Ecamsat and Biosentinel: Autonomous Bio Nanosatellites Addressing Strategic Knowledge Gaps for Manned Spaceflight Beyond LEO

https://ntrs.nasa.gov/search.jsp?R=20170009842 2019-08-31T01:54:47+00:00Z

EcAMSat and BioSentinel: Autonomous Bio Nanosatellites Addressing Strategic Knowledge Gaps for Manned Spaceflight Beyond LEO

Mike Padgen, Ph.D. Millennium Engineering and Integration Co. NASA Ames Research Center

SUNY CNSE Colloquium March 24, 2017 Ames Research Center

• 1 of 9 NASA Field Centers • Second oldest NASA facility • Located in Silicon Valley • Est. 1939 as part of National Advisory Committee for SF Bay Aeronautics (NACA) • Absorbed into NASA in 1958 Google • ARC ~2300 employees

Moffett Air Field Oakland San Francisco Ames

NASA Ames San Jose

Image taken aboard ISS What We Do at Ames

• National Full-Scale Aerodynamics Complex • 80x120’ wind tunnel is largest in the world • Unitary Plan Wind Tunnel • 3 test sections, most commercial & military aircraft tested here 80x120’ Wind Tunnel • Mercury, Gemini, Apollo capsules

• NextGen Air Transportation

Hangar 1 What We Do at Ames

• ARC Jet • Creates super hot plasmas to help simulate re-entry • Only facility capable of modeling re-entry from interplanetary space

• Pleiades supercomputer th • 13 fastest in world Space shuttle wing tests • Appeared in The Martian! What We Do at Ames: Flight Missions

• Pioneer missions (1970s)

• LADEE and LCROSS • Measured structure and composition of the thin lunar atmosphere Inscription on Pioneer 9 and 10 • Kepler and K2 Missions • ~2500 confirmed exoplanets

• ISS Life Sciences • Wet-Lab 2, molecular biology in space! • Rodent research • Fruit fly lab

Kepler Ames Has a History of Successful Bio Nanosats • Nanosats are small, autonomous, free-flying satellites • Less reliance on human-tended experiments • Access to space: Low-cost launches as secondary payloads • Multiple flights possible - test, learn, iterate • Excellent education vehicle: Significant academic participation worldwide • Peer-reviewed science experiments

PharmaSat

GeneSat-1 O/OREOS

5.1 kg, 3U 5.5 kg, 3U (2010) 4.4 kg, 3U (2006) (2009) 1U = 10x10x10 cm A Quick Intro to AlamarBlue

• Metabolic indicator dye • Reduced by FAD, NADPH, cytochromes • Color change from blue to pink • Increase in fluorescence • Shift in absorbance

• Used by PharmaSat, O/Oreos, EcAMSat and BioSentinel GeneSat-1: 1st biological nanosatellite in Earth orbit, 1st real- time, in-situ gene expression measurement in space • Launch: December 2006 Fluorescence • Nutrient deprivation in dormant state (GFP) • Nutrient delivered upon orbit stabilization • GFP to measure gene expression • OD to measure cell population

Fluorescence (GFP)

E. coli PharmaSat: Effect of Microgravity on Yeast Susceptibility to Antifungal Drugs • Grow yeast in multiwell fluidics card in µ-gravity • Measure inhibition of growth by antifungal • Optical absorbance (turbidity: cell density) S. cerevisiae • Metabolism indicator dye: Alamar Blue • Control + 3 concentrations of antifungal 19 May 2009

Antonio Ricco, et al. Proc. SPIE 7929, Microfluidics, BioMEMS, and Medical Microsystems IX, 79290T (2011) O/OREOS: Effects of Space exposure on biological organisms (6 mo) and organic molecules (18 mo) • 2 x 1U payloads • SESLO: Monitor survival, growth, metabolism of Bacillus subtilis: in-situ OD / colorimetry • SEVO Track changes in organic molecules and biomarkers: UV / visible / NIR spectroscopy

O/OREOS: Organism/Organic Response to Orbital Stress SESLO: Space Environment Survival of Living Organisms SEVO: Space Environment Viability of Organics Wayne L. Nicholson, et al. Astrobiology, Vol. 11, Issue 10, Dec. 2011, pp. 951-958 Andrew Mattioda, et al. Astrobiology, Vol. 12, No 9, 2012 Nathan E. Bramall, et al. Planetary and Space Science, Vol. 60, 2012, pp: 121–130 Amanda M. Cook, et al. Astrobiology. February 2014, Vol. 14, Issue 2, pp: 87-101 Addressing Strategic Knowledge Gaps

