The objective of this study is to ascertain how evolutionary processes in bacteria change in response to the spaceflight environment, and specifically to microgravity. We propose to use growth rate as a proxy for fitness, and to ‘race’ a non-motile mutant of Bacillus subtilis along a membrane wetted with growth media and bounded by impassable printed wax barriers. As cells grow into the fresh media, they will create a front of newly divided cells. These ‘racetracks’ will be imaged as the cells propagate, and we will be able to observe changes in growth rate over time for treatments in microgravity, 1-g onboard the International Space Station (ISS), and 1-g on the ground. Deep-sequencing of winning lines will identify what genetic changes occurred with respect to the ancestral cells. Following a successful Compliance Review in April, 2018, to allow transition into the Techshot Multi-use Variable-g Platform (MVP), the Experimental Requirements Document (ERD) Review was completed on October 11, 2018, and the Science Verification Tests (SVT) began in earnest. These tasks included development of a spore protocol, selecting mutant lines, DNA types, and finalizing media composition. Upon receipt of a 3D-printed mock-up MVP module and sufficient cell cassettes from Techshot in winter 2018, we were able to perform growth runs in approximately flight-like conditions, and able to further close out tasks, including confirming growth and biocompatibility in flight-like hardware, proper materials selection (e.g., capillary mat under the PES (polyethersulfone) membrane, switch to red ink for improved hydrophobicity). We finalized sterilization and assembly protocols, and were able to do imaging from within the mock MVP module as part of a flight-like growth run.
Subsequent to the conclusion of the SVT tasks May 1, 2019, the Experimental Verification Test (EVT) Readiness Review was successfully completed on May 9, and EVT began on May 15, 2019. For EVT, a full mock-up in 6 flight-like MVP modules (akin to the ground control) was performed. 42 cell cassettes were assembled and loaded with spores and media (7 per module) in a semi-randomized nature, with the full experimental matrix (2 mutants, three media types, 7 replicates per treatment). A 27-day growth experiment was initiated with each camera (2 per module) taking images every two hours, at three light intensities for the duration. In all, almost 12,000 high-resolution images of the bacterial tracks were taken as they propagated. At the completion of the EVT growth experiment, all 42 cassettes were frozen at -80°C to replicate on-station storage. After two days, 18 cassettes (n=3 for each treatment) were thawed, and used for bacterial isolation efforts and DNA extractions. All isolations were successful, and all DNA extractions produced sufficient mass and quality of DNA for downstream sequencing analysis. EVT concluded on June 12, 2019, with all acceptable success criteria, as outlined in the ERD, being met or exceeded. The Flight Readiness Review is currently scheduled for June 24, 2019. If the review is successful, this experiment is anticipated to launch aboard SpaceX-18 (no earlier than July 21, 2019) with sample return to occur no earlier than SpaceX-19 reentry (January 2020).
The overall experimental framework and results from our science validation tests were presented as an oral presentation at the Joint CSA/ESA/JAXA/NASA (Canadian Space Agency/European Space Agency/Japan Aerospace Exploration Agency/NASA) Increments 59 and 60 Science Symposium in February, 2019.
Abstracts for Journals and Proceedings
Everroad RC. "Long-term multi-generational evolutionary studies of bacteria in the spaceflight environment (MVP-Cell-02).
" Talk presented at the Joint CSA/ESA/JAXA/NASA Increments 59 and 60 Science Symposium, Houston, Texas, USA, February 2019. (remote participation)
Joint CSA/ESA/JAXA/NASA Increments 59 and 60 Science Symposium, Houston, Texas, February 2019. , Feb-2019