Developing Technologies for Biological Experiments in Deep Space

Home , ArgoMoon, Artemis 1, BioSentinel, Cislunar Explorers, Lunar IceCube

Developing technologies for biological experiments in deep space

Sergio R. Santa Maria Elizabeth Hawkins Ada Kanapskyte

sergio.santamaria@nasa.gov NASA’s life science programs

STS-1 (1981)

STS-135 (2011)

1973 – 1974 1981 - 2011 2000 – 2006 – Space Shuttle International Skylab Bio CubeSats Program Space Station Microgravity effects

- Nausea / vomit - Disorientation & sleep loss - Body fluid redistribution - Muscle & bone loss - Cardiovascular deconditioning - Increase pathogenicity in microbes Interplanetary space radiation

What type of radiation are we going to encounter beyond low Earth orbit (LEO)?

Galactic Cosmic Rays (GCRs): - Interplanetary, continuous, modulated by the 11-year solar cycle - High-energy protons and highly charged, energetic heavy particles (Fe-56, C-12) - Not effectively shielded; can break up into lighter, more penetrating pieces Challenges: biology effects poorly understood (but most hazardous) Interplanetary space radiation Solar Particle Events (SPEs)

- Interplanetary, sporadic, transient (several min to days) - High proton fluxes (low and medium energy) - Largest doses occur during maximum solar activity

Challenges: unpredictable; large doses in a short time Space radiation effects

Space radiation is the # 1 risk to astronaut health on extended space exploration missions beyond the Earth’s magnetosphere

• Immune system suppression, learning and memory impairment have been observed in animal models exposed to mission-relevant doses (Kennedy et al. 2011; Britten et al. 2012) • Low doses of space radiation are causative of an increased incidence and early appearance of cataracts in astronauts (Cuccinota et al. 2001) • Cardiovascular disease mortality rate among Apollo lunar astronauts is 4-5-fold higher than in non-flight and LEO astronauts (Delp et al. 2016)

• Astronauts have shown an increase in chromosomal abnormalities, even in LEO, during ISS, Mir & STS (Hubble shuttle) missions • GCR will be much more abundant as astronauts go to higher orbits beyond Earth’s protective magnetic field

Cucinotta et al. 2008 Chromosomes 1, 2, 4 in red, green & yellow (ISS) h iiso iei pc sw nwi is12.5daysonalunarroundtripor1.5yearsinLEO. As we asweknowit– The limitsoflifeinspace – send peoplefurtherintospace,wecanusemodelorganisms and/orbiosensorstounderstandthe 36 million mi 180 240,000 mi 240,000 Millions Millions mi - 300 mi 62 mi Distance from

Minutes Earth 12.5 days Known biological risksandhowtheycanbeaddressed • 6 months L2 25 million mi NEA 12 months Mission Duration Extended ISS 18 months 65 million mi Unknown Mars 3 years Beyond What’s next for NASA?

Artemis-1 mission & BioSentinel (launch 2021/2022)

Artemis-1: secondary payloads (6U CubeSats) CubeSats CubeSats

CubeSat configurations

CubeSats: toys, tools, or debris cloud?

Poghosyan & Golkar, 2017 CubeSat technologies (Artemis-1)

Lunar Lunar IceCube Cislunar Explorers Flashlight

SkyFire

ArgoMoon NEA Scout Biological missions using CubeSats (NASA Ames Research Center, 2006 – 2022) NASA Ames pioneering biological space missions

E. coli GeneSat-1 (2006 / 3U): gene expression EcAMSat (2017 / 6U): antibiotic resistance

S. cerevisiae PharmaSat (2009 / 3U): drug dose response BioSentinel (~2022 / 6U): DNA damage response

B. subtilis O/OREOS* (2010 / 3U): survival, metabolism *Organism/Organic Response to Orbital Stress

C. richardii SporeSat-1 (2014 / 3U): ion channel sensors, microcentrifuges GeneSat mission: NASA’s 1st CubeSat

1st bio nanosatellite in Earth’s orbit, 1st real-time, in-situ gene expression measurement in space