• If humans are to go further into space for longer: • Model organisms can help us understand and mitigate key biological risks • Direct impact on human health or performance • Impact on the biota that accompany humans • Impact on organisms used to process waste or produce food

• Deep space radiation • Long-term exposure to microgravity E. coli Anti-Microbial Satellite EcAMSat Team

• PI: AC Matin, Stanford University • Stevan Spremo, PM

• Current Team: Matt Chin, Tori Chinn, Aaron Cohen, Charlie Friedericks, Mike Henschke, Chris Kitts, Mike Miller, Mike Padgen, Macarena Parra, Mario Perez, Mike Rasay, Tony Ricco, Tim Snyder

• Support from NASA’s Human Exploration and Operations Mission Directorate (HEOMD) as a Small Complete Mission of Opportunity in Fundamental Space Biology. For Astronaut Health During Long Term Spaceflight, Many Questions Remain

• Immediate need to understand antibiotic resistance in microgravity • Evidence that bacteria become more virulent and resistant than in normal gravity • Astronaut’s immune system is compromised in microgravity

• Model Organism: uropathogenic Escherichia coli (UPEC) • Urinary tract infections have been reported in astronauts • Gentamicin has been used to treat UTI • WT and ΔrpoS mutant strains • rpoS responsible for activating general stress response (GSR) • GSR increases antibiotic resistance Gentamicin Resistance Increases in Low-Shear Cellular Suspensions High-aspect-ratio vessels Gentamicin (16 µg/mL) (HARVs)

Low-shear

Control suspension % Survival

shear suspension shear

control

low

low control • Control: rotation about a vertical axis • Low-shear suspension: rotation about a wildtype ∆rpoS horizontal axis • HARVs used to simulate microgravity, although experimental results in actual microgravity can vary EcAMSat is a Re-flight of PharmaSat Hardware • EcAMSat will measure growth and metabolic activity of E. coli treated with antibiotic in microgravity compared to the ground control • PharmaSat measured growth and metabolic activity of yeast treated with antifungal in microgravity compared to ground control

• Yeast  E. coli • Filter pore size: 1.2 µm  0.2 µm • Higher pressure in fluidic system • Experiment temperature: 27°C  37°C • More power required • Storage: dry  wet EcAMSat is the First* NASA 6U Mission

• Overall size: 10 x 22 x 34 cm • Passive magnetic orientation • UHF Beacon and S-band radio • Extensive thermal power management testing and modeling

• Mission Ops run by Santa Clara University

* See slide 24 Experiment Design

Load cells Growth ~48 hr Challenge ~48 hr Measure ~48hr in lab Add Cells in Add Grow to Add Measure Alamar buffer media stationary antibiotic viability orbital Blue stabilzn.

Pre-launch Temp control ON: stasis Incubate at 37°C

Optics ON: Readings every 15 min

Data telemetry

6-8 weeks ~4 days ~150 hours time Note: A 24 hr delayed synchronous ground control will be run EcAMSat Fluidic Card

• WT and mutant strains loaded into alternating wells prior to sealing • Each bank is independent and will receive a different dose of antibiotic during the experiment Fluidic System Schematic Metering PC Board Pump LED Heater Layer SV SV Thermal Spreader & T Sensors Diaphragm Capping Layer Pump Gas Perm. Membrane Optical Quality/Clear Channel 1/6 LB 4 mm 0.2 µm Filter Nutrient Acrylic Bubble Outlet Trap 7.7 mm SV Inlet E. coli Acrylic Gm-dilution 0.2 µm Filter loop SV Channel Gentamicin Gas Perm. Membrane Optical Quality/Clear in M9 Capping Layer SV Main Thermal Spreader &T Sensors Waste Detector Heater Layer M9 Dilution Cross-SectionPC of Board Card Stack SV Bag Waste H SV High SV Waste M Alamar Medium Blue in M9 Check Valve Low SV Waste L SV Control

SV 48 Well Fluidic Card Waste C SV: Solenoid valve Assembled Payload

Fluidic Payload Bags and Valve Board Heaters

Hermetic Payload Can Bubble Trap

Pumps

Can is 2U or roughly 10x10x20 cm Experiment Verification on Ground

• Payload can rotated on rotisserie to prevent settling of cells • Experiment run through “flight like” commanding • Optics data calibrated and normalized • Growth of wild type and mutant are similar

