BioSentinel: Developing a Space Radiation Biosensor

BioSentinel is an autonomous fully self-contained science mission that will conduct the first study of the biological response to space radiation outside low Earth orbit (LEO) in over 40 years. The 4-unit (4U) BioSentinel biosensor system, is housed within a 6-Unit (6U) spacecraft, and uses yeast cells in multiple independent microfluidic cards to detect and measure DNA damage that occurs in response to ambient space radiation. Cell growth and metabolic activity will be measured using a 3-color LED detection system and a metabolic indicator dye with a dedicated thermal control system per fluidic card

Biosentinel: Improving Desiccation Tolerance of Yeast Biosensors for Deep-Space Missions

BioSentinel is one of 13 secondary payloads to be deployed on Exploration Mission 1 (EM-1) in 2019. We will use the budding yeast Saccharomyces cerevisiae as a biosensor to determine how deep-space radiation affects living organisms and to potentially quantify radiation levels through radiation damage analysis. Radiation can damage DNA through double strand breaks (DSBs), which can normally be repaired by homologous recombination. Two yeast strains will be air-dried and stored in microfluidic cards within the payload: a wild-type control strain and a radiation sensitive rad51 mutant that is deficient in DSB repairs. Throughout the mission, the microfluidic cards will be rehydrated with growth medium and an indicator dye. Growth rates of each strain will be measured through LED detection of the reduction of the indicator dye, which correlates with DNA repair and the amount of radiation damage accumulated. Results from BioSentinel will be compared to analog experiments on the ISS and on Earth. It is well known that desiccation can damage yeast cells and decrease viability over time. We performed a screen for desiccation-tolerant rad51 strains. We selected 20 re-isolates of rad51 and ran a weekly screen for desiccation-tolerant mutants for five weeks. Our data shows that viability decreases over time, confirming previous research findings. Isolates L2, L5 and L14 indicate desiccation tolerance and are candidates for whole-genome sequencing. More time is needed to determine whether a specific strain is truly desiccation tolerant. Furthermore, we conducted an intracellular trehalose assay to test how intracellular trehalose concentrations affect or protect the mutant strains against desiccation stress. S. cerevisiae cell and reagent concentrations from a previously established intracellular trehalose protocol did not yield significant absorbance measurements, so we tested varying cell and reagent concentrations and determined proper concentrations for successful protocol use

BioSentinel: An Adaptable Platform for Studying the Biological Effects of Deep Space Radiation

NASA's BioSentinel mission is a 6U nanosatellite with autonomous life support that will utilize the budding yeast Saccharomyces cerevisiae to study the DNA damage response to the deep space radiation environment. BioSentinel is planned to launch in 2019 as a secondary payload on the Space Launch System's first Exploration Mission (EM-1), and will undergo a lunar fly-by and enter heliocentric orbit after deployment. As the first biological mission beyond Low Earth Orbit (LEO) in nearly half a century, this mission will help fill critical gaps in knowledge about the effects of uniquely composed, chronic, low-flux deep space radiation on biological systems. Yeast is well-suited for this mission due to its desiccation tolerance and space-flight heritage. As a eukaryotic model organism, it also serves as a robust analog for human cells. Data gathered on this mission will thus inform us of the hazards involved in long-duration human exploration in deep space, and the protections necessary to mitigate them. Due to its low-cost, flexible and advanced technology, the 4U BioSensor payload contained within the nanosatellite is adaptable to other model microorganisms, exploration platforms and environments relevant to human exploration, such as the ISS, the Lunar Orbital Platform - Gateway and future lunar landers. In order to query the DNA damage response to deep space radiation, BioSentinel contains a wild type yeast strain as a positive control, and a radiation sensitive rad51 mutant strain that is defective for DNA repair. Yeast cells are desiccated in microfluidic cards, and rehydrated with growth medium and metabolic indicator dye at the desired time points during the mission. A thermal control system supports these stasis and growth states, and an optical system continuously measures cell growth and metabolism. An onboard radiation spectrometer and dosimeter allows us to correlate the dose, energy and particle-type of deep space radiation to the biological response. Data received from the deep space biosensor will be compared to control payloads on Earth and the ISS. Ongoing science testing for the BioSentinel project includes optimization for cell viability, desiccation tolerance, and long-term biocompatibility, as well as radiation experiments to understand the sensitivity and responsiveness of cells to varying radiation doses and particle types

