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.

A fluorescence viewer for rapid molecular assay readout in space and low-resource terrestrial environments.

Fluorescence-based assays provide sensitive and adaptable methods for point of care testing, environmental monitoring, studies of protein abundance and activity, and a wide variety of additional applications. Currently, their utility in remote and low-resource environments is limited by the need for technically complicated or expensive instruments to read out fluorescence signal. Here we describe the Genes in Space Fluorescence Viewer (GiS Viewer), a portable, durable viewer for rapid molecular assay readout that can be used to visualize fluorescence in the red and green ranges. The GiS Viewer can be used to visualize any assay run in standard PCR tubes and contains a heating element. Results are visible by eye or can be imaged with a smartphone or tablet for downstream quantification. We demonstrate the capabilities of the GiS Viewer using two case studies-detection of SARS-CoV-2 RNA using RT-LAMP and quantification of drug-induced changes in gene expression via qRT-PCR on Earth and aboard the International Space Station. We show that the GiS Viewer provides a reliable method to visualize fluorescence in space without the need to return samples to Earth and can further be used to assess the results of RT-LAMP and qRT-PCR assays on Earth.

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.

A CRISPR-based assay for the study of eukaryotic DNA repair onboard the International Space Station.

As we explore beyond Earth, astronauts may be at risk for harmful DNA damage caused by ionizing radiation. Double-strand breaks are a type of DNA damage that can be repaired by two major cellular pathways: non-homologous end joining, during which insertions or deletions may be added at the break site, and homologous recombination, in which the DNA sequence often remains unchanged. Previous work suggests that space conditions may impact the choice of DNA repair pathway, potentially compounding the risks of increased radiation exposure during space travel. However, our understanding of this problem has been limited by technical and safety concerns, which have prevented integral study of the DNA repair process in space. The CRISPR/Cas9 gene editing system offers a model for the safe and targeted generation of double-strand breaks in eukaryotes. Here we describe a CRISPR-based assay for DNA break induction and assessment of double-strand break repair pathway choice entirely in space. As necessary steps in this process, we describe the first successful genetic transformation and CRISPR/Cas9 genome editing in space. These milestones represent a significant expansion of the molecular biology toolkit onboard the International Space Station.

Nucleic acid detection aboard the International Space Station by colorimetric loop-mediated isothermal amplification (LAMP).

Human spaceflight endeavors present an opportunity to expand our presence beyond Earth. To this end, it is crucial to understand and diagnose effects of long-term space travel on the human body. Developing tools for targeted, on-site detection of specific DNA sequences will allow us to establish research and diagnostics platforms that will benefit space programs. We describe a simple DNA diagnostic method that utilizes colorimetric loop-mediated isothermal amplification (LAMP) to enable detection of a repetitive telomeric DNA sequence in as little as 30 minutes. A proof of concept assay for this method was carried out using existing hardware on the International Space Station and the results were read instantly by an astronaut through a simple color change of the reaction mixture. LAMP offers a novel platform for on-orbit DNA-based diagnostics that can be deployed on the International Space Station and to the broader benefit of space programs.

Gene expression studies using a miniaturized thermal cycler system on board the International Space Station.

The distance and duration of human spaceflight missions is set to markedly increase over the coming decade as we prepare to send astronauts to Mars. However, the health impact of long-term exposure to cosmic radiation and microgravity is not fully understood. In order to identify the molecular mechanisms underpinning the effects of space travel on human health, we must develop the capacity to monitor changes in gene expression and DNA integrity in space. Here, we report successful implementation of three molecular biology procedures on board the International Space Station (ISS) using a miniaturized thermal cycler system and C. elegans as a model organism: first, DNA extraction-the initial step for any type of DNA analysis; second, reverse transcription of RNA to generate complementary DNA (cDNA); and third, the subsequent semi-quantitative PCR amplification of cDNA to analyze gene expression changes in space. These molecular procedures represent a significant expansion of the budding molecular biology capabilities of the ISS and will permit more complex analyses of space-induced genetic changes during spaceflight missions aboard the ISS and beyond.

Erratum: Author Correction: Successful amplification of DNA aboard the International Space Station.

[This corrects the article DOI: 10.1038/s41526-017-0033-9.].

Successful amplification of DNA aboard the International Space Station.