3U CubeSat

• Model organism: E. coli (~ 0.5 x 2 µm bacteria) • Nutrient deprivation in dormant state (6 weeks) • Launch: Dec 2006 to low Earth orbit (440 km) 12-well fluidic card • Nutrient solution feed upon orbit stabilization, grow E. coli in microgravity • Monitor gene expression via GFP • Monitor optical density: cell population

Fluorescence Fluorescence (GFP) (GFP) Telemeter Compare to data to Earth ground data

Dec 16, 2006 PharmaSat mission

Effect of microgravity on yeast susceptibility to antifungal drug • Launch: May 2009 to LEO (~450 km) • Grow yeast in multiwell fluidics card in microgravity • Measure inhibition of growth by antifungal S. cerevisiae • Optical absorbance (turbidity: cell density) • Metabolism indicator dye: alamarBlue (3-LED optical detection) • Control + 3 concentrations of antifungal 3U CubeSat

48-well fluidic card + Ground Spaceflight

May 19, 2009 O/OREOS mission

Organism / Organic Response to Orbital Stress (1st astrobiology CubeSat) • Effects of space exposure on biological organisms (6 mo) & organic molecules (18 mo) • SESLO (Space Environment Survival of Living Organisms): monitor survival, growth, and metabolism of B. subtilis using in-situ optical density /colorimetry Nov 19, 2010 • SEVO (Space Environment Viability of Organics): track changes in organic molecules and biomarkers: UV / visible / NIR spectroscopy

B. subtilis

107 spores / well 3U CubeSat (75 μL per well) SporeSat mission

Gravitational response of fern spores via Ca2+ ion channel response

• Model organism: Ceratopteris richardii (aquatic fern spores) • 2U payload (3U total) • Launch: April 18, 2014 to low Earth orbit • Variable gravity in microgravity environment using 50-mm microcentrifuges • 32 ion-specific [Ca2+] electrode pairs

BioCDs

SpaceX CRS-3

2U payload 3U CubeSat A.C. Matin et al.

UPEC viability, the wild type and the ∆rpoS strains were grown in with pressure-sensitive acrylic adhesive (9471LE on 51-µm-thick Meli- conventional laboratory flasks shaken overnight at 200 rpm in 1/6- nex 455 polyester carrier, 3 M; St. Paul, MN). Each well (4.0 mm strength LB at 37 °C. As before (Zgurskaya et al., 1997), growth under diameter x 7.8 mm long; 100 µL volume) is fitted at its inlet and outlet these conditions was complete within 6 h, allowing some 8 h in with 0.2-µm filters (nylon fiber; Sterlitech, Kent, WA) to prevent cell

stationary phase, permitting activation of GSR in the wild type, but leakage. Well tops and bottoms are sealed by 50-µm-thick air-and-CO2- not in the mutant missing the rpoS gene, which, as stated, is required for permeable optical-quality poly(styrene) membranes. Attached to both the activation of GSR (Matin, 2014). The cultures were then diluted to sides of the card are thermal spreaders (thin aluminum plates), each

