Validation of Cell-Free Protein Synthesis Aboard the International Space Station.

Cell-free protein synthesis (CFPS) is a rapidly maturing in vitro gene expression platform that can be used to transcribe and translate nucleic acids at the point of need, enabling on-demand synthesis of peptide-based vaccines and biotherapeutics as well as the development of diagnostic tests for environmental contaminants and infectious agents. Unlike traditional cell-based systems, CFPS platforms do not require the maintenance of living cells and can be deployed with minimal equipment; therefore, they hold promise for applications in low-resource contexts, including spaceflight. Here, we evaluate the performance of the cell-free platform BioBits aboard the International Space Station by expressing RNA-based aptamers and fluorescent proteins that can serve as biological indicators. We validate two classes of biological sensors that detect either the small-molecule DFHBI or a specific RNA sequence. Upon detection of their respective analytes, both biological sensors produce fluorescent readouts that are visually confirmed using a hand-held fluorescence viewer and imaged for quantitative analysis. Our findings provide insights into the kinetics of cell-free transcription and translation in a microgravity environment and reveal that both biosensors perform robustly in space. Our findings lay the groundwork for portable, low-cost applications ranging from point-of-care health monitoring to on-demand detection of environmental hazards in low-resource communities both on Earth and beyond.

Implementing Hands-On Molecular and Synthetic Biology Education Using Cell-Free Technology.

Active, hands-on learning has been shown to improve educational outcomes in STEM subjects. However, implementation of hands-on activities for teaching biology has lagged behind other science disciplines due to challenges associated with the use of living cells. To address this limitation, we developed BioBits®: biology education activities enabled by freeze-dried cell-free reactions that can be activated by just adding water. Here, we describe detailed protocols for labs designed to teach the central dogma, biomaterial formation, an important mechanism of antibiotic resistance, and CRISPR-Cas9 gene editing via cell-free synthesis of proteins with visual outputs. The activities described are designed for a range of educational levels and time/resource requirements, so that educators can select the demonstrations that best fit their needs. We anticipate that the availability of BioBits® activities will enhance biology instruction by enabling hands-on learning in a variety of educational settings.

Wearable materials with embedded synthetic biology sensors for biomolecule detection.

Integrating synthetic biology into wearables could expand opportunities for noninvasive monitoring of physiological status, disease states and exposure to pathogens or toxins. However, the operation of synthetic circuits generally requires the presence of living, engineered bacteria, which has limited their application in wearables. Here we report lightweight, flexible substrates and textiles functionalized with freeze-dried, cell-free synthetic circuits, including CRISPR-based tools, that detect metabolites, chemicals and pathogen nucleic acid signatures. The wearable devices are activated upon rehydration from aqueous exposure events and report the presence of specific molecular targets by colorimetric changes or via an optical fiber network that detects fluorescent and luminescent outputs. The detection limits for nucleic acids rival current laboratory methods such as quantitative PCR. We demonstrate the development of a face mask with a lyophilized CRISPR sensor for wearable, noninvasive detection of SARS-CoV-2 at room temperature within 90 min, requiring no user intervention other than the press of a button.

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.

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

Development of a bioassay as a measure of drozitumab-mediated apoptosis induced by soluble Fc gamma receptors.

Drozitumab is an agonistic therapeutic monoclonal antibody (mAb) against the pro-apoptotic death receptor 5 (DR5). In vitro cell killing assays using drozitumab have traditionally required cross-linking with anti-Fc antibody to amplify the pro-apoptotic signal, although drozitumab shows activity in in vivo tumor models without artificial cross-linking. Recently it has been shown that FcγR expressing cells play an important role in the activity of drozitumab by mediating cross-linking in vivo (Wilson et al., 2011). To provide a more biologically relevant alternative to cross-linking with anti-Fc antibody in in vitro bioassays, methods for cross-linking with soluble FcγR extracellular domain (ECD) were developed in this work. FcγR cross-linking methods developed in this work were assessed in solution, bead-bound, and plate-bound assay formats, as well as a cell-based assay format. The assays showed reproducible drozitumab dose-response curves in the concentration range of 5-20,000ng/mL and had acceptable precision and accuracy. The assays are also able to detect degradative changes in drozitumab samples subjected to thermal stress. The data suggest that FcγR cross-linking of drozitumab is a viable alternative to anti-Fc cross-linking of drozitumab to measure effector mediated apoptosis of drozitumab in vitro.

The extracellular matrix and focal adhesion kinase signaling regulate cancer stem cell function in pancreatic ductal adenocarcinoma.

Cancer stem cells (CSCs) play an important role in the clonogenic growth and metastasis of pancreatic ductal adenocarcinoma (PDAC). A hallmark of PDAC is the desmoplastic reaction, but the impact of the tumor microenvironment (TME) on CSCs is unknown. In order to better understand the mechanisms, we examined the impact of extracellular matrix (ECM) proteins on PDAC CSCs. We quantified the effect of ECM proteins, ß1-integrin, and focal adhesion kinase (FAK) on clonogenic PDAC growth and migration in vitro and tumor initiation, growth, and metastasis in vivo in nude mice using shRNA and overexpression constructs as well as small molecule FAK inhibitors. Type I collagen increased PDAC tumor initiating potential, self-renewal, and the frequency of CSCs through the activation of FAK. FAK overexpression increased tumor initiation, whereas a dominant negative FAK mutant or FAK kinase inhibitors reduced clonogenic PDAC growth in vitro and in vivo. Moreover, the FAK inhibitor VS-4718 extended the anti-tumor response to gemcitabine and nab-paclitaxel in patient-derived PDAC xenografts, and the loss of FAK expression limited metastatic dissemination of orthotopic xenografts. Type I collagen enhances PDAC CSCs, and both kinase-dependent and independent activities of FAK impact PDAC tumor initiation, self-renewal, and metastasis. The anti-tumor impact of FAK inhibitors in combination with standard chemotherapy support the clinical testing of this combination.