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  1. null (Ed.)
    Abstract Cell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for biomanufacturing and synthetic biology. However, activating membrane-dependent functionality of cell-derived vesicles in bacterial CFE systems has been limited. Here, we address this limitation by characterizing native membrane vesicles in Escherichia coli- based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. As a model, we focus on bacterial glycoengineering. We first use multiple, orthogonal techniques to characterize vesicles and show how extract processing methods can be used to increase concentrations of membrane vesicles in CFE systems. Then, we show that extracts enrichedmore »in vesicle number also display enhanced concentrations of heterologous membrane protein cargo. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving cell-free N- linked and O -linked glycoprotein synthesis. We anticipate that these methods will facilitate on-demand glycoprotein production and enable new CFE systems with membrane-associated activities.« less
    Free, publicly-accessible full text available December 1, 2022
  2. Background Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) diagnostic tests for SARS-CoV-2 are the cornerstone of the global testing infrastructure. However, these tests require cold-chain shipping to distribute, and the labor of skilled technicians to assemble reactions and interpret the results. Strategies to reduce shipping and labor costs at the point-of-care could aid in diagnostic testing scale-up and response to the COVID-19 outbreak, as well as in future outbreaks. Methods In this study we test both lab-developed and commercial SARS-CoV-2 diagnostic RT-qPCR mixes for the ability to be stabilized against elevated temperature by lyophilization. Fully assembled reactions were lyophilized and storedmore »for up to a month at ambient or elevated temperature and were subsequently assayed for their ability to detect dilutions of synthetic SARS-CoV-2 RNA. Results Of the mixes tested, we show that one commercial mix can maintain activity and sensitivity after storage for at least 30 days at ambient temperature after lyophilization. We also demonstrate that lyoprotectants such as disaccharides can stabilize freeze-dried diagnostic reactions against elevated temperatures (up to 50°C) for at least 30 days. Conclusion We anticipate that the incorporation of these methods into SARS-CoV-2 diagnostic testing will improve testing pipelines by reducing labor at the testing facility and eliminating the need for cold-chain shipping.« less
  3. Cell-free synthetic biology is a maturing field that aims to assemble biomolecular reactions outside cells for compelling applications in drug discovery, metabolic engineering, biomanufacturing, diagnostics, and education. Cell-free systems have several key features. They circumvent mechanisms that have evolved to facilitate species survival, bypass limitations on molecular transport across the cell wall, enable high-yielding and rapid synthesis of proteins without creating recombinant cells, and provide high tolerance towards toxic substrates or products. Here, we analyze ~750 published patents and ~2000 peer-reviewed manuscripts in the field of cell-free systems. Three hallmarks emerged. First, we found that both patent filings and manuscriptmore »publications per year are significantly increasing (five-fold and 1.5-fold over the last decade, respectively). Second, we observed that the innovation landscape has changed. Patent applications were dominated by Japan in the early 2000s before shifting to China and the USA in recent years. Finally, we discovered an increasing prevalence of biotechnology companies using cell-free systems. Our analysis has broad implications on the future development of cell-free synthetic biology for commercial and industrial applications.« less
  4. Abstract Protein glycosylation, the enzymatic modification of amino acid sidechains with sugar moieties, plays critical roles in cellular function, human health, and biotechnology. However, studying and producing defined glycoproteins remains challenging. Cell-free glycoprotein synthesis systems, in which protein synthesis and glycosylation are performed in crude cell extracts, offer new approaches to address these challenges. Here, we review versatile, state-of-the-art systems for biomanufacturing glycoproteins in prokaryotic and eukaryotic cell-free systems with natural and synthetic N-linked glycosylation pathways. We discuss existing challenges and future opportunities in the use of cell-free systems for the design, manufacture, and study of glycoprotein biomedicines.
  5. Abstract

    Ribosome-mediated polymerization of backbone-extended monomers into polypeptides is challenging due to their poor compatibility with the translation apparatus, which evolved to use α-L-amino acids. Moreover, mechanisms to acylate (or charge) these monomers to transfer RNAs (tRNAs) to make aminoacyl-tRNA substrates is a bottleneck. Here, we rationally design non-canonical amino acid analogs with extended carbon chains (γ-, δ-, ε-, and ζ-) or cyclic structures (cyclobutane, cyclopentane, and cyclohexane) to improve tRNA charging. We then demonstrate site-specific incorporation of these non-canonical, backbone-extended monomers at the N- and C- terminus of peptides using wild-type and engineered ribosomes. This work expands the scope ofmore »ribosome-mediated polymerization, setting the stage for new medicines and materials.

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  6. Conjugate vaccines are among the most effective methods for preventing bacterial infections. However, existing manufacturing approaches limit access to conjugate vaccines due to centralized production and cold chain distribution requirements. To address these limitations, we developed a modular technology for in vitro conjugate vaccine expression (iVAX) in portable, freeze-dried lysates from detoxified, nonpathogenic Escherichia coli. Upon rehydration, iVAX reactions synthesize clinically relevant doses of conjugate vaccines against diverse bacterial pathogens in 1 hour. We show that iVAX-synthesized vaccines against Francisella tularensis subsp. tularensis (type A) strain Schu S4 protected mice from lethal intranasal F. tularensis challenge. The iVAX platform promisesmore »to accelerate development of new conjugate vaccines with increased access through refrigeration-independent distribution and portable production.« less
  7. We demonstrate in vitro incorporation of cyclic β-amino acids into peptides by the ribosome through genetic code reprogramming. Further, we show that incorporation efficiency can be increased through the addition of elongation factor P.