an A600 of 0.45 in M9. Gm (Sigma-Aldrich, St. Louis, MO) was added to containing three embedded AD590 temperature sensors that provide both the wild type and the mutant cultures to a final concentration of output current directly proportional to absolute temperature (Analog 16 µg/mL; a parallel aliquot of cell suspension of each strain without Devices, Norwood, MA); a thin-film heater fabricated from kapton tape the drug served as control. Following 24-h incubation without shaking, and patterned metal conductors (Minco, Minneapolis, MN) is affixed to 1.8 mL of the cultures were transferred to test tubes to which 200 µL of the opposite side of each spreader plate, relative to the fluidic card, and 10x AB (ThermoFisher Scientific, Grand Island, NY) were added. To controlled in closed-loop fashion using the temperature-sensor outputs. monitor changes in AB absorption, the cultures were dispensed in Each wellEcAMSat is equipped with its ownmission 3-color LED (LTST-C17FB1WT; Lite- microtiter plate wells (Thermo Scientific, Waltham, MA), each well On Technology Corp., Taiwan); a photodetector (Model no. TSL237T; receiving 0.25 mL. Appropriate control solutions in other rows of the AMS-TAOS USA, Plano, TX) at the opposite end of each well converts plates were also in 0.25 mL quantities, and five wells were used for each the transmitted light intensity to a proportional frequency, from which condition. Absorption changes at 470, 525, and 615 nm were measured absorbance values can be calculated. (No moving parts are associated in a microplate reader (Biochrom US, Holliston,E. coli MA); AntiMicrobial data were with theSatellite optical measurements.) mission The card, thermal spreaders, and 6U CubeSat acquired by DigiRead software (ASYS Hitech, Holliston, MA) and printed-circuit (PC) boards supporting the LEDs and photodetectors, transferred to Excel (Microsoft, Redmond,• Antibiotic WA) for analysis. resistance in whichmicrogravity are placed on vs. opposite dose sides in of uropathogenic the fluidic card, constituteE. thecoli “card stack” (see the cross section, upper right in Fig. 1). • 6U format provided 50% moreThe card solar-panelfluid-delivery system power (Fig. 1) to includes keep eleven payload electrically- 2.3. EcAMSat payload system experiment at 37 °C foractuated extended solenoid durations valves (SVs, LHDA0531315HA; The Lee Co., West- brook, CT); a diaphragm pump for high-flow-rate fluid mixing, circula- The E. coli in this setup are placed in the payload hardware in a 48- • Launch: Nov 12, 2017 (ISStion, and deployment: priming of tubing Nov (NF 5S; 20, KNF 2017) Neuberger, Trenton, NJ); a well fluidic card (Fig. 1; Micronics, Redmond, WA). The cards are made st precisionst metering pump (LPVX0502600BC; The Lee Co.) to prepare from laser-cut layers of poly(methylmethacrylate),•16U bio bonded CubeSat together and 1 bio satellite to be deployed from ISS

A.C. Matin et al. and deliver the desired concentrations and volumes of antibiotic and other reagents; three 35-mL and six 25-mL reagent bags (fluorinated ethylene propylene, FEP; American Fluoroseal/Saint-Gobain, Gaithers- burg, MD); a bubble trap (custom fabricated by NASA Ames); and a check valve (Smart Products; Morgan Hill, CA) to prevent waste fluids from flowing back into the system. The 48 wells are configured in 4 fluidically independent rows or “banks” of 12 each (labeled “High”, “Medium”, “Low”, and “Control” in Fig. 1, indicating the relative Gm concentrations that were administered). Each bank on the inlet side of the card is connected to the normally-closed port of one SV and, on the outlet side, to a 25-mL waste bag partially filled with M9; pressurization (∼7 kPa) of these waste bags by means of a spring-loaded metal plate replaces any fluid that evaporates over time from the wells through their permeable membrane cover. The nutrient (1/6-strength LB), antibiotic (Gm), antibiotic dilution medium (M9), and AB bags are also Deployment from ISS attached to SV normally-closed ports (Fig. 1). A Gm-dilution loop is created by attaching the M9 bag via another SV near the outlet of the bubble trap, which is placed ahead of the point of fluid delivery to the card. The main waste bag collects the previous contents of the tubing each time it is filled with a new reagent (see below) prior to delivery to the card. Fig. 2A and B show, respectively, the assembled EcAMSat payload fluidic system hardware and the hermetically sealed containment vessel (internal volume ∼1.2 L) in which the system is housed. The sealed payload containment vessel is integrated with the spacecraft “bus”, which includes the power, communications, data-handling, and control functions. The completed nanosatellite has overall dimensions of 10 × 22 × 36 cm.

