Stanford Advisors


All Publications


  • On-demand biomanufacturing of protective conjugate vaccines SCIENCE ADVANCES Stark, J. C., Jaroentomeechai, T., Moeller, T. D., Hershewe, J. M., Warfel, K. F., Moricz, B. S., Martini, A. M., Dubner, R. S., Hsu, K. J., Stevenson, T. C., Jones, B. D., DeLisa, M. P., Jewett, M. C. 2021; 7 (6)

    Abstract

    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 promises to accelerate development of new conjugate vaccines with increased access through refrigeration-independent distribution and portable production.

    View details for DOI 10.1126/sciadv.abe9444

    View details for Web of Science ID 000615369000039

    View details for PubMedID 33536221

    View details for PubMedCentralID PMC7857678

  • Synthetic Siglec-9 Agonists Inhibit Neutrophil Activation Associated with COVID-19. ACS central science Delaveris, C. S., Wilk, A. J., Riley, N. M., Stark, J. C., Yang, S. S., Rogers, A. J., Ranganath, T., Nadeau, K. C., Blish, C. A., Bertozzi, C. R. 2021; 7 (4): 650-657

    Abstract

    Severe cases of coronavirus disease 2019 (COVID-19), caused by infection with SARS-CoV-2, are characterized by a hyperinflammatory immune response that leads to numerous complications. Production of proinflammatory neutrophil extracellular traps (NETs) has been suggested to be a key factor in inducing a hyperinflammatory signaling cascade, allegedly causing both pulmonary tissue damage and peripheral inflammation. Accordingly, therapeutic blockage of neutrophil activation and NETosis, the cell death pathway accompanying NET formation, could limit respiratory damage and death from severe COVID-19. Here, we demonstrate that synthetic glycopolymers that activate signaling of the neutrophil checkpoint receptor Siglec-9 suppress NETosis induced by agonists of viral toll-like receptors (TLRs) and plasma from patients with severe COVID-19. Thus, Siglec-9 agonism is a promising therapeutic strategy to curb neutrophilic hyperinflammation in COVID-19.

    View details for DOI 10.1021/acscentsci.0c01669

    View details for PubMedID 34056095

    View details for PubMedCentralID PMC8009098

  • BioBits Health: Classroom Activities Exploring Engineering, Biology, and Human Health with Fluorescent Readouts ACS SYNTHETIC BIOLOGY Stark, J. C., Huang, A., Hsu, K. J., Dubner, R. S., Forbrook, J., Marshalla, S., Rodriguez, F., Washington, M., Rzimicky, G. A., Nguyen, P. Q., Hasselbacher, B., Jabri, R., Kamran, R., Koralewski, V., Wightkin, W., Martinez, T., Jewett, M. C. 2019; 8 (5): 1001-1009

    Abstract

    Recent advances in synthetic biology have resulted in biological technologies with the potential to reshape the way we understand and treat human disease. Educating students about the biology and ethics underpinning these technologies is critical to empower them to make informed future policy decisions regarding their use and to inspire the next generation of synthetic biologists. However, hands-on, educational activities that convey emerging synthetic biology topics can be difficult to implement due to the expensive equipment and expertise required to grow living cells. We present BioBits Health, an educational kit containing lab activities and supporting curricula for teaching antibiotic resistance mechanisms and CRISPR-Cas9 gene editing in high school classrooms. This kit links complex biological concepts to visual, fluorescent readouts in user-friendly freeze-dried cell-free reactions. BioBits Health represents a set of educational resources that promises to encourage teaching of cutting-edge, health-related synthetic biology topics in classrooms and other nonlaboratory settings.

    View details for DOI 10.1021/acssynbio.8b00381

    View details for Web of Science ID 000468697000011

    View details for PubMedID 30925042

  • A cell-free biosynthesis platform for modular construction of protein glycosylation pathways. Nature communications Kightlinger, W. n., Duncker, K. E., Ramesh, A. n., Thames, A. H., Natarajan, A. n., Stark, J. C., Yang, A. n., Lin, L. n., Mrksich, M. n., DeLisa, M. P., Jewett, M. C. 2019; 10 (1): 5404

