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ACS Synth Biol
ACS Synth Biol
sb
asbcd6
ACS Synthetic Biology
2161-5063
American Chemical Society

39150229
10.1021/acssynbio.3c00672
Research Article
Desiccated Cyanobacteria Serve As Efficient Plasmid DNA Carriers in Space Flight
Kakouridis Anne †
Diamond Spencer ‡
Eng Thomas †
Mills Heath J. §
Gámez Holzhaus Olivia §
Summers Michael L. ∥
https://orcid.org/0000-0002-4974-3863
Garcia-Pichel Ferran ⊥
https://orcid.org/0000-0002-6513-7425
Mukhopadhyay Aindrila *†⊥
† Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States
‡ Innovative Genomics Institute, University of California, Berkeley, California 94720, United States
§ Rhodium Scientific, Houston, Texas 77058, United States
∥ Department of Biology, California State University, Northridge, California 91330, United States
⊥ School of Life Sciences, Arizona State University, Tempe, Arizona 85281, United States
* Aindrila Mukhopadhyay, amukhopadhyay@lbl.gov.
16 08 2024
20 09 2024
13 9 27332741
14 11 2023
30 07 2024
30 07 2024
© 2024 The Authors. Published by American Chemical Society
2024
The Authors
https://creativecommons.org/licenses/by-nc-nd/4.0/ Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/).

Effective transport of biological systems as cargo during space travel is a critical requirement to use synthetic biology and biomanufacturing in outer space. Bioproduction using microbes will drive the extent to which many human needs can be met in environments with limited resources. Vast repositories of biological parts and strains are available to meet this need, but their on-site availability requires effective transport. Here, we explore an approach that allows DNA plasmids, ubiquitous synthetic biology parts, to be safely transported to the International Space Station and back to the Kennedy Space Center without low-temperature or cryogenic stowage. Our approach relied on the cyanobacterium Nostoc punctiforme PC73102, which is naturally tolerant to prolonged desiccation. Desiccated N. punctiforme was able to carry the non-native pSCR119 plasmid as intracellular cargo safely to space and back. Upon return to the laboratory, the extracted plasmid showed no DNA damage or additional mutations and could be used as intended to transform the model synbio host Escherichia coli to bestow kanamycin resistance. This proof-of-concept study provides the foundation for a ruggedized transport host for DNA to environments where there is a need to reduce equipment and infrastructure for biological parts stowage and storage.

Cyanobacteria
International Space Station
Nostoc punctiforme
Space Flight
Space Travel
Biological and Environmental Research 10.13039/100006206 DE-AC02-05CH11231 Rhodium Scientific, LLC NA CWMD1918-008 document-id-old-9sb3c00672
document-id-new-14sb3c00672
ccc-price
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pmcIntroduction

Recent advances in synthetic biology have led to the development of DNA-based systems for information storage and biomanufacturing,1 holding great promise for space applications. DNA is a stable and efficient molecule for storing genetic information and is particularly attractive for space-based biomanufacturing, where products can be synthesized when and where they are needed.2 However, the space environment poses significant challenges to the stability and functionality of DNA-based systems because exposure to cosmic radiation, microgravity, and extreme temperatures can cause DNA damage, strand breaks, and other genetic changes.3−7 In addition, space environmental factors can also affect the gene expression and protein synthesis machinery of cells, impacting the functionality and efficiency of DNA-based systems.8,9 Current practices for space environments involve the use of cryopreservation for transport and storage of DNA,10,11 limiting the number of samples for synthetic biology applications. Advancing our knowledge in this area will enable the development of advanced DNA-based technologies for space applications, including the production of materials, medicines, and other biomolecules required for sustaining human exploration and habitation in space. In this study, we specifically address the need for robust methods to safely transport DNA to space stations and its storage.

To protect DNA from the stresses associated with space flight while safely carrying it at room temperature, we experimented with the use of cyanobacteria, well-known for native desiccation tolerance,12 as the DNA carriers. Nostoc punctiforme is a model cyanobacterial system that is sufficiently genetically tractable to examine its potential to serve as transport chassis for DNA.13 Several attributes of this organism make it an appropriate choice for this and future space biomanufacturing-related applications. As a fully autotrophic and diazotrophic microorganism, it enables a simpler feedstock logistical supply chain that does not require provision of fixed carbon or nitrogen sources.14N. punctiforme has evolved remarkable adaptations in its soil habitat of origin to withstand harsh environmental conditions, some of which may be relevant for space travel. These include a natural resistance to strong desiccation in vegetative cells, the formation of specialized spore-like cells called akinetes to enhance survival under nutrient limitation, and multigene responses to reactive oxygen radicals.15 Importantly for this study, it is resistant to ultraviolet radiation exposure through the synthesis of various ultraviolet sunscreen compounds and through motility/developmental responses, which can be deployed simultaneously to hedge its bets for survival.16 While many cyanobacteria demonstrate these capabilities,17N. punctiforme is an ideal choice as a model system with available genetic tools.