1.4 0.20 Nutrient Antibiotic Alamar Blue 1.2 WT OD 1.0 Stationary Mut OD Phase ~24 hr 0.8 WT Pink

0.6 Mut Pink OD Mut Blue 0.4

0.2 WT Blue WT OD LB pumping starts 0.0 Mut OD 0.02 Rel. Conc. Alamar Rel. Conc. Alamar Blue; OD 0 10 20 30 40 50 0 50 100 150 Time, hr Time, hr Evaluating the Effect of Gentamicin on E. coli

• Clear differences between wild type and mutant in control • At each dose there was a significant difference in the ability of the cells to reduce alamarBlue relative to the control case

1.4 1.4 100% Control High Dose * WT 1.2 1.2 **** 80% 1.0 1.0 *** Mut Blue 0.8 WT Pink 0.8 60% 0.6 Mut Pink 0.6 WT Pink 40% Mut Blue WT Blue

0.4 0.4 Treated/Untreated

Mut Pink - AB Absorbance; AB OD AB Absorbance; AB OD 0.2 WT Blue 0.2 20% WT OD WT OD Gm 0.0 Mut OD 0.0 Mut OD 0 10 20 30 40 50 0 10 20 30 40 50 0% Time, hr Time, hr Low Medium High EcAMSat Summary

• EcAMSat leverages hardware from successful PharmaSat mission • Fluidic system provides accurate dilution of antibiotic and consistent delivery to the fluidics card housing the biology

• Uropathogenic E. coli model organism for studying changes in antibiotic resistance in microgravity • Extensive ground control tests already performed in addition to a 24 hr delayed synchronous ground control • Differences between WT and mutant strain missing key stress response gene, rpoS, can yield insight into mechanisms responsible for these changes • Help determine if therapies developed on ground will be successful in flight Where are we now?

• Original scheduled launch: July 2015 • Falcon 9 (SpaceX) FormoSat 5 + SHERPA • 6 launch delays • SHERPA de-manifested Jan 2017

• Current launch: Falcon 9 date TBD

BioSentinel Team

• Bob Hanel, PM; Dawn McIntosh, Deputy PM • Sharmila Bhattacharya, PI • BioSensor Payload Team: Chetan Angadi, Josh Benton, Rich Bielawski, Justin Blaich, Travis Boone, James Chartres, Michael Dougherty, Lance Ellingson, Charlie Friedericks, Diana Gentry, Sarah Hayes, Mike Henschke, Liz Hyde, Ben Klamm, Diana Marina, Eddy Mazmanian, Griffin McCutcheon, Mike Padgen, Macarena Parra, Mario Perez, Abe Rademacher, Tony Ricco, Sergio Santa Maria, Aaron Schooley, Ann-Sofie Schreurs, Ming Tan, Eric Tapio, Huyen Tran • NASA/Ames, NASA/JSC-Radworks, Loma Linda U. Med. Ctr., U. Saskatchewan • Support from NASA’s Advanced Exploration Systems and Human Exploration and Operations Mission Directorate BioSentinel Space Radiation BioSensor Experiment & Mission

• BioSentinel is a 6U free-flying satellite to be delivered by NASA’s Exploration Mission 1 (EM-1) to a heliocentric interplanetary orbit • First biology experiment beyond LEO since Apollo!

• Model organism, S. cerevisae (brewer’s yeast) will be used to measure effects of deep space radiation on DNA damage • Help to understand, quantify, and potentially mitigate key biological risk 100 225 330 385,000 km 385,000 Millions Millions million million - 435 km 435 100 km km km Distance km from Earth Moon Known • 6 Months L2 ISS 40 NEA million km 12Months BioSentinel its interplanetary radiation environment 12 Unknown 18 100 - to 18 Months million will operate in an BioSentinel - Mission Duration km month (deep mission. - space) space) over 3 Years Mars BioSentinel Science Mission: “Canary in a Coal Mine” • Quantify DNA damage from space radiation environment • Space environment cannot be reproduced on Earth: omnidirectional, continuous, low flux, variety of particle types • Health risk for humans spending long durations beyond LEO • Radiation flux can spike 1000x during a solar particle event (SPE) • Correlate biological response with physical radiation measurements • Linear Energy Transfer (LET) spectrometer bins and counts particle events by LET • Total Ionizing Dose (TID) sensor measures integrated deposited energy

30 BioSentinel Science Mission: “Canary in a Coal Mine” • Yeast assay: microfluidic arrays monitor DSB/repair • Three strains of S. cerevisae: 1 wild type, 1 radiation sensitve mutant, and 1 engineered strain • engineered strain quantifies double strand breaks (DSBs) • Wet and activate multiple banks of microwells over mission lifetime • Double strand break & associated repair enable cell growth & division • Reserve wells activated autonomously in case of SPE