    Abstract

    Glycosylation plays important roles in cellular function and endows protein therapeutics with beneficial properties. However, constructing biosynthetic pathways to study and engineer precise glycan structures on proteins remains a bottleneck. Here, we report a modular, versatile cell-free platform for glycosylation pathway assembly by rapid in vitro mixing and expression (GlycoPRIME). In GlycoPRIME, glycosylation pathways are assembled by mixing-and-matching cell-free synthesized glycosyltransferases that can elaborate a glucose primer installed onto protein targets by an N-glycosyltransferase. We demonstrate GlycoPRIME by constructing 37 putative protein glycosylation pathways, creating 23 unique glycan motifs, 18 of which have not yet been synthesized on proteins. We use selected pathways to synthesize a protein vaccine candidate with an α-galactose adjuvant motif in a one-pot cell-free system and human antibody constant regions with minimal sialic acid motifs in glycoengineered Escherichia coli. We anticipate that these methods and pathways will facilitate glycoscience and make possible new glycoengineering applications.

    View details for DOI 10.1038/s41467-019-12024-9

    View details for PubMedID 31776339

  • BioBits (TM) Bright: A fluorescent synthetic biology education kit SCIENCE ADVANCES Stark, J. C., Huang, A., Nguyen, P. Q., Dubner, R. S., Hsu, K. J., Ferrante, T. C., Anderson, M., Kanapskyte, A., Mucha, Q., Packett, J. S., Patel, P., Patel, R., Qaq, D., Zondor, T., Burke, J., Martinez, T., Miller-Berry, A., Puppala, A., Reichert, K., Schmid, M., Brand, L., Hill, L. R., Chellaswamy, J. F., Faheem, N., Fetherling, S., Gong, E., Gonzalzles, E., Granito, T., Koritsaris, J., Binh Nguyen, Ottman, S., Palffy, C., Patel, A., Skweres, S., Slaton, A., Woods, T., Donghia, N., Pardee, K., Collins, J. J., Jewett, M. C. 2018; 4 (8): eaat5107

    Abstract

    Synthetic biology offers opportunities for experiential educational activities at the intersection of the life sciences, engineering, and design. However, implementation of hands-on biology activities in classrooms is challenging because of the need for specialized equipment and expertise to grow living cells. We present BioBits™ Bright, a shelf-stable, just-add-water synthetic biology education kit with easy visual outputs enabled by expression of fluorescent proteins in freeze-dried, cell-free reactions. We introduce activities and supporting curricula for teaching the central dogma, tunable protein expression, and design-build-test cycles and report data generated by K-12 teachers and students. We also develop inexpensive incubators and imagers, resulting in a comprehensive kit costing

    View details for DOI 10.1126/sciadv.aat5107

    View details for Web of Science ID 000443498100062

    View details for PubMedID 30083609

    View details for PubMedCentralID PMC6070313

  • BioBits (TM) Explorer: A modular synthetic biology education kit SCIENCE ADVANCES Huang, A., Nguyen, P. Q., Stark, J. C., Takahashi, M. K., Donghia, N., Ferrante, T., Dy, A. J., Hsu, K. J., Dubner, R. S., Pardee, K., Jewett, M. C., Collins, J. J. 2018; 4 (8): eaat5105

    Abstract

    Hands-on demonstrations greatly enhance the teaching of science, technology, engineering, and mathematics (STEM) concepts and foster engagement and exploration in the sciences. While numerous chemistry and physics classroom demonstrations exist, few biology demonstrations are practical and accessible due to the challenges and concerns of growing living cells in classrooms. We introduce BioBits™ Explorer, a synthetic biology educational kit based on shelf-stable, freeze-dried, cell-free (FD-CF) reactions, which are activated by simply adding water. The FD-CF reactions engage the senses of sight, smell, and touch with outputs that produce fluorescence, fragrances, and hydrogels, respectively. We introduce components that can teach tunable protein expression, enzymatic reactions, biomaterial formation, and biosensors using RNA switches, some of which represent original FD-CF outputs that expand the toolbox of cell-free synthetic biology. The BioBits™ Explorer kit enables hands-on demonstrations of cutting-edge science that are inexpensive and easy to use, circumventing many current barriers for implementing exploratory biology experiments in classrooms.