Identification of Novel Desiccation-Tolerant S. cerevisiae Strains for Deep Space Biosensors

NASA's BioSentinel mission, a secondary payload that will fly on the Space Launch Systems first Exploration Mission (EM-1), utilizes the budding yeast S. cerevisiae to study the biological response to the deep space radiation environment. Yeast samples are desiccated prior to launch to suspend growth and metabolism while the spacecraft travels to its target heliocentric orbit beyond Low Earth Orbit. Each sample is then rehydrated at the desired time points to reactivate the cells. A major risk in this mission is the loss of cell viability that occurs in the recovery period following the desiccation and rehydration process. Cell survival is essential for the detection of the biological response to features in the deep space environment, including ionizing radiation.The aim of this study is to mitigate viable cell loss in future biosensors by identifying mutations and genes that confer tolerance to desiccation stress in rad51, a radiation-sensitive yeast strain. We initiated a screen for desiccation-tolerance after rehydrating cells that were desiccated for three years, and selected various clones exhibiting robust growth. To verify retention of radiation sensitivity in the isolated clonesa crucial feature for a successful biosensorwe exposed them to ionizing radiation. Finally, to elucidate the genetic and molecular bases for observed desiccation-tolerance, we will perform whole-genome sequencing of those rad51 clones that exhibit both robust growth and radiation sensitivity following desiccation. The identification and characterization of desiccation-tolerant strains will allow us to engineer a biological model that will be resilient in face of the challenges of the deep space environment, and will thus ensure the experimental success of future biosensor missions

BioSentinel: Optimizing Growth Conditions for Improved Yeast Cell Viability After Long-Term Desiccation

NASA's BioSentinel mission is one of thirteen secondary payloads to be deployed on the Space Launch System Exploration Mission-1 (SLS EM-1). The BioSentinel nanosatellite will be sent into a heliocentric orbit beyond Low Earth Orbit (LEO), to study the effects of deep space radiation on the budding yeast, Saccharomyces cerevisiae. Ionizing radiation encountered in deep space can create damaging lesions in DNA, including double strand breaks (DSBs). Budding yeast is suitable as a biological model to study these effects, as it is eukaryotic, and can be desiccated for prolonged periods while retaining viability, thus serving as a robust analog for human cells. On the ground, yeast cells are grown in liquid medium, then loaded into the wells of microfluidic cards and air dried prior to integration into the payload. Once the spacecraft reaches its target heliocentric orbit, a mixture of growth medium and metabolic indicator dye will be pumped into the microwells at specific time points to rehydrate the cells and allow them to grow. A 3-color LED detection system will measure changes in growth and metabolism resulting from ionizing radiation exposure. BioSentinel contains a wild type control strain and a rad51 mutant that is defective for DNA damage repair. In this study, we will determine the optimal amount of time to grow diploid yeast cells in liquid culture before they are desiccated for space flight. After an extended time in stationary phase, they become more tolerant to desiccation due to stress caused by nitrogen starvation. However, excessive exposure can lead to loss of viability and to a heterogeneous cell population due to sporulation. Since viability loss during desiccation poses a risk to mission success, a stress preconditioning process during initial growth may increase long-term cell viability. To determine the growth period that improves desiccation tolerance but allows for retention of uniform radiation sensitivity, we will grow both strains in liquid medium for a varying number of days (4 to 7), desiccate the cells, and then observe changes to cell viability and ionizing radiation sensitivity over time. Supported by the Space Life Sciences Training Program at NASA Ames Research Center

BioSentinel: Mission Summary and Lessons Learned From the First Deep Space Biology CubeSat Mission

