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Project Title:  Experimental Evolution of Bacillus subtilis Populations in Space; Mutation, Selection and Population Dynamics Reduce
Images: icon  Fiscal Year: FY 2019 
Division: Space Biology 
Research Discipline/Element:
Cell & Molecular Biology | Microbiology 
Start Date: 07/01/2015  
End Date: 09/30/2020  
Task Last Updated: 06/20/2019 
Download report in PDF pdf
Principal Investigator/Affiliation:   Everroad, Craig  Ph.D. / NASA Ames Research Center 
Address:  Exobiology Branch 
Mail Stop 239-4; Bldg 239/ Room 367 
Moffett Field , CA 94035-0001 
Email: craig.everroad@nasa.gov 
Phone: 650-604-4997  
Congressional District: 18 
Web:  
Organization Type: NASA CENTER 
Organization Name: NASA Ames Research Center 
Joint Agency:  
Comments: NOTE: PI previously at Bay Area Environmental Research Institute until 2018 
Co-Investigator(s)
Affiliation: 
Bebout, Brad  Ph.D. NASA Ames Research Center 
Koehne, Jessica  Ph.D. NASA Ames Research Center 
Ricco, Antonio  Ph.D. NASA Ames Research Center 
Bergstrom, Robert  Ph.D. CoPI: Bay Area Environmental Research Institute, grant NNX15AM68A 
Key Personnel Changes / Previous PI: Ed. Note 8/8/18: Principal Investigator (PI) Craig Everroad is now civil servant at NASA Ames and Robert Bergstrom, Ph.D., Bay Area Environmental Research Institute (BAERI), is CoPI at the BAERI for grant number NNX15AM68A.
Project Information: Grant/Contract No. Internal Project ; NNX15AM68A 
Responsible Center: NASA ARC 
Grant Monitor: Sato, Kevin  
Center Contact: 650-604-1104 
kevin.y.sato@nasa.gov 
Solicitation: 2014 Space Biology Flight NNH14ZTT001N 
Grant/Contract No.: Internal Project ; NNX15AM68A 
Project Type: FLIGHT 
Flight Program: ISS 
No. of Post Docs:
No. of PhD Candidates:
No. of Master's Candidates:
No. of Bachelor's Candidates:
No. of PhD Degrees:
No. of Master's Degrees:
No. of Bachelor's Degrees:
Space Biology Element: (1) Cell & Molecular Biology
(2) Microbiology
Space Biology Cross-Element Discipline: (1) Reproductive Biology
Space Biology Special Category: None
Flight Assignment/Project Notes: NOTE: Extended to 9/30/2020 per F. Hernandez/ARC (Ed., 7/23/19)

NOTE: Extended to 9/30/2019 per F. Hernandez/ARC (Ed., 4/2/19)

NOTE: Extended to 6/30/2019 per F. Hernandez/ARC and NSSC information (Ed., 8/8/18)

NOTE: Period of performance changed to 7/01/2015-6/30/2018 per NSSC (Ed., 9/14/16)

NOTE: End date change to 6/30/2018 per A. Chu/ARC and NSSC; start date to remain at 11/1/2014 per A. Chu/ARC (Ed., 9/23/15)

Task Description: The proposed research aims to understand the effects of the space environment on evolutionary processes in the bacterium Bacillus subtilis. Different mutant lines will be ‘raced’ along solid surfaces to allow continuous selection in the cultures and to maximize the number of generations possible. Deep sequencing of winners will identify evolutionary rates, mechanisms, and targets of selection. We propose printing wax barriers to make paths along a growth surface (agar, membranes) and spotting each starting position of each path with dormant spores of the experimental bacteria to ‘race’ different mutants. Once on orbit, the material is wetted with growth medium, allowing the individual spots of B. subtilis to grow along their determined paths. This approach provides an opportunity for exponential growth only along the propagating edges, generating continuous bottlenecking thus amplifying selective pressures on the experimental populations. By monitoring the respective growth rate of different mutant lines maintained in each of these experimental conditions, we can estimate relative fitness of the lines. Long-term changes in relative growth rate indicate adaptation. Deep-sequencing of DNA from adapted cells (‘winners’ at the end of runs) will identify genetic changes within the respective populations. We expect that rates of mutation will differ between microgravity, 1-g, and ground controls, and that the targets of these mutations will differ as the different populations of bacteria adapt to their respective conditions. This research will also utilize the native ability of B. subtilis to uptake foreign DNA. Information-rich environmental DNA is added into the growth medium, and the populations are raced as above. By sampling the winners, and identifying if/what foreign genes are assimilated in each treatment, this experiment will identify potential genes of interest for future studies of genetic adaptation to the space environment. Our approach maximizes the number of generations possible in the 60-day window for this call, and maximizes the potential for evolutionary processes to occur. By performing multi-generational experimental evolution on bacteria on the International Space Station (ISS), the work proposed here aims to advance understanding of the evolutionary processes and challenges facing biological systems in long-term space exploration and habitation.