2.4. AB-mediated assessment of Gm effect in the EcAMSat payload Fig. 1. Schematic diagram of EcAMSat fluidic system (at left) connected to EcAMSat 48-well fluidic card (at lower right). A single fluidic well is also shown in cross section (top right). The wild type and ∆rpoS mutant of UPEC were grown as described SV = 3-way solenoid valve; arrows show direction of fluid flow; Waste H, M, L, C collect the flow-through from the High, Medium, Low, and Control banks of 12 wells each; other above, rinsed with M9 (3x), and diluted in M9 to an A600 of 1.0. In a components are as marked. sterile biosafety cabinet, 5 µL aliquots of each strain were loaded in alternating wells of the 48-well fluidic card so that six of the 12 wells per bank contained the wild type and six wells, the mutant. The card fi was sealed and purged with CO2 to facilitate bubble-free lling of the channels and wells: any CO2 bubbles remaining after priming with degassed M9 dissolved readily as additional M9 flowed through the wells. The card was manually primed with a syringe containing degassed M9 connected to the outlet. A second empty syringe at the inlet with its plunger drawn back served to generate a slight vacuum (which assists bubble-free filling of wells). After filling and until Fig. 2. (A) Fully assembled EcAMSat biological/fluidic/optical/thermal payload system; connection was made to the fluidic system, the card remained under (B) its hermetic payload containment vessel with electrical interface board; overall size ∼10 × 10 × 20 cm. (C) Chronological summary of the sequence of operations and pressure (∼4.4 kPa), arising from a bag containing M9 hanging fl fl measurements for the ground experiments conducted to date; the space ight system will approximately 45 cm above the card. This served to replace any uid follow the same timeline. lost by evaporation through the permeable membranes, thereby fl preventing bubble formation in the wells. The rest of the sterile uidic considered in subsequent sections. fi system was lled with the appropriate solutions (see Fig. 1), assembled To start the viability measurements, the 3-color LEDs with emissions with the rest of the payload hardware, and then sealed in the hermetic at the above-mentioned wavelengths were sequentially energized, one containment vessel; as explained above, slightly pressurized M9 in the color and one well at a time. The photodetector of each well converted waste bags continued to compensate for any evaporation. As placement the transmitted light intensity of the individual colors to proportional in the containment vessel eliminated further need for a sterile environ- frequencies, permitting calculation of absorbance. (During the space- ment, the assembled payload system was removed from the biosafety flight experiments, the stored frequencies will be telemetrically trans- cabinet and attached to a benchtop “rotisserie” apparatus; this rotated ferred to Earth from the satellite.) The measurements for each well were the payload successively clockwise and counterclockwise by nearly one taken every 15 min. The payload system was warmed to 37 °C for ∼3h full rotation with a period of ∼80 s, preventing cell settling. The by the heaters and thermal spreaders with closed-loop temperature experiments were run using ground-support equipment, i.e., a desktop control using the mean value from the six temperature sensors. 1/6- fl computer and power supply, and employed a “space- ight-like” strength LB was pumped into each bank in turn, starting with the command sequence. control bank, replacing the M9. The pumping phase lasted for 2 h per In experiments involving the microtiter plates (see above), cells bank (see Results section for total durations of the various phases). The were incubated without shaking only when being treated with the drug cells were allowed to grow to stationary phase, in which the GSR is fi and in the absence of nutrients. A bio lm formation by the cells under activated. Next, the metering pump delivered M9 to the control bank, ff these conditions does not occur and none was found. Given the di erent each well receiving ∼4x its 100 µL volume to reach at least 90% fi setup of the EcAMSat payload, whether a bio lm could form in this exchange. The metering pump then extracted a small, measured platform and its potential effects on the interpretation of the results are

BioSentinel mission: NASA’s 1st interplanetary bio satellite

Main objective: develop a tool with autonomous life support technologies to study the biological effects of the space radiation environment at different orbits • First biological study beyond low Earth orbit (LEO) in 50 years - First biological 6U CubeSat to fly beyond LEO - First CubeSat to combine biological studies with autonomous capability & physical dosimetry beyond LEO - Secondary payload in SLS ARTEMIS-1 (launch in 2021/2022) – 13 payloads - Far beyond the protection of Earth’s magnetosphere (~0.3 AU from Earth at 6 months; ~40 million km) - BioSentinel will allow to compare different radiation and gravitational environments (free space, ISS, Lunar surface…)

BioSentinel escapes into Lunar transit a heliocentric orbit (3-7 days) Lunar Transfer & Fly-by Distance to ISS: ~ 350 km Distance to the Moon: ~385,000 km Distance at 6 months: ~40’000,000 km Launch

Secondary payload deployment (L+4-5 hrs) What is BioSentinel?