Launched on Artemis I, BioSentinel carries a biology experiment into deep space for the first time in 50 years. A 6U CubeSat form factor was utilized for the spacecraft, which included technologies newly developed or adapted for operations beyond Earth orbit. The spacecraft carries onboard budding yeast, Saccharomyces cerevisiae, as an analog to human cells to test the biological response to deep space radiation. This was the maiden deep-space voyage for many of the subsystems, and the first time to evaluate their performance in flight operation. Flying a CubeSat beyond LEO comes with unique challenges with respect to trajectory uncertainty and mission operations planning. The nominal plan was a lunar fly-by, followed by an insertion into heliocentric orbit. However, some possible scenarios included lunar eclipses that could have severely impacted the power budget during that phase of the mission, while others could have resulted in a “retrograde” hyperbola at swing-by resulting in the spacecraft traveling inward toward Earth or even towards a collision with the lunar surface. The commissioning phase of the mission was successful and completed a week ahead of schedule. It did not come without its exciting moments and challenges. First contact with the spacecraft uncovered that the vehicle was unexpectedly tumbling after deployment, a situation that needed to be corrected urgently. The mission operations team executed a contingency plan to stabilize the spacecraft, with just moments to spare before the battery ran out of power. The BioSensor payload onboard the spacecraft is a complex instrument that includes microfluidics, optical systems, sensor control electronics, as well as the living yeast cells. BioSentinel also includes a TimePix radiation sensor implemented by JSC’s RadWorks group. Total dose and Linear Energy Transfer (LET) spectrum data are compared to the rate of cell growth and metabolic activity measured in the S. cerevisiae cells. BioSentinel mature nanosatellite technologies included: deep space communications and navigation, autonomous attitude control and momentum management, and micro-propulsion systems, to provide an adaptable nanosatellite platform for deep space uses. This paper discusses the performance of the BioSentinel spacecraft through the mission phase, and includes lessons learned from challenges and anomalies. BioSentinel had many successes and will be a pathfinder for future deep space CubeSats and biology missions

Biosentinel: Developing a Space Radiation Biosensor

Ionizing radiation presents a major challenge to human exploration and long-term residence in space. The deep-space radiation spectrum includes highly energetic particles that generate double strand breaks (DSBs), deleterious DNA lesions that are usually repaired without errors via homologous recombination (HR), a conserved pathway in all eukaryotes. While progress identifying and characterizing biological radiation effects using Earth-based facilities has been significant, no terrestrial source duplicates the unique space radiation environment.We are developing a biosensor-based nanosatellite to fly aboard NASAs Space Launch System Exploration Mission 1, expected to launch in 2017 and reach a 1AU (astronomic unit) heliocentric orbit. Our biosensor (called BioSentinel) uses the yeast S. cerevisiae to measure DSBs in response to ambient space radiation. The BioSentinel strain contains engineered genetic defects that prevent growth until and unless a radiation-induced DSB near a reporter gene activates the yeasts HR repair mechanisms. Thus, culture growth and metabolic activity directly indicate a successful DSB-and-repair event. In parallel, HR-defective and wild type strains will provide survival data. Desiccated cells will be carried within independent culture microwells, built into 96-well microfluidic cards. Each microwell set will be activated by media addition at different time points over 18 months, and cell growth will be tracked continuously via optical density. One reserve set will be activated only in the occurrence of a solar particle event. Biological measurements will be compared to data provided by onboard physical dosimeters and to Earth-based experiments.BioSentinel will conduct the first study of biological response to space radiation outside Low Earth Orbit in over 40 years. BioSentinel will thus address strategic knowledge gaps related to the biological effects of space radiation and will provide an adaptable platform to perform human-relevant measurements in multiple space environments. We hope that it can therefore be used on the ISS, on and around other planetary bodies as well as other exploration platforms as a self-contained system that will allow us to compare and calibrate different radiation environments.BioSentinels results will be critical for improving interpretation of the effects of space radiation exposure, and for reducing the risk associated with long-term human exploration

Analyses of the yeast Rad51 recombinase A265V mutant reveal different in vivo roles of Swi2-like factors

The Saccharomyces cerevisiae Swi2-like factors Rad54 and Rdh54 play multifaceted roles in homologous recombination via their DNA translocase activity. Aside from promoting Rad51-mediated DNA strand invasion of a partner chromatid, Rad54 and Rdh54 can remove Rad51 from duplex DNA for intracellular recycling. Although the in vitro properties of the two proteins are similar, differences between the phenotypes of the null allele mutants suggest that they play different roles in vivo. Through the isolation of a novel RAD51 allele encoding a protein with reduced affinity for DNA, we provide evidence that Rad54 and Rdh54 have different in vivo interactions with Rad51. The mutant Rad51 forms a complex on duplex DNA that is more susceptible to dissociation by Rdh54. This Rad51 variant distinguishes the in vivo functions of Rad54 and Rdh54, leading to the conclusion that two translocases remove Rad51 from different substrates in vivo. Additionally, we show that a third Swi2-like factor, Uls1, contributes toward Rad51 clearance from chromatin in the absence of Rad54 and Rdh54, and define a hierarchy of action of the Swi2-like translocases for chromosome damage repair