Research Impact/Earth Benefits: Improved understanding of the evolutionary process and in the dynamics of adaptive evolution in a model bacterium.

Task Progress & Bibliography Information FY2019 
Task Progress: 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.

Bibliography Type: Description: (Last Updated: 06/20/2019)  Show Cumulative Bibliography Listing
 
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

Project Title:  Experimental Evolution of Bacillus subtilis Populations in Space; Mutation, Selection and Population Dynamics Reduce
Images: icon  Fiscal Year: FY 2018 
Division: Space Biology 
Research Discipline/Element:
Cell & Molecular Biology | Microbiology 
Start Date: 07/01/2015  
End Date: 09/30/2019  
Task Last Updated: 05/03/2018 
Download report in PDF pdf
Principal Investigator/Affiliation:   Everroad, Craig  Ph.D. / NASA Ames Research Center 
Address:  Exobiology Branch 
Mail Stop 239-4; Bldg 239/ Room 367 
Moffett Field , CA 94035-0001 
Email: craig.everroad@nasa.gov 
Phone: 650-604-4997  
Congressional District: 18 
Web:  
Organization Type: NASA CENTER 
Organization Name: NASA Ames Research Center 
Joint Agency:  
Comments: NOTE: PI previously at Bay Area Environmental Research Institute until 2018 
Co-Investigator(s)
Affiliation: 
Bebout, Brad  Ph.D. NASA Ames Research Center 
Koehne, Jessica  Ph.D. NASA Ames Research Center 
Ricco, Antonio  Ph.D. NASA Ames Research Center 
Bergstrom, Robert  Ph.D. CoPI: Bay Area Environmental Research Institute, grant NNX15AM68A 
Key Personnel Changes / Previous PI: Ed. Note 8/8/18: PI Craig Everroad is now civil servant at NASA Ames and Robert Bergstrom, Ph.D., Bay Area Environmental Research Institute (BAERI), is CoPI at the BAERI for grant number NNX15AM68A.
Project Information: Grant/Contract No. Internal Project ; NNX15AM68A 
Responsible Center: NASA ARC 
Grant Monitor: Sato, Kevin  
Center Contact: 650-604-1104 
kevin.y.sato@nasa.gov 
Solicitation: 2014 Space Biology Flight NNH14ZTT001N 
Grant/Contract No.: Internal Project ; NNX15AM68A 
Project Type: FLIGHT 
Flight Program: ISS 
No. of Post Docs:
No. of PhD Candidates:
No. of Master's Candidates:
No. of Bachelor's Candidates:
No. of PhD Degrees:
No. of Master's Degrees:
No. of Bachelor's Degrees:
Space Biology Element: (1) Cell & Molecular Biology
(2) Microbiology
Space Biology Cross-Element Discipline: (1) Reproductive Biology
Space Biology Special Category: None
Flight Assignment/Project Notes: NOTE: Extended to 9/30/2019 per F. Hernandez/ARC (Ed., 4/2/19)

NOTE: Extended to 6/30/2019 per F. Hernandez/ARC and NSSC information (Ed., 8/8/18)

NOTE: Period of performance changed to 7/01/2015-6/30/2018 per NSSC (Ed., 9/14/16)

NOTE: End date change to 6/30/2018 per A. Chu/ARC and NSSC; start date to remain at 11/1/2014 per A. Chu/ARC (Ed., 9/23/15)