BioSentinel is a yeast radiation biosensor that will measure the DNA damage response caused by space radiation, and will provide a tool to study the true biological effects of the space environment at different orbits.

Why? Space radiation environment’s unique spectrum cannot be duplicated on Earth. It includes high-energy particles, is omnidirectional, continuous, and of low flux.

How? Lab-engineered S. cerevisiae cells will sense & repair direct (and indirect) damage to their DNA. Yeast cells will remain dormant until rehydrated and grown using a microfluidic and optical detection system.

S. cerevisiae (budding yeast) What is BioSentinel?

Why? Space radiation environment’s unique spectrum cannot be duplicated on Earth. It includes high-energy particles, is omnidirectional, continuous, and of low flux.

S. cerevisiae Why budding yeast? (budding yeast) It is an eukaryote; easy genetic & physical manipulation; assay availability; flight heritage; ability to be stored in dormant state While it is a simple model organism, yeast cells are the best for the job given the limitations & constraints of spaceflight BioSentinel: a 6U nanosatellite for deep space

16-well fluidic card (x18) Card stack Budding yeast 9-card fluidic manifold (x2)

~5 µm ~5 um 4~5 cm cm

6U BioSentinel spacecraft 4U BioSensor payload BioSentinel: microfluidics card

3-LED emitter

Microfluidic Optical card (x18) Source

Heater layer

Fluidic card

Photodiode detector array Heater layer

Optical detector Repair of proton-induced DNA damage c-ray and MMS spot dilutions were conducted twice. Plates were scanned using plates were removed. The colonies on all plates were then manually counted an Epson Perfection 1650. Genetic analyses were performed as previously and the number of surviving coloniesRepair on exposed of proton-induced plates was compared DNA damage to the described (44). Four full tetrads from rad52D control crosses and five from the number of colonies present on unexposed plates (of the same number of cells 200-Gy proton survivor crosses (YTH3223) were picked. The data shown are plated) to determine survival percentage. Most experiments were done at least crepresentative-ray and MMS of spot the dilutions data obtained. were conducted twice. Plates were scanned using platesthree times were in removed. duplicate. The The colonies curve presentedon all plates in Figure were then 1B was manually typical counted of the an Epson Perfection 1650. Genetic analyses were performed as previously andresults the obtained number fromof surviving that experiment. colonies on exposed plates was compared to the describedProton irradiation (44). Four full tetrads from rad52D control crosses and five from the number of colonies present on unexposed plates (of the same number of cells 200-GyThe proton proton accelerator survivor at crosses LLUMC (YTH3223) was the sourcewere picked. of protons The data utilized shown in this are plated)Multiple to exposure determine protocol survival percentage. Most experiments were done at least representativestudy. The experimental of the data obtained. arrangement of the proton accelerator used was threeTreatment times of in cells duplicate. with moreThe curve than presented one stress in was Figure conducted 1B was as typical follows. of theIn- Repair of proton-induced DNA damage described previously (13). Control plates were manipulated similar to irradiated resultsdividual obtained colonies from that that survived experiment. the primary proton irradiation were selected, Protonplates. The irradiation dose rate was 0.6 Gy/min at an energy of 250 MeV (237 MeV at cultured and stored as permanent glycerol stocks at –80°C. These cells were Thetargetc-ray proton with and a acceleratorMMS LET spot of 0.41 dilutions at LLUMC keV/ werem). was LETconducted the values source twice. at of LLUMC Plates protons were were utilized scanned previously in thisusing Multiplethenplates repropagated were exposure removed. on protocol YPD The and colonies exposed on to all UV, plates-rays, were elevated then manually temperatures counted or an Epson Perfection 1650. Geneticl analyses were performed as previously and the number of surviving colonies on exposedc plates was compared to the study. The experimental arrangement of the proton accelerator used was Treatment of cells with more than one stress was conducted as follows. In- reporteddescribed as 0.39 (44). keV/ Fourlm full for tetrads an energy from ofrad52 249D MeVcontrol (43) crosses and 0.5 and keV/ fivelm from for an the protonsnumber as of described colonies present above. on Again, unexposed individual plates colonies (of the same that survived number of these cells describedenergy200-Gy of previously172 proton MeV, survivor in(13). which Control crosses a 38-mm plates (YTH3223) polycarbonate were manipulated were picked. absorber similar The was data to used irradiated shown (13). are dividualtreatmentsplated) colonies