Synthetic circuits for tracking human cell fate
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE38189 | GEO | 2012/05/24
SECONDARY ACCESSION(S): PRJNA167441
REPOSITORIES: GEO
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Project description:A variety of biological phenomena, from disease progression to stem cell differentiation, are typified by a prolonged cellular response to a transient environmental cue. While biologically relevant, heterogeneity in these long-term responses is difficult to assess at the population level, necessitating the development of biological tools to track cell fate within subpopulations. Here we present a novel synthetic biology approach for identifying and tracking mammalian cell subpopulations. We constructed three genomically integrated circuits that use bistable autoregulatory transcriptional feedback to retain memory of exposure to brief stimuli. These "memory devices" are used to isolate and track the progeny of cells that responded differentially to doxycycline, hypoxia, or DNA-damaging agents. Following hypoxic or ultraviolet radiation exposure, strongly responding cells activate the memory device and exhibit changes in gene expression, growth rates, and viability for multiple generations after the initial stimulus. Taken together, these results indicate that a heritable memory of hypoxia and DNA damage exists in subpopulations that differ in long-term cell behavior.
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Project description:Synthetic biology has focused on engineering genetic modules that operate orthogonally from the host cells. A synthetic circuit, however, can be designed to reprogram the host proteome, which in turn enhances the function of the synthetic circuit. Here, we apply this holistic synthetic biology concept by exploiting the crosstalk between metabolic networks in cells, leading to a protein environment more favorable for protein synthesis. Specifically, we show that a local module expressing translation machinery can reprogram the bacterial proteome, changing the expression levels of more than 780 proteins. The integration of the proteins synthesized by the local modules and the reprogramed proteome generate a cell-free system that can synthesize a diverse set of proteins in different reaction formats, with up to 5-fold higher expression level than classical cell-free systems. Our work demonstrates a holistic approach that integrates synthetic and systems biology concepts. This approach has the potential to achieve outcomes not possible by only local, orthogonal circuits.
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Project description:The present study aims to characterise proteins bound to circuits collected from children on extracorporeal membrane oxygenation (ECMO). ECMO circuits were collected from 6 patients. Quantification of concentrations for proteins bound to the ECMO circuit samples was performed using bicinchoninic acid (BCA) protein assay, whilst characterisation of the bound proteome was performed using Data-independent acquisition Mass Spectrometry (DIA-MS). Reactome Over-representation Pathway Analyses tool was used to identify functional pathways corresponding to the common proteins bound to circuits across all patients.
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Project description:Differential responses to stimuli can affect how cells succumb to disease. In yeast, DNA damage can create heterogeneous responses. To delineate how a response contributes to a cell's future behavior, we constructed a transcription-based memory circuit that detects DNA repair to isolate subpopulations with heritable damage responses. Strongly responsive cells show multigenerational effects, including growth defects and iron-associated gene expression. Less-responsive cells exhibit increased mutation frequencies but resume wild-type behavior. These two subpopulations remain distinct for multiple generations, indicating a transmissible memory of damage. Collectively, this work demonstrates the efficacy of using synthetic biology to define how environmental exposure contributes to distinct cell fates.
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