As the range and duration of human ventures into space increase, it becomes imperative that we understand the effects of the cosmic environment on astronaut health. Molecular technologies now widely used in research and medicine will need to become available in space to ensure appropriate care of astronauts. The polymerase chain reaction (PCR) is the gold standard for DNA analysis, yet its potential for use on-orbit remains under-explored. We describe DNA amplification aboard the International Space Station (ISS) through the use of a miniaturized miniPCR system. Target sequences in plasmid, zebrafish genomic DNA, and bisulfite-treated DNA were successfully amplified under a variety of conditions. Methylation-specific primers differentially amplified bisulfite-treated samples as would be expected under standard laboratory conditions. Our findings establish proof of concept for targeted detection of DNA sequences during spaceflight and lay a foundation for future uses ranging from environmental monitoring to on-orbit diagnostics.

Escape behavior elicited by single, channelrhodopsin-2-evoked spikes in zebrafish somatosensory neurons.

Somatosensory neurons in teleosts and amphibians are sensitive to thermal, mechanical, or nociceptive stimuli [1, 2]. The two main types of such cells in zebrafish--Rohon-Beard and trigeminal neurons--have served as models for neural development [3-6], but little is known about how they encode tactile stimuli. The hindbrain networks that transduce somatosensory stimuli into a motor output encode information by using very few spikes in a small number of cells [7], but it is unclear whether activity in the primary receptor neurons is similarly efficient. To address this question, we manipulated the activity of zebrafish neurons with the light-activated cation channel, Channelrhodopsin-2 (ChR2) [8, 9]. We found that photoactivation of ChR2 in genetically defined populations of somatosensory neurons triggered escape behaviors in 24-hr-old zebrafish. Electrophysiological recordings from ChR2-positive trigeminal neurons in intact fish revealed that these cells have extremely low rates of spontaneous activity and can be induced to fire by brief pulses of blue light. Using this technique, we find that even a single action potential in a single sensory neuron was at times sufficient to evoke an escape behavior. These results establish ChR2 as a powerful tool for the manipulation of neural activity in zebrafish and reveal a degree of efficiency in coding that has not been found in primary sensory neurons.

OFF ganglion cells cannot drive the optokinetic reflex in zebrafish.

Whereas the zebrafish retina has long been an important model system for developmental and genetic studies, little is known about the responses of the inner retinal neurons. Here we report single-unit ganglion cell recordings from 5- to 6-day-old zebrafish larvae. In wild-type larvae we identify at least five subtypes of ganglion cell responses to full-field illumination, with ON-OFF and ON-type cells predominating. In the nrc mutant retina, in which the photoreceptor terminals develop abnormally, we observe normal OFF responses but abnormal ON-OFF responses and no ON responses. Previously characterized as blind, these mutants lack an optokinetic reflex (OKR), but in another behavioral assay nrc mutant fish have near-normal responses to the offset of light and slow and sluggish responses to the onset of light. Pharmacological block of the ON pathway mimics most of the nrc visual defects. We conclude that the abnormal photoreceptor terminals in nrc mutants predominantly perturb the ON pathway and that the ON pathway is necessary to drive the OKR in larval zebrafish.

A role for cardiotrophin-like cytokine in the circadian control of mammalian locomotor activity.

The suprachiasmatic nucleus (SCN) drives circadian rhythms of locomotor behavior by releasing factors that act on receptor sites near the third ventricle. Here we show that cardiotrophin-like cytokine (CLC) satisfies multiple criteria for a circadian regulator of locomotor activity. In the mouse, CLC is expressed in a subpopulation of SCN vasopressin neurons with a circadian rhythm that peaks during the daily period of locomotor quiescence. CLC receptors flank the third ventricle, and acute infusion of CLC into the third ventricle produced a transient blockade of locomotor activity without affecting the circadian clock. The hypothalamic infusion of neutralizing antibodies to the CLC receptor produced extra daily locomotor activity at the time when CLC is maximally expressed. These results suggest that CLC is probably an SCN output signal important for shaping daily rhythms of behavior; moreover, they indicate an unexpected role for a cytokine in adult brain function.

A screen for secreted factors of the suprachiasmatic nucleus.

The circadian clock in the suprachiasmatic nucleus (SCN) drives daily locomotor activity rhythms presumably by secreting diffusible factors whose target sites are accessible from the third ventricle of the hypothalamus. This article describes the methodology of a systematic molecular and behavioral screen to identify "locomotor factors" of the SCN. To find SCN-secreted factors not previously documented, a hamster SCN cDNA library was screened in a yeast signal sequence trap. In a subsequent behavioral screen, newly identified and previously documented SCN factors were tested for an effect on locomotor activity rhythms by chronic infusion into the third ventricle of hamsters. Using this approach combined with further experiments, we identified transforming growth factor-alpha (TGF-alpha) as a likely SCN inhibitor of locomotion.