Task Description: The proposed research aims to understand the effects of the space environment on evolutionary processes in the bacterium Bacillus subtilis. Different mutant lines will be ‘raced’ along solid surfaces to allow continuous selection in the cultures and to maximize the number of generations possible. Deep sequencing of winners will identify evolutionary rates, mechanisms, and targets of selection. We propose printing wax barriers to make paths along a growth surface (agar, membranes) and spotting each starting position of each path with dormant spores of the experimental bacteria to ‘race’ different mutants. Once on orbit, the material is wetted with growth medium, allowing the individual spots of B. subtilis to grow along their determined paths. This approach provides an opportunity for exponential growth only along the propagating edges, generating continuous bottlenecking thus amplifying selective pressures on the experimental populations. By monitoring the respective growth rate of different mutant lines maintained in each of these experimental conditions, we can estimate relative fitness of the lines. Long-term changes in relative growth rate indicate adaptation. Deep-sequencing of DNA from adapted cells (‘winners’ at the end of runs) will identify genetic changes within the respective populations. We expect that rates of mutation will differ between microgravity, 1-g, and ground controls, and that the targets of these mutations will differ as the different populations of bacteria adapt to their respective conditions. This research will also utilize the native ability of B. subtilis to uptake foreign DNA. Information-rich environmental DNA is added into the growth medium, and the populations are raced as above. By sampling the winners, and identifying if/what foreign genes are assimilated in each treatment, this experiment will identify potential genes of interest for future studies of genetic adaptation to the space environment. Our approach maximizes the number of generations possible in the 60-day window for this call, and maximizes the potential for evolutionary processes to occur. By performing multi-generational experimental evolution on bacteria on the International Space Station (ISS), the work proposed here aims to advance understanding of the evolutionary processes and challenges facing biological systems in long-term space exploration and habitation.

Research Impact/Earth Benefits: Improved understanding of the evolutionary process and in the dynamics of adaptive evolution in a model bacterium.

Task Progress & Bibliography Information FY2018 
Task Progress: 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. This year’s progress has been minimal due to limited budgets and the unavailability of the flight hardware of the European Modular Cultivation System (EMCS) onboard the ISS. Efforts have focused on transitioning the experiment into new hardware using the Techshot Multi-use Variable-g Platform (MVP). These include several discussions and planning sessions with Techshot representatives and the Space Biology Project Science team as new modules are designed for the MVP. A draft experimental requirements document (ERD) was provided to Techshot, and a Space Biology Compliance Review was conducted in April, 2018.

The overall experimental framework and results from our science validation tests were presented as an oral presentation at the 33rd American Society for Gravitational and Space Research (ASGSR) meeting held in Renton, Washington in October, 2017.

Bibliography Type: Description: (Last Updated: 06/20/2019)  Show Cumulative Bibliography Listing
 
Abstracts for Journals and Proceedings Everroad RC, Detweiler AM, Koehne J, Ricco A. "Long-term multi-generational evolutionary studies of bacteria in the spaceflight environment." Talk given at the 33rd Annual Meeting of the American Society for Gravitational and Space Research, Seattle, WA, October 25-28, 2017.

33rd Annual Meeting of the American Society for Gravitational and Space Research, Seattle, WA, October 25-28, 2017. , Oct-2017

