology (such as understanding endogenous signaling pathways and feedback circuits) and new frontiers in health and medicine (such as identification of diseased cells and targeted therapeutics).
Temperature Responsive Biopolymer for Mercury Remediation.
Kostal, J, Mulchandani, A, Gropp, KE, and Chen, WA. Environmental Science & Technology. 2003.
Abstract
Tunable biopolymers based on elastin-like polypeptides (ELP) were engineered for the selective removal of mercury. ELP undergoes a reversible thermal precipitation within a wide range of temperatures and was exploited to enable easy recovery of the sequestered mercury. A bacterial metalloregulatory protein, MerR, which binds mercury with an unusually high affinity and selectivity, was fused to the ELP to provide the highly selective nature of the biopolymers. Selective binding of mercury was demonstrated at an expected ratio of 0.5 mercury/biopolymer, and minimal binding of competing heavy metals (cadmium, nickel, and zinc), even at 100-fold excess, was observed. The sequestered mercury was extracted easily, enabling continuous reuse of the biopolymers. In repeating cycles, mercury concentration was reduced to ppb levels, satisfying even drinking water limits. Utility of the biopolymers with mercury-contaminated Lake Elsinore water was demonstrated with no decrease in efficiency. The nanoscale biopolymers reported here using metalloregulatory proteins represent a “green” technology for environmentally benign mercury removal. As nature offers a wide selection of specific metalloregulatory proteins, this technology offers promising solutions to remediation of other important pollutants such as arsenic or chromium.