31 BioSensor DSB Detection Mechanism

BioSentinel: a Biosensor in Space • What: BioSentinel is a yeast radiation biosensor that will measure the DNA damage caused by space radiation, specifically double strand

breaks (DSBs). IR Ionizing Homologous radiation (IR) Non-homologous recombination (HR) DNA end joining (NHEJ) HR DSB NHEJ

MutagenicEnd joini nrepairg

ErrorErro-r-ffreeree repairrepair In yeast diploid cells, NHEJ is inhibited. Thus, HR is the main repair pathway for DSBs One of our yeast strains is engineered in such a way that only the repaired cells will grow actively in the media provided and will be detected by optical density (OD) & colorimetric S. cerevisiae (budding yeast) measurements Low-Gain Antenna Spacecraft Configuration

Command & Data Propulsion Handling/Power/Transponder System Stack (CF3)2CH2 Batteries

Integrated Guidance Solar Arrays Navigation & Control Solar Array Gimbal Unit Star Tracker Cutout

Medium- Gain Radiation Sensors: Bio Payload Container (4U, 4.5 kg) Antenna TID + LET spectrometer Experiment Design Repeat Load in AR, AR, lab Lunar 1 hr 2 weeks 1 hr 2 weeks Fly-By weeks weeks Desiccated Fill First 2 Fill Next 2 Measure Optics Stasis Measure Optics Stasis Yeast System Cards Cards Check- out Temp control ON: Temp control ON: 6 mo pre- launch stasis Active Card at 23°C Active Card at 23°C All bags and non-active cards: Keep Alive at 5°C

Optics ON: Readings Optics ON: Readings every 2 min every 2 min

Radiation measurements

Data telemetry 6 mo 2 weeks 6-18 mo time

Note: A delayed synchronous ground control will be run Bio-fluidic-opto-thermal Configuration thermal link (gasket) optical PCB/source heater layer 16-well card fluidic card = 1 “set” thermal spreader (18 sets total) optical PCB/detection Bio-Fluidic-Opto-Thermal Cross Section Yeast growth with flight-like prototype fluidic card + optics

Pink

RTD sensor (embedded) T

• Yeast dried onto µwell walls Blue • 3 LEDs + detector, per well, track growth via optical density and cell metabolic activity via dye OD color changes. • LEDs: 570, 630, 850 nm BioSentinel Payload: Design/Function Highlights

KEY • Two card manifolds Fluidic and two bag manifolds card • Each card controlled 3-way independently and has valve its own desiccant FEP tubing Internal channels Check valve Desiccant trap Polishing bubble trap Calibration cell Hermetic feed-thru Desiccant

carbon cloth

37 SPE Autonomous Trigger for BioSensor • Onboard TID monitor • Ionizing EM radiation (gamma) precedes particles by ~ hours • SPE differentiable from smaller coronal mass ejections • Activate “designated SPE set” of fluidic wells • measure radiation biological damage under wet conditions • expected to generate more damage wet than in dry state (OH•) Radiation Sensitivity of Yeast Cells in Fluidics Cards

• Gamma, protons, iron, oxygen, silicon radiation sources have been used to quantify effects • 2-year old air-dried cells are responsive to high-energy particle irradiation at 250 cGy (expected dose in a 12-month mission).

300 MeV Fe-56 dry IR 300 MeV Fe-56 dry IR wt diploid rad51 diploid 100 100

80 80

60 60

40 40

20 20 % reduced % alamarBlue % reduced % alamarBlue 0 0 0 3 6 9 121518212427303336394245485154576063 0 3 6 9 121518212427303336394245485154576063 Hour Hour

0 cGy-dry 250 cGy-dry 0 cGy-dry 250 cGy-dry Summary of BioSentinel

• BioSentinel will be the first biology experiment outside of LEO since Apollo • Heliocentric orbit will provide a deep space radiation environment for a 12 month mission • S. cerevisae as a model organism will measure the effect of deep space radiation on DNA damage • Help to understand, quantify, and potentially mitigate key biological risk • Demonstration of a deep space biosensor • Potential follow-on missions: Mars Sentinel, radiation shielding Where are We Now?

• Building engineering development units for qualification and testing • Long-term biocompatibility tests in progress • Radiation testing, including SPE simulation

• Launch for SLS EM-1: Sept 30, 2018

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