    View details for DOI 10.1126/sciadv.aat5105

    View details for Web of Science ID 000443498100061

    View details for PubMedID 30083608

    View details for PubMedCentralID PMC6070312

  • A cell-free platform for rapid synthesis and testing of active oligosaccharyltransferases BIOTECHNOLOGY AND BIOENGINEERING Schoborg, J. A., Hershewe, J. M., Stark, J. C., Kightlinger, W., Kath, J. E., Jaroentomeechai, T., Natarajan, A., DeLisa, M. P., Jewett, M. C. 2018; 115 (3): 739–50

    Abstract

    Protein glycosylation, or the attachment of sugar moieties (glycans) to proteins, is important for protein stability, activity, and immunogenicity. However, understanding the roles and regulations of site-specific glycosylation events remains a significant challenge due to several technological limitations. These limitations include a lack of available tools for biochemical characterization of enzymes involved in glycosylation. A particular challenge is the synthesis of oligosaccharyltransferases (OSTs), which catalyze the attachment of glycans to specific amino acid residues in target proteins. The difficulty arises from the fact that canonical OSTs are large (>70 kDa) and possess multiple transmembrane helices, making them difficult to overexpress in living cells. Here, we address this challenge by establishing a bacterial cell-free protein synthesis platform that enables rapid production of a variety of OSTs in their active conformations. Specifically, by using lipid nanodiscs as cellular membrane mimics, we obtained yields of up to 420 μg/ml for the single-subunit OST enzyme, "Protein glycosylation B" (PglB) from Campylobacter jejuni, as well as for three additional PglB homologs from Campylobacter coli, Campylobacter lari, and Desulfovibrio gigas. Importantly, all of these enzymes catalyzed N-glycosylation reactions in vitro with no purification or processing needed. Furthermore, we demonstrate the ability of cell-free synthesized OSTs to glycosylate multiple target proteins with varying N-glycosylation acceptor sequons. We anticipate that this broadly applicable production method will advance glycoengineering efforts by enabling preparative expression of membrane-embedded OSTs from all kingdoms of life.

    View details for DOI 10.1002/bit.26502

    View details for Web of Science ID 000423672800020

    View details for PubMedID 29178580

  • Single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery. Nature communications Jaroentomeechai, T. n., Stark, J. C., Natarajan, A. n., Glasscock, C. J., Yates, L. E., Hsu, K. J., Mrksich, M. n., Jewett, M. C., DeLisa, M. P. 2018; 9 (1): 2686

    Abstract

    The emerging discipline of bacterial glycoengineering has made it possible to produce designer glycans and glycoconjugates for use as vaccines and therapeutics. Unfortunately, cell-based production of homogeneous glycoproteins remains a significant challenge due to cell viability constraints and the inability to control glycosylation components at precise ratios in vivo. To address these challenges, we describe a novel cell-free glycoprotein synthesis (CFGpS) technology that seamlessly integrates protein biosynthesis with asparagine-linked protein glycosylation. This technology leverages a glyco-optimized Escherichia coli strain to source cell extracts that are selectively enriched with glycosylation components, including oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs). The resulting extracts enable a one-pot reaction scheme for efficient and site-specific glycosylation of target proteins. The CFGpS platform is highly modular, allowing the use of multiple distinct OSTs and structurally diverse LLOs. As such, we anticipate CFGpS will facilitate fundamental understanding in glycoscience and make possible applications in on demand biomanufacturing of glycoproteins.

    View details for DOI 10.1038/s41467-018-05110-x

    View details for PubMedID 30002445

    View details for PubMedCentralID PMC6043479

  • A Pipeline for Studying and Engineering Single-Subunit Oligosaccharyltransferases CHEMICAL GLYCOBIOLOGY, PT A: SYNTHESIS, MANIPULATION AND APPLICATIONS OF GLYCANS Jaroentomeechai, T., Zheng, X., Hershewe, J., Stark, J. C., Jewett, M. C., DeLisa, M. P., Imperiali, B. 2017; 597: 55-81

    Abstract

    Asparagine-linked (N-linked) protein glycosylation is one of the most abundant types of posttranslational modification, occurring in all domains of life. The central enzyme in N-linked glycosylation is the oligosaccharyltransferase (OST), which catalyzes the covalent attachment of preassembled glycans to specific asparagine residues in target proteins. Whereas in higher eukaryotes the OST is comprised of eight different membrane proteins, of which the catalytic subunit is STT3, in kinetoplastids and prokaryotes the OST is a monomeric enzyme bearing homology to STT3. Given their relative simplicity, these single-subunit OSTs (ssOSTs) have emerged as important targets for mechanistic dissection of poorly understood aspects of N-glycosylation and at the same time hold great potential for the biosynthesis of custom glycoproteins. To take advantage of this utility, this chapter describes a multipronged approach for studying and engineering ssOSTs that integrates in vivo screening technology with in vitro characterization methods, thereby creating a versatile and readily adaptable pipeline for virtually any ssOST of interest.

