On-demand biomanufacturing of protective conjugate vaccines.

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.

BioBits Health: Classroom Activities Exploring Engineering, Biology, and Human Health with Fluorescent Readouts.

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.

Author Correction: Single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery.

The original version of this Article contained an error in Figure 2, wherein the bottom right western blot panel in Figure 2a was blank. This has now been corrected in both the PDF and HTML versions of the Article.

BioBits™ Explorer: A modular synthetic biology education kit.

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.

BioBits™ Bright: A fluorescent synthetic biology education kit.

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

Single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery.

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.