In this study, we probed the capability of desiccated N. punctiforme to act as a rugged DNA carrier in a real space flight test involving travel as cargo to the International Space Station, subsequent month-long orbit, and eventual return to the laboratory to be examined for revival and analyses of DNA integrity and rate of mutations (Figure 1).

Figure 1 Workflow (a) to transform N. punctiforme with plasmid pSCR119, (b) to prepare flight or ground samples, and (c) to process returning flight and ground samples. In (a) and (b), (1) growth conditions are 7–10 days, 50 mL BG11 2 mM NaHCO3, 30 °C 150 rpm, and 16 μE/s·m2 light; (2) recovery conditions are overnight incubation, 50 mL BG11 2 mM NaHCO3, 30 °C 100 rpm, and 8 μE/s·m2 light; (3) electroporation conditions are 400 μL cells and 10 μg plasmid DNA; and (4) growth conditions are 14 days incubation, 50 mL BG11 20 mM MgCl2, 2.5 μg/mL neomycin, 30 °C 80 rpm, and 4 μE/s·m2 light. (5) Ground and flight samples traveled to the Kennedy Space Center (KSC), and (6) flight samples spent 34 days at the International Space Station (ISS). Figure made using BioRender.

Results

We used the innate nature of cyanobacteria for desiccation and resuscitation to provide a potential rugged transport workflow, where no refrigeration or freezing is required. N. punctiforme provided an ideal model system because it has published cultivation and transformation methods. However, prior to conducting DNA transformation or desiccation workflows, it was necessary to optimize strain cultivation and maintenance in the laboratory using the reported conditions. While many different reports exist to cultivate N. punctiforme (e.g., Meeks,35 Nowruzi et al.,36 and Guljamow et al.37), the condition that worked best in our hands was supplementing BG11 media with NaHCO3 (final concentration 2 mM) to produce the most robust biomass. Results from supplementing BG11 with different concentrations of NaHCO3 are provided in Table S-1.

Next, we optimized the conditions that allow the best transformation outcomes. To test this, we explored published protocols (e.g., ref (33)) with some variations described in the Methods section. We found that among the conditions tested as reported in the Methods section and Table S-2, the following conditions produced the greatest transformation efficiency: 3 × 5 s bursts sonication, 10 μg plasmid DNA, and either 2.5 or 5 μg/mL kanamycin or neomycin. We found that maximal growth was impacted by the transformation and conditions required for the maintenance of plasmid DNA. Here also we tested several regimes of antibiotic amendments to obtain the ideal trade-off between cell mass and retention of plasmid DNA (Table S-2).

Rehydration and DNA extraction after a period of desiccation were the key aspects of discovery for this study. A priori, it was not known what length of time could be accommodated before sample revival was no longer observed. Further, the quantity of cells required for successful rehydration was also unknown; thus, we explored desiccation rehydration workflows using both transformed and WT N. punctiforme. All approaches attempted are described in detail in the Methods section. Samples were prepared at the Lawrence Berkeley National Laboratory (LBNL) in California and sent to the Kennedy Space Center (KSC) in Florida, USA. The ground samples remained at KSC while another identical set of flight samples were launched to the International Space Station (ISS) on the SpaceX Crew Dragon Commercial Resupply Service Mission 24 (CRS-24) on December 21, 2021. The flight samples remained on the ISS for 34 days before returning to the KSC on January 24, 2022. Flight and ground samples were then shipped back to LBNL. In our preflight tests, we were able to revive both transformant and WT cells stored up to 4 weeks of desiccation on filters. However, we were not able to revive transformants or WT desiccated cells from flight or ground samples using any of the protocols we attempted. We discuss potential routes to optimize this aspect in the Discussion section.

We were successful in extracting DNA from transformant and WT desiccated cells from flight and ground samples. Among the different methods we tested as detailed in the Methods section (Table S-3), we achieved the greatest DNA yield and quality using the following steps: (1) cut the filters into 4 × 4 mm size pieces, (2) place the pieces in the first tube of the DNeasy PowerSoil Pro Kit with 500 μL of buffer (10 mM Tris-Cl and 0.5 mM EDTA; pH 8.0), (3) incubate the tubes for 24 h in the dark at room temperature, (4) vortex the tubes for 15 min at half speed, and (5) follow the rest of the kit manufacturer’s instructions. Other conditions (e.g., larger pieces of filter, shorter incubation times in buffer, and different vortexing regimes) resulted in lower DNA yield and quality. For the methods tested, we observed that ∼3,000,000 cells on a 4 cm2 piece of filter provided the lower limit for successful DNA extraction regardless of the method used. Understanding the lower limits of cells that allow for successful transport and extraction will assist in conditions selected for future exploration. These thresholds were unclear a priori and benefited from testing across a range of cell quantities.