Project Title:  Experimental Evolution of Bacillus subtilis Populations in Space; Mutation, Selection and Population Dynamics Reduce
Images: icon  Fiscal Year: FY 2017 
Division: Space Biology 
Research Discipline/Element:
Cell & Molecular Biology | Microbiology 
Start Date: 07/01/2015  
End Date: 06/30/2018  
Task Last Updated: 05/02/2017 
Download report in PDF pdf
Principal Investigator/Affiliation:   Everroad, Craig  Ph.D. / NASA Ames Research Center 
Address:  Exobiology Branch 
Mail Stop 239-4; Bldg 239/ Room 367 
Moffett Field , CA 94035-0001 
Email: craig.everroad@nasa.gov 
Phone: 650-604-4997  
Congressional District: 18 
Web:  
Organization Type: NASA CENTER 
Organization Name: NASA Ames Research Center 
Joint Agency:  
Comments: NOTE: PI previously at Bay Area Environmental Research Institute until 2018 
Co-Investigator(s)
Affiliation: 
Bebout, Brad  Ph.D. NASA Ames Research Center 
Koehne, Jessica  Ph.D. NASA Ames Research Center 
Ricco, Antonio  Ph.D. NASA Ames Research Center 
Bergstrom, Robert  Ph.D. CoPI: Bay Area Environmental Research Institute, grant NNX15AM68A 
Key Personnel Changes / Previous PI: Ed. Note 8/8/18: PI Craig Everroad is now civil servant at NASA Ames and Robert Bergstrom, Ph.D., Bay Area Environmental Research Institute (BAERI), is CoPI at the BAERI for grant number NNX15AM68A
Project Information: Grant/Contract No. Internal Project ; NNX15AM68A 
Responsible Center: NASA ARC 
Grant Monitor: Sato, Kevin  
Center Contact: 650-604-1104 
kevin.y.sato@nasa.gov 
Solicitation: 2014 Space Biology Flight NNH14ZTT001N 
Grant/Contract No.: Internal Project ; NNX15AM68A 
Project Type: FLIGHT 
Flight Program: ISS 
No. of Post Docs:
No. of PhD Candidates:
No. of Master's Candidates:
No. of Bachelor's Candidates:
No. of PhD Degrees:
No. of Master's Degrees:
No. of Bachelor's Degrees:
Space Biology Element: (1) Cell & Molecular Biology
(2) Microbiology
Space Biology Cross-Element Discipline: (1) Reproductive Biology
Space Biology Special Category: None
Flight Assignment/Project Notes: NOTE: Period of performance changed to 7/01/2015-6/30/2018 per NSSC (Ed., 9/14/16)

NOTE: End date change to 6/30/2018 per A. Chu/ARC and NSSC; start date to remain at 11/1/2014 per A. Chu/ARC (Ed., 9/23/15)

Task Description: The proposed research aims to understand the effects of the space environment on evolutionary processes in the bacterium Bacillus subtilis. Different mutant lines will be ‘raced’ along solid surfaces to allow continuous selection in the cultures and to maximize the number of generations possible. Deep sequencing of winners will identify evolutionary rates, mechanisms, and targets of selection. We propose printing wax barriers to make paths along a growth surface (agar, membranes) and spotting each starting position of each path with dormant spores of the experimental bacteria to ‘race’ different mutants. Once on orbit, the material is wetted with growth medium, allowing the individual spots of B. subtilis to grow along their determined paths. This approach provides an opportunity for exponential growth only along the propagating edges, generating continuous bottlenecking thus amplifying selective pressures on the experimental populations. By monitoring the respective growth rate of different mutant lines maintained in each of these experimental conditions, we can estimate relative fitness of the lines. Long-term changes in relative growth rate indicate adaptation. Deep-sequencing of DNA from adapted cells (‘winners’ at the end of runs) will identify genetic changes within the respective populations. We expect that rates of mutation will differ between microgravity, 1-g, and ground controls, and that the targets of these mutations will differ as the different populations of bacteria adapt to their respective conditions. This research will also utilize the native ability of B. subtilis to uptake foreign DNA. Information-rich environmental DNA is added into the growth medium, and the populations are raced as above. By sampling the winners, and identifying if/what foreign genes are assimilated in each treatment, this experiment will identify potential genes of interest for future studies of genetic adaptation to the space environment. Our approach maximizes the number of generations possible in the 60-day window for this call, and maximizes the potential for evolutionary processes to occur. By performing multi-generational experimental evolution on bacteria on the International Space Station (ISS), the work proposed here aims to advance understanding of the evolutionary processes and challenges facing biological systems in long-term space exploration and habitation.

Research Impact/Earth Benefits: Improved understanding of the evolutionary process and in the dynamics of adaptive evolution in a model bacterium.

Task Progress & Bibliography Information FY2017 
Task Progress: 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. This year’s progress has been primarily related to advancing and refining experimental conditions and protocols to sustain long-term growth, and to adapt our experimental system to the flight hardware of the European Modular Cultivation System (EMCS) onboard the ISS, including into five non-flight seed cassettes provided to the science team.

For the current reporting period, our primary objectives have been to finalize appropriate experimental procedures with respect to conditions onboard ISS, including growth under elevated CO2 , testing for background DNA contamination, maintaining sterility in the context of EMCS assembly, growth under EMCS-like conditions including long-term growth of 20-days or more, and optimization of media and track design. Experiments underway include final track design tests, and DNA-uptake tests.