to were determine selected, that survival survived cultured percentage. the and primary stored Most proton at –80 experiments° irradiationC. were were done selected, at least plates.Calibrationrepresentative The dose and charge of rate the was data readings 0.6 obtained. Gy/min were at performed an energy by of placing 250 MeV an (237 ion chamber MeV at culturedthree times and storedin duplicate. as permanent The curve glycerol presented stocks in Figureat –80° 1BC.These was typical cells wereof the target(PTW with Markus a LET parallel of 0.41 plate) keV/ at thelm). target. LET This values was at performed LLUMC and were calculated previously at Statisticalthenresults repropagated obtained analysis fromon YPD that and experiment. exposed to UV, c-rays, elevated temperatures or Proton irradiation reportedleast three as 0.39 times keV/ orl untilm for the an energy readings of 249 agreed. MeV These (43) and readings 0.5 keV/ werelm for then an Resultsprotons were as described graphed above.and error Again, bars were individual determined colonies using that standard survived error these of The proton accelerator at LLUMC was the source of protons utilized in this Multiple exposure protocol energycompared of 172 to a MeV, detector in which upstream, a 38-mm which polycarbonate detected the absorber number was of counts used (13). up- thetreatments mean. Generally,were selected, mean culturedstandard and stored error at (for –80 the°C. number of experiments study. The experimental arrangement of the proton accelerator used was Treatment of cells with moreÆ than one stress was conducted as follows. In- Calibrationstreamdescribed that andequal previously charge 1 Gy (13). readings at the Control target. were plates After performed were calculation, manipulated by placing the ionsimilar an chamber ion to chamber irradiated was designated)dividual colonies was reported. that survived In some the cases, primary the error proton bars irradiation were smaller were than selected, the (PTWremovedplates. Markus and The the dose parallel plates rate plate) were was placed0.6 at the Gy/min target. at the at target.This an energywas The performed region of 250 before MeV and calculated the (237 entrance MeV at at Statisticalsymbolcultured representing analysisand stored the as curve.permanent glycerol stocks at –80°C. These cells were leastoftarget the three Bragg with times curve a LET or was of until used. 0.41 the keV/The readings peaklm). of LET the agreed. values Bragg These at curve LLUMC readings was monoenergetic, were were previously then Resultsthen repropagated were graphed on and YPD error and exposedbars were to determined UV, c-rays, using elevated standard temperatures error of or comparedmeaningreported there to as a0.39 was detector keV/ no rangelm upstream, for shifting an energy which and of thedetected 249 peak MeV thewas (43) number not and spread 0.5 of keV/ counts out.lm Yeast for up- an theprotons mean. as Generally, described mean above.standard Again, individual error (for colonies the number that of survived experiments these energy of 172 MeV, in which a 38-mm polycarbonate absorber was used (13). Resultstreatments were selected, culturedÆ and stored at –80°C. streamplatesCalibration were that equal exposed and 1 charge Gy to at protons readings the target. in were at After least performed calculation, three separate by placing the experimentsion an chamber ion chamber with was designated) was reported. In some cases, the error bars were smaller than the removedreproducible(PTW Markusand results.the platesparallel It were was plate) not placed at necessary the at target. the target. to This grow The was cells region performed in beforethe dark and the calculated following entrance at symbolStatistical representing analysis the curve. ofexposureleast the Bragg three to curveprotons times was or and used. untilc-rays The the as peak readings photoreactivation of the agreed. 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InÆ some cases, the error bars were smaller than the plates were exposed to protons in at least three separate experiments with We employed S.cerevisiae in our analysis of the DNA repair reproducibleSurvivalremoved curves and results. the plates It was were not placed necessary at the to target. grow The cells region in the before dark the following entrance symbol representing the curve. of the Bragg curve was used. The peak of the Bragg curve was monoenergetic, mechanisms used to repair damage arising from proton Downloaded from exposureOvernight to cultures protons (2 and ml) ofc-rays yeast as strains photoreactivation were grown at does 30 C not in repair YPD liquid. strand Yeast cells lacking HR and PRR repair pathways are sensitive meaning there was no range shifting and the peak was not° spread out. Yeast irradiation. 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BioSentinel: optical detection system

