RESUMO
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.
Assuntos
Técnicas Biossensoriais , Voo Espacial , Proteínas , Técnicas Biossensoriais/métodos , Sistemas Automatizados de Assistência Junto ao Leito , Sistema Livre de CélulasRESUMO
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.
Assuntos
COVID-19 , Humanos , SARS-CoV-2/genética , RNA Viral/genética , Técnicas de Diagnóstico Molecular/métodos , Testes Imediatos , Técnicas de Amplificação de Ácido Nucleico/métodos , Bioensaio , Sensibilidade e EspecificidadeRESUMO
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.
Assuntos
Sistemas CRISPR-Cas/genética , Radiação Cósmica/efeitos adversos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA/efeitos da radiação , Exposição Ocupacional/efeitos adversos , Astronautas , DNA Fúngico/genética , DNA Fúngico/efeitos da radiação , Edição de Genes , Humanos , Mutagênese , Mutação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/genética , AstronaveRESUMO
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.
Assuntos
Caenorhabditis elegans/genética , DNA de Helmintos/genética , Eletroforese em Gel de Ágar/instrumentação , Expressão Gênica , RNA de Helmintos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/instrumentação , Animais , Astronautas , Caenorhabditis elegans/efeitos da radiação , Radiação Cósmica/efeitos adversos , DNA de Helmintos/isolamento & purificação , Eletroforese em Gel de Ágar/métodos , Humanos , RNA de Helmintos/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Voo Espacial , Ausência de PesoRESUMO
[This corrects the article DOI: 10.1038/s41526-017-0033-9.].
RESUMO
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.