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.
As previously reported, the Experimental Verification Test (EVT) Readiness Review was successfully completed on May 9, 2019. EVT began on May 15, 2019 and was completed on June 12, 2019, with all acceptable success criteria being met or exceeded. The Flight Readiness Review (FRR) was successfully presented and approved at NASA Ames Research Center (ARC) on June 24, 2019. Members of the Science team first arrived at Kennedy Space Center (KSC), Merritt Island, FL, on July 12, 2019 to assemble the flight and ground control experiments. All of the work was performed in the Space Station Processing Facility (SSPF) at KSC. The first 3 days involved laboratory set-up, safety training, inventory and assembly of the 84 flight cassettes (2 strains, 3 media types, 7 replicates per treatment, 2 gravity treatments (microgravity, 1-g)) for the flight experiment. On day 4 the different media types were prepared with the associated foreign DNA (high complexity, low complexity, DNA-free), loaded into syringes, and integrated into the 84 flight cassettes (Figure 1). On day 5, 42 cassettes for the ground control experiment were assembled and the different media types were loaded into syringes and integrated to the cassettes, as per the flight experiment set-up. QA tests were performed on all of the cassettes, before and after integration of the media. Serial numbers and other metadata associated with cassette assembly and media loading operations were recorded. Loaded cassettes were placed in the 25°C incubator and checked at 24, 48, and 62 hours to assess potential contamination of the media. No contamination was observed on any of the flight or ground control media. Flight and control cassettes were delivered to payload developer (PD) for integration on July 17 and 20, respectively. Handover of the integrated flight hardware for flight occurred on July 18, 2019.
The flight experiment launched aboard SpaceX CRS-18 on July 25, 2019 and was activated onboard the ISS on August 27, 2019. The experiment ended on September 15 after 19 days of growth, and the samples were moved into cold stowage at 4°C. ISS crew imaged each cassette as they were transferred to cold stowage. Samples were subsequently transferred to -80°C on September 24, 2019. The ground control experiment was activated by the PD on August 30, 2019 and the ground control experiment ended on September 24, 2019.
No contamination occurred in the flight or ground control samples, and all but 8 of the 126 replicates germinated (94%). Some challenges occurred during the experiment, causing loss of some replicates as well as imaging data. However, a sample size of at least n=3 was preserved for all treatments. Flight samples returned to Earth on SpaceX CRS-20 on April 7, 2020. Ground control samples are currently stored at -80°C by the PD, and flight samples are currently stored at -80°C at NASA Johnson Space Center (JSC), awaiting return to Ames Research Center (ARC) for isolation and genomics work to proceed. Preliminary imaging results from the ground control experiment indicate by-strain differences in growth, as well as a treatment effect from the different DNA types in the growth media.
The overall experimental framework and preliminary results from our flight and ground experiments were presented as an oral presentation at the American Society for Gravitational and Space Research 35th Annual meeting, Denver, CO, USA, Nov. 20-23, 2019.
Abstracts for Journals and Proceedings
Everroad RC, Bebout B, Chang C, Detweiler AM, Harshfield N, Karouia F, Koehne J, Kost D, Logan S, Martin KR, Ricco AJ, Thomas N. "Experimental evolution of Bacillus subtilis 168 in the spaceflight environment." Oral presentation presented at the 35th Annual Meeting of the American Society for Gravitational and Space Research, Denver, CO, November 20-23, 2019.
Abstracts. 35th Annual Meeting of the American Society for Gravitational and Space Research, Denver, CO, November 20-23, 2019. , Nov-2019