A. A. C. Dose (Gy) Dose (Gy) Dose (Gy) Dedicated 3-color opticalA. system at each well to track growth via 0 2.5 5 10 20 30 0 2.5 5 10 20 30 0 2.5 5 10 20 30 optical density and cell metabolic activity via dye color changes rad52Δ A. rad52Δ LEDs: 570 nm (green, measures pink) rad52Δ

630 nm (red, measures blue) wild type no cells 850 nm (infrared, measures growth) rad52Δ Time: 0 hours Time: 19 hours Time: 24 hours

Survival (%) 250 MeV protons 250 MeVMeV protonsH-1 Proton (150 MeV) irradiation sensitivity: rad51Δ mutant strain Brookhaven National Lab 110 Gamma irradiation sensitivity: rad52 HR defective strain 100 250 MeV protons 90 Dose (Gy) 90 0 Gy 80 2.5 Gy Fig. 1. Yeast strains lacking proteins involvedB. in repair of DNA strand breaks are sensitive to proton irradiation. (A) Yeast80 strains lacking proteins involved in BER Fig. 1. Yeast strains lacking proteins involvedB.B. in repair 100 of DNA strand breaks are sensitive to proton irradiation. (A) Yeast strains lacking proteins involved in BER 5 Gy (apn1D apn2D), NER (rad1D), PRR (rad18D), HR100 ( rad52D), cell cycle checkpoints (mec1D) and the isogenic wild-type7070 strains, DBY747 (for apn1D apn2D, ( ), NER ( ), PRR ( ), HR ( ), cell cycle checkpoints ( ) and the isogenic wild-type strains, DBY747 (for , 10 Gy apn1rad1DDapn2, rad18D D and rad52rad1D) and YMP10650rad18D (for mec1rad52D),D after exposure to 150-Gy protonsmec1D generated from the proton accelerator at Loma Lindaapn1 University.D apn2D (B) 20 Gy 100 6060 rad1TheD, strainsrad18D shownand rad52 in (AD)) were and treated YMP10650 with increasing (for mec1D doses), after of exposure proton irradiation to 150-Gy in protons order to generated generate afrom survival the proton curve. accelerator A dilution atseries Loma of Lindacells was University. prepared (B and) 30 Gy Thevolumes strains according shown in (toA) 100 were and treated 1000 cells with were increasing plated doses onto10 YPD of proton plates. irradiation The plates in were order then to generate exposed a to survival the proton curve. dosages50 A dilution shown series and then of cells incubated was prepared at 30°C andfor 3 days. The number of colonies that grew onB. each plate was compared to the untreated plates to determine percent survival50 for each proton dose. Single rad52D Fig.volumes 1. Yeast according strains to lacking 100 and proteins 1000 cells involved were plated in repair100 onto of YPD DNA plates. strand Thebreaks plates are sensitivewere then to exposed proton irradiation. towild%type%wild the!"#$%&'()%wild type proton type (A dosages) Yeast strains shown lacking and then proteins incubated involved at 30° inC BERfor 3 colonies that survived 150 and 200-Gy proton irradiation10 were selected, cultured and treated as above for inclusion in this40 survival curve. The curve shown is typical (days.apn1D Theapn2 numberD), NER of colonies (rad1D), that PRR grew (rad18 on eachD), HR plate (10rad52 wasD compared), cell cycle to the checkpoints untreated (mec1 platesD to) and determine therad51%rad51 isogenic*+$,-%rad51Δ percent Δ wild-type survival40 strains, for each DBY747 proton dose. (for apn1 SingleD apn2D, 0 Gy !"#$%&'()% rad52D of the results obtained. (C) Yeast strains lacking proteins involved in HR, rad50D, rad51D, rad54D,wildrad55 typeD, rad5730D and xrs2D and the isogenic wild type 2.5 Gy rad1coloniesD, rad18 that survivedD and rad52 150D and) and 200-Gy YMP10650 proton irradiation(for mec1D were), after selected, exposure cultured to 150-Gy and treated protons as generated above forrad52%rad52 from inclusion*+$,.%rad52Δ theΔ proton in this acceleratorsurvival curve. at Loma The curve Linda shown University. is typical (B) (LYS390), were treated as in (A). 5 Gy 1 *+$,-%rad51Δ 30