Printing and Sterilization –We have advanced our printing protocols to extend bacterial growth, based on the ‘dumbbell pattern’ previously reported, by narrowing the path for growth, removing the starting circle for growth, and adding a grid pattern to the wax to assist with imaging analysis. Some final experiments are underway to optimize growth duration and propagation speed.

One of the challenges of adapting the seed cassettes to heterotrophic bacteria and complex media is the risk of contamination and a need for sterilization of the hardware pre-flight (excepting the bacterial spores). We have solved this challenge with an ultraviolet light (UV) protocol, with multiple, extended exposures, which has resulted in minimal to no contamination. In combination with autoclave-sterilization of the seed cassette base and inserts, we developed reliable assembly procedures with sterilized hardware (excluding the seed cassette cover, which must be rendered aseptic-only).

Growth and Propagation –We have subsequently determined growth under elevated ambient CO2 conditions, as found onboard ISS. CO2 appeared to have no effect on bacterial growth. We have also solved the challenge of extensive growth on very small volumes of medium by designing a highly rich, buffered medium with added nutrients to extend growth and induce catabolite repression.

Stasis – We have demonstrated growth from spores after long-term storage (dried 10 months), using SBM medium dried for six months, under EMCS-like conditions. Dried medium/spore combinations, when rewetted with sterile, distilled water, after extensive periods of time in an inert/dry state, began growth in less than 24-hours post-wetting. We are now working to optimize the speed and distances capable for a given volume of medium, with growth currently continuing along the tracks for 20+ days.

Bibliography Type: Description: (Last Updated: 06/20/2019)  Show Cumulative Bibliography Listing
 
 None in FY 2017
Project Title:  Experimental Evolution of Bacillus subtilis Populations in Space; Mutation, Selection and Population Dynamics Reduce
Images: icon  Fiscal Year: FY 2016 
Division: Space Biology 
Research Discipline/Element:
Cell & Molecular Biology | Microbiology 
Start Date: 07/01/2015  
End Date: 06/30/2018  
Task Last Updated: 09/02/2015 
Download report in PDF pdf
Principal Investigator/Affiliation:   Everroad, Craig  Ph.D. / NASA Ames Research Center 
Address:  Exobiology Branch 
Mail Stop 239-4; Bldg 239/ Room 367 
Moffett Field , CA 94035-0001 
Email: craig.everroad@nasa.gov 
Phone: 650-604-4997  
Congressional District: 18 
Web:  
Organization Type: NASA CENTER 
Organization Name: NASA Ames Research Center 
Joint Agency:  
Comments: NOTE: PI previously at Bay Area Environmental Research Institute until 2018 
Co-Investigator(s)
Affiliation: 
Bebout, Brad  Ph.D. NASA Ames Research Center 
Koehne, Jessica  Ph.D. NASA Ames Research Center 
Ricco, Antonio  Ph.D. NASA Ames Research Center 
Key Personnel Changes / Previous PI: None
Project Information: Grant/Contract No. NNX15AM68A 
Responsible Center: NASA ARC 
Grant Monitor: Sato, Kevin  
Center Contact: 650-604-1104 
kevin.y.sato@nasa.gov 
Solicitation: 2014 Space Biology Flight NNH14ZTT001N 
Grant/Contract No.: NNX15AM68A 
Project Type: FLIGHT 
Flight Program: ISS 
No. of Post Docs:
No. of PhD Candidates:
No. of Master's Candidates:
No. of Bachelor's Candidates:
No. of PhD Degrees:
No. of Master's Degrees:
No. of Bachelor's Degrees:
Space Biology Element: (1) Cell & Molecular Biology
(2) Microbiology
Space Biology Cross-Element Discipline: (1) Reproductive Biology
Space Biology Special Category: None
Flight Assignment/Project Notes: NOTE: Period of performance changed to 7/01/2015-6/30/2018 per NSSC (Ed., 9/14/16)

NOTE: End date change to 6/30/2018 per A. Chu/ARC and NSSC; start date to remain at 11/1/2014 per A. Chu/ARC (Ed., 9/23/15)