A portion of the plasmid DNA extracted from the desiccated cell was sent for sequencing. Another portion of the extracted DNA was used for transformation into E. coli. The transformed E. coli cells were able to grow on media containing kan/neo (final concentration of 50 μg/mL), indicating that the plasmid had retained its functions in terms of replication potential and conferring kan/neo resistance. We then pooled all of the colonies per plate and extracted the plasmids from E. coli to obtain more DNA. This DNA was also sent for sequencing.

Sequencing of plasmids successfully recovered directly from N. punctiforme and transformed E. coli cells was used to evaluate if single nucleotide substitutions (SNS) or single nucleotide variants (SNV), together referred to as divergent sites, occurred with an altered frequency between plasmid pairs that remained on the ground or went into flight using inStrain.18 As it is common for divergent sites to exist in the DNA of plasmids present in a specific laboratory relative to its deposited reference sequence, we initially quantified the number of background divergent sites present in our pSCR119 plasmid before the experiment and after using it in an E. coli transformation. We identified 35 divergent sites (three SNS and 32 SNV) in our pSCR119 plasmid relative to the reference before the experiment and 34 divergent sites (four SNS and 30 SNV) in the plasmid after using it to transform E. coli cells. Subsequently, we evaluated the divergent site count between paired sets of pSCR119 plasmid DNA used in the experiment with sufficient sequencing coverage (see the Methods section) that were either directly extracted from desiccated N. punctiforme cells or transformed into E. coli and subsequently extracted. For ground–flight plasmid pairs directly extracted from desiccated N. punctiforme cells, we identified an average of 31.6 ± 0.5 divergent sites (SNS = 4 ± 0; SNV = 27.6 ± 0.5) in samples that remained on the ground and an average of 32.6 ± 0.9 divergent sites (SNS = 4 ± 0; SNV = 28.6 ± 0.9) in samples that went to flight (Figure 2A). We found no statistically significant difference in the number of divergent sites between N. punctiforme extracted paired samples (p-value = 0.142; n = 5 pairs). For ground–flight plasmid pairs transformed into E. coli, we identified an average of 33.6 ± 1.0 divergent sites (SNS = 3.6 ± 0.5; SNV = 30.0 ± 1.2) in samples that remained on the ground and an average of 33.9 ± 1.5 divergent sites (SNS = 3.4 ± 0.5; SNV = 30.4 ± 1.7) in samples that went to flight (Figure 2B). Again, we found no statistically significant difference in the number of divergent sites between E. coli extracted paired samples (p-value = 0.569; n = 7 pairs) (Figure 2 and Figures S-2, S-4, and S-5).

Figure 2 Comparison of divergent site counts (sum of SNV and SNS) for plasmids of paired flight and ground samples extracted directly from N. punctiforme (n = 5) and sequenced directly (a) or (b) transformed into E. coli then extracted and sequenced (n = 7 pairs). (a and b) Divergent site counts from paired sets of flight and ground samples were compared using the paired t test with no statistically significant differences detected for plasmids extracted directly from N. punctiforme (p-value = 0.142) or recovered from E. coli transformations (p-value = 0.567). Each alphabet represents a ground and flight sample pair. Some of the samples (dots) overlap in the figure due to having the same number of divergent sites.

Discussion

The main goal of this study was to establish a model cyanobacteria such as N. punctiforme as an efficient DNA transport system for travel where cargo capacity and infrastructure need to be minimal. One such configuration is space travel, where the use of large cooling equipment can be both capacity and cost prohibitive. The cyanobacterial strain N. punctiforme was chosen for this endeavor due to its natural tolerance to desiccation. Known to be the primary components in biological soil crusts (BSCs)19,20 in arid and semiarid regions worldwide,21 a unique feature of BSCs is the ability of the constituent microorganisms, particularly cyanobacteria, to withstand extended periods of drought and to revive rapidly upon rehydration.22,23 Such cyanobacteria have evolved various physiological and morphological adaptations that enable them to survive desiccation and persist for long periods in a dormant state24 and rapidly resume metabolic activity and photosynthesis when hydrated.23 In this context, we examined the transported and desiccated Nostoc strains for resuscitation and growth. In our workflow, the desiccated N. punctiforme that remained in the desiccated state for 34 days at the ISS and 60 days in total were not able to grow upon hydration and cultivation in the laboratory. Since the ground control samples for these also could not be resuscitated, we concluded that the length of time was too long with the methods employed. However, our limited samples only allowed a subset of conditions to be tested to attempt cell resuscitation. In the future, when more such opportunities are available (this study was limited to one journey to the ISS), the use of different desiccation parameters (e.g., via slower desiccation or pelleting vs filtration) and resuscitation approaches (e.g., in a wider range of cultivation media) could provide methods that revive the carrier Nostoc strains.