Survival (%) 20 Theof the strains results shown obtained. in (A ()C were) Yeast treated strains with lacking increasing proteins doses involved of proton in irradiationHR, rad50D in, orderrad51 toD, generaterad54D, arad55 survivalD, rad57 curve.DPercentage of reduced alamarBlue Aand dilutionxrs2D seriesand the of cells isogenic was wild prepared type and 10 Gy volumes according to 100 and 1000 cells were plated onto1 YPD plates. The plates were then exposed to*+$,.%rad52 the protonΔ dosages shown and then incubated at 30 C for 3 (LYS390), were treated as in (A). Survival (%) 10 137Cs gamma irradiation 2010 ° 121

1 Percentage of reduced alamarBlue days. The number of colonies that grew on each plateSurvival (%) was compared to the untreated plates to determine percent survival for each proton dose. Single rad52D !"#$%&'()%wild type 0 10 colonies that survived 150 and 200-Gy proton irradiation0.1 were selected, cultured and treated as above for inclusion in this survival0 2 4 curve.6 8 10The 12 14 curve 16 18 shown 20 22 24 is typical26 28 30 32 34 36 38 40 42 44 46 48 137Cs gamma irradiation *+$,-%rad51Δ 121 of the results obtained. (C) Yeast strains lacking proteins0.1 involved0 in5 HR, 10rad50 D25, rad5150D , rad54100 D, rad55D, rad57D and xrs2D and the isogenicTime wild (hours) type 0 20 Gamma40 dose60 (Gy) 80 100 *+$,.%rad52Δ 0 (LYS390), were treated as in (A). 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 1 Survival (%) 0.1 Gamma dose (Gy) HR repair defective cells showTime sensitivity (hours) to ionizing radiation Yeast growth with flight-like 0 5 10 25 50 100 optical unit 137Cs gammaGamma irradiation dose (Gy) 121

0.1 0 5 10 25 50 100 Gamma dose (Gy) BioSentinel: future & ongoing objectives

A flexible design that can (and will be) used on different space platforms Examples of future biosensor technologies

Dielectric spectroscopy biosensor Miniaturized bio sequencing devices

Goals: Design and develop MinION instruments with integrated bio 5mm growth, sample extraction, and sequencing capabilities to study Fluidic well effects of space environment on microorganisms (e.g., DNA Cells in solution mutations, gene expression)

“Fringing” capacitance Credit: Oxford Nanopore Technologies

SENSOR SIDE 1 SENSOR SIDE 2 Repurpose proven LEO technologies for deep space CIRCUIT BOARD SporeSat SHIELD Fluorescence microscopy Goal: Develop miniaturized instrument for continuous GeneSat non-invasive monitoring of the biological response to deep space radiation via bio electrical signatures. TOP: Dielectric sensing prototype with electrodes connecting to microwell containing bio organisms. BOTTOM: Rendering of prototype and cross-sectional Microcentrifuges to Organ / tissue on chip illustration of contactless sensing mechanism. generate artificial gravity microfluidics Conclusions

• Nanosatellites like CubeSats can do real science in low Earth orbit (LEO) and in interplanetary deep space o Instrument miniaturization & new micro/nano technologies o Fully automated instruments o Adaptable technologies for different platforms (ISS, free-flyers, Lunar landers & gateway) o Real-time, in-situ experiments provide insights on dynamics not available from expose-and-return strategies

• Heritage of astrobiology and fundamental space biology experiments in LEO is a major enabler for interplanetary biological missions o Flying biology in desiccated form, filling microfluidic cards/wells in microgravity o Long-term material & reagent biocompatibility (long-duration pre-launch preparation) o Radiation-tolerant design o High-heritage components: microfluidics, optical measurements, environmental sensors