Task Description: The proposed research aims to understand the effects of the space environment on evolutionary processes in the bacterium Bacillus subtilis. Different mutant lines will be ‘raced’ along solid surfaces to allow continuous selection in the cultures and to maximize the number of generations possible. Deep sequencing of winners will identify evolutionary rates, mechanisms, and targets of selection. We propose printing wax barriers to make paths along a growth surface (agar, membranes) and spotting each starting position of each path with dormant spores of the experimental bacteria to ‘race’ different mutants. Once on orbit, the material is wetted with growth medium, allowing the individual spots of B. subtilis to grow along their determined paths. This approach provides an opportunity for exponential growth only along the propagating edges, generating continuous bottlenecking thus amplifying selective pressures on the experimental populations. By monitoring the respective growth rate of different mutant lines maintained in each of these experimental conditions, we can estimate relative fitness of the lines. Long-term changes in relative growth rate indicate adaptation. Deep-sequencing of DNA from adapted cells (‘winners’ at the end of runs) will identify genetic changes within the respective populations. We expect that rates of mutation will differ between microgravity, 1-g, and ground controls, and that the targets of these mutations will differ as the different populations of bacteria adapt to their respective conditions. This research will also utilize the native ability of B. subtilis to uptake foreign DNA. Information-rich environmental DNA is added into the growth medium, and the populations are raced as above. By sampling the winners, and identifying if/what foreign genes are assimilated in each treatment, this experiment will identify potential genes of interest for future studies of genetic adaptation to the space environment. Our approach maximizes the number of generations possible in the 60-day window for this call, and maximizes the potential for evolutionary processes to occur. By performing multi-generational experimental evolution on bacteria on the International Space Station (ISS), the work proposed here aims to advance understanding of the evolutionary processes and challenges facing biological systems in long-term space exploration and habitation.

Research Impact/Earth Benefits: Improved understanding of the evolutionary process and in the dynamics of adaptive evolution in a model bacterium.

Task Progress & Bibliography Information FY2016 
Task Progress: The objective of this study is to ascertain how evolutionary processes in bacteria change in response to the spaceflight environment and 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 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 on-board 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. This year’s progress has been primarily related to defining experimental conditions and protocols to be used for our proposed flight experiment using the European Modular Cultivation System (EMCS) hardware on-board the ISS.

Our primary objectives thus far in this science flight definition phase have been to select appropriate experimental mutants of Bacillus subtilis strain 168, determine biocompatibility and types of growth media, growth surfaces, and materials/patterns to be used for track printing. Efforts are underway towards developing suitable sterilization protocols of growth surfaces post-printing. We have begun work to determine growth rates, and establish reliable ‘wake-up’ of Bacillus spores, and propagation along printed paths. We have also worked to develop methods for the image analysis under EMCS-like conditions.

Printing and Sterilization – Preliminary printing trials revealed no problems with either chromatography paper, or the polyethersulfone (PES) membranes similar to the materials used in the EMCS seed cassette containers. We have tested several track patterns, and are currently optimizing track width. Post printing sterilization has also been demonstrated successfully with ethanol washes of the printed filters. Ethanol causes minimal distortion to the wax, or membrane materials, and currently is superior to other approached such as autoclaving, which deform/melt the materials.

Growth and Propagation – Biocompatibility tests have shown that Bacillus appears to grow well on the PES membranes, as well as on chromatography paper, and glass fiber filter paper, although the rates do vary between materials. There does not seem to be any inhibition of growth due to the wax, though tests continue to determine this. Initial media tests are underway, with as are procedures for sporulation / wake-up protocols for preparing inert cells for spaceflight. Growth rate experiments are underway for our preliminary mutant lines to determine optimal growth temperature, as well as effects of materials on overall growth rate. We are also working to optimize track widths, media concentrations and experimental conditions for consistent propagation at reliable speeds.

Imaging – We have developed methods for correlating biomass with image data as can be collected with the EMCS hardware. Using open source software for pixel analysis, we have successfully demonstrated that correlations between image pixel value, and total biomass, can be made. It is our hope that such data will be valuable, in conjunction with growth speed along the printed paths, for determining number of generations and growth rate.