    View details for DOI 10.1016/bs.mie.2017.07.011

    View details for Web of Science ID 000414418800004

    View details for PubMedID 28935112

  • Cell-Free Synthetic Biology: Engineering Beyond the Cell COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY Perez, J. G., Stark, J. C., Jewett, M. C. 2016; 8 (12)

    Abstract

    Cell-free protein synthesis (CFPS) technologies have enabled inexpensive and rapid recombinant protein expression. Numerous highly active CFPS platforms are now available and have recently been used for synthetic biology applications. In this review, we focus on the ability of CFPS to expand our understanding of biological systems and its applications in the synthetic biology field. First, we outline a variety of CFPS platforms that provide alternative and complementary methods for expressing proteins from different organisms, compared with in vivo approaches. Next, we review the types of proteins, protein complexes, and protein modifications that have been achieved using CFPS systems. Finally, we introduce recent work on genetic networks in cell-free systems and the use of cell-free systems for rapid prototyping of in vivo networks. Given the flexibility of cell-free systems, CFPS holds promise to be a powerful tool for synthetic biology as well as a protein production technology in years to come.

    View details for DOI 10.1101/cshperspect.a023853

    View details for Web of Science ID 000390367600004

    View details for PubMedID 27742731

    View details for PubMedCentralID PMC5131772

  • Energizing eukaryotic cell-free protein synthesis with glucose metabolism FEBS LETTERS Anderson, M. J., Stark, J. C., Hodgman, C., Jewett, M. C. 2015; 589 (15): 1723-1727

    Abstract

    Eukaryotic cell-free protein synthesis (CFPS) is limited by the dependence on costly high-energy phosphate compounds and exogenous enzymes to power protein synthesis (e.g., creatine phosphate and creatine kinase, CrP/CrK). Here, we report the ability to use glucose as a secondary energy substrate to regenerate ATP in a Saccharomyces cerevisiae crude extract CFPS platform. We observed synthesis of 3.64±0.35 μg mL(-1) active luciferase in batch reactions with 16 mM glucose and 25 mM phosphate, resulting in a 16% increase in relative protein yield (μg protein/$ reagents) compared to the CrP/CrK system. Our demonstration provides the foundation for development of cost-effective eukaryotic CFPS platforms.

    View details for DOI 10.1016/j.febslet.2015.05.045

    View details for Web of Science ID 000358096200003

    View details for PubMedID 26054976

    View details for PubMedCentralID PMC4651010

  • Repurposing a Bacterial Quality Control Mechanism to Enhance Enzyme Production in Living Cells JOURNAL OF MOLECULAR BIOLOGY Boock, J. T., King, B. C., Taw, M. N., Conrado, R. J., Siu, K., Stark, J. C., Walker, L. P., Gibson, D. M., DeLisa, M. P. 2015; 427 (6): 1451-1463

    Abstract

    Heterologous expression of many proteins in bacteria, yeasts, and plants is often limited by low titers of functional protein. To address this problem, we have created a two-tiered directed evolution strategy in Escherichia coli that enables optimization of protein production while maintaining high biological activity. The first tier involves a genetic selection for intracellular protein stability that is based on the folding quality control mechanism inherent to the twin-arginine translocation pathway, while the second is a semi-high-throughput screen for protein function. To demonstrate the utility of this strategy, we isolated variants of the endoglucanase Cel5A, from the plant-pathogenic fungus Fusarium graminearum, whose production was increased by as much as 30-fold over the parental enzyme. This gain in production was attributed to just two amino acid substitutions, and it was isolated after two iterations through the two-tiered approach. There was no significant tradeoff in activity on soluble or insoluble cellulose substrates. Importantly, by combining the folding filter afforded by the twin-arginine translocation quality control mechanism with a function-based screen, we show enrichment for variants with increased protein abundance in a manner that does not compromise catalytic activity, providing a highly soluble parent for engineering of improved or new function.

    View details for DOI 10.1016/j.jmb.2015.01.003

    View details for Web of Science ID 000351798700018

    View details for PubMedID 25591491

    View details for PubMedCentralID PMC4576832