Bibliography Type: Description: (Last Updated: 06/20/2019)  Show Cumulative Bibliography Listing
 
 None in FY 2016
Project Title:  Experimental Evolution of Bacillus subtilis Populations in Space; Mutation, Selection and Population Dynamics Reduce
Images: icon  Fiscal Year: FY 2015 
Division: Space Biology 
Research Discipline/Element:
Cell & Molecular Biology | Microbiology 
Start Date: 11/01/2014  
End Date: 10/31/2017  
Task Last Updated: 12/17/2014 
Download report in PDF pdf
Principal Investigator/Affiliation:   Everroad, Craig  Ph.D. / NASA Ames Research Center 
Address:  Exobiology Branch 
Mail Stop 239-4; Bldg 239/ Room 367 
Moffett Field , CA 94035-0001 
Email: craig.everroad@nasa.gov 
Phone: 650-604-4997  
Congressional District: 18 
Web:  
Organization Type: NASA CENTER 
Organization Name: NASA Ames Research Center 
Joint Agency:  
Comments: NOTE: PI previously at Bay Area Environmental Research Institute until 2018 
Co-Investigator(s)
Affiliation: 
Bebout, Brad  Ph.D. NASA Ames Research Center 
Koehne, Jessica  Ph.D. NASA Ames Research Center 
Ricco, Antonio  Ph.D. NASA Ames Research Center 
Project Information: Grant/Contract No. Internal Project 
Responsible Center: NASA ARC 
Grant Monitor: Smith, Jeffrey  
Center Contact: 650-604-0880 
jeffrey.d.smith2@nasa.gov 
Solicitation: 2014 Space Biology Flight NNH14ZTT001N 
Grant/Contract No.: Internal Project 
Project Type: FLIGHT 
Flight Program: ISS 
No. of Post Docs:  
No. of PhD Candidates:  
No. of Master's Candidates:  
No. of Bachelor's Candidates:  
No. of PhD Degrees:  
No. of Master's Degrees:  
No. of Bachelor's Degrees:  
Space Biology Element: (1) Cell & Molecular Biology
(2) Microbiology
Space Biology Cross-Element Discipline: (1) Reproductive Biology
Space Biology Special Category: None
Task Description: The proposed research aims to understand the effects of the space environment on evolutionary processes in the bacterium Bacillus subtilis. Different mutant lines will be ‘raced’ along solid surfaces to allow continuous selection in the cultures and to maximize the number of generations possible. Deep sequencing of winners will identify evolutionary rates, mechanisms, and targets of selection. We propose printing wax barriers to make paths along a growth surface (agar, membranes) and spotting each starting position of each path with dormant spores of the experimental bacteria to ‘race’ different mutants. Once on orbit, the material is wetted with growth medium, allowing the individual spots of B. subtilis to grow along their determined paths. This approach provides an opportunity for exponential growth only along the propagating edges, generating continuous bottlenecking thus amplifying selective pressures on the experimental populations. By monitoring the respective growth rate of different mutant lines maintained in each of these experimental conditions, we can estimate relative fitness of the lines. Long-term changes in relative growth rate indicate adaptation. Deep-sequencing of DNA from adapted cells (‘winners’ at the end of runs) will identify genetic changes within the respective populations. We expect that rates of mutation will differ between microgravity, 1-g, and ground controls, and that the targets of these mutations will differ as the different populations of bacteria adapt to their respective conditions. This research will also utilize the native ability of B. subtilis to uptake foreign DNA. Information-rich environmental DNA is added into the growth medium, and the populations are raced as above. By sampling the winners, and identifying if/what foreign genes are assimilated in each treatment, this experiment will identify potential genes of interest for future studies of genetic adaptation to the space environment. Our approach maximizes the number of generations possible in the 60-day window for this call, and maximizes the potential for evolutionary processes to occur. By performing multi-generational experimental evolution on bacteria on the International Space Station, the work proposed here aims to advance understanding of the evolutionary processes and challenges facing biological systems in long-term space exploration and habitation.

Research Impact/Earth Benefits: 0

Task Progress & Bibliography Information FY2015 
Task Progress: New project for FY2015.

Bibliography Type: Description: (Last Updated: 06/20/2019)  Show Cumulative Bibliography Listing
 
 None in FY 2015