The fidelity of the plasmid DNA was conserved with few to no mutations and is consistent with our ability to transform this plasmid into another synbio host, E. coli, and demonstrate function. However, the lack of revival of the strain itself could potentially be due to epigenetic causes. Given the benefits of using fully autotrophic and diazotrophic microorganisms, it would be useful to further enhance the recovery of N. punctiforme. Based on the methods used in this study and the outcomes, future studies should use slower dry periods with controlled reduction in humidity to allow N. punctiforme to engage its native desiccation machinery fully. Laboratory evolution experiments could also be performed to improve desiccation robustness and recovery phenotypes. Other cyanobacteria have been examined to pinpoint cultivation conditions such as nitrogen limitation25 and cellular responses such as the role of the pigments26 as beneficial for resuscitation. These concepts could be implemented for developing an enhanced N. punctiforme chassis for transport and storage applications. Overall, for this study we conclude that while the revival of N. punctiforme cultures requires further optimization, the current workflow protects the plasmid DNA from any deleterious mutations during space travel.

Transport and storage of DNA has been examined using other routes, of which the most appealing are workflows that use dry purified DNA samples or purified DNA stored in a buffer system.27,28 Studies that assess the DNA stability of such approaches have also been conducted.29 Of these, encapsulation to maintain inert conditions30 has been reported as a common approach and could provide an alternate way to obtain a long-term storage of nucleic acid samples. Such approaches need to be tested for their low gravity and potential impact from radiation exposure. Use of a radiation tolerant desiccated cell provides benefits due to its simplicity in both the preparation and extraction of the samples without the need for specialized equipment. In future studies, where a much larger total number of samples can be tested, a side-by-side comparison of several of these approaches can be attempted.

This study represents a key step toward a much larger set of goals being envisioned for microbiology-enabled synthetic biology in space.5,8 Cyanobacteria, including N. punctiforme, have been studied in the context of their ability to survive in space and interesting native metabolomic capabilities.31,32 In this study, we explored the basic but essential molecular biology workflow; that of DNA parts transport and availability. The ability to transport a plasmid without mutations, without the need for refrigeration or cryo-storage, and successfully transform a common synthetic biology workhorse after exposure to space conditions sets the stage for many synbio workflows. The use of N. punctiforme was important and showed that microbes like these can remain desiccated for extended periods in the presence of radiation and also permit minimal damage to the harbored plasmid DNA. This study provides an immediately usable workflow for non-native DNA transport using desiccated Nostoc strains and also future applications that use the Nostoc itself in space synbio applications.

Materials and Methods

Cultivation Conditions

N. punctiforme (ATCC 29133/PCC 73102) (Figure 3a) was obtained as a cryostock (5% DMSO final concentration) from the Garcia-Pichel laboratory at Arizona State University, Tempe, AZ, USA. Each 1 mL cryostock was thawed and transferred to a 250 mL baffled flask containing 50 mL of BG11 minimal medium containing nitrate. The 1X BG11 medium used in this study was made by diluting 50X Cyanobacteria BG-11 Freshwater solution (cat. no. C3061, Sigma-Aldrich, Inc., St. Louis, MO, USA) with autoclaved Milli-Q water. Flasks were placed on a shaker at 150 rpm, 30 °C, under 24 h lights emitting 16 μE/s·m2 (Mosthink LED Plant Grow Lights Strips Full Spectrum, Amazon, Seattle, WA, USA). The light intensity the cultures experienced was checked weekly by measuring photosynthetically active radiation (PAR) with a PAR meter (PHOTOBIO Advanced Quantum PAR Meter by Phantom, Amazon, Seattle, WA, USA). To test different growing media amendments, some N. punctiforme cryostocks were thawed and grown in BG11 supplemented with NaHCO3 (final concentrations 1, 2, 4, 6, 8, and 10 mM) (Table S-1).

Figure 3 (a) Microscopy image of WT N. punctiforme. (b) Plasmid map of pSCR119 (plasmid map based on commercial source Novoprolabs34). For validated sequence data of the plasmid used, see the Supporting Information. (c) N. punctiforme growing in BG11 supplemented with kanamycin (2.5 μg/mL final concentration) 10 days after transformation when pSCR119 was not added (left) vs when it was added (right).

Dense cultures were obtained after 7–10 days of growth. Cell density was measured by chlorophyll-a (Chl a) extraction following a protocol from the Summers laboratory at California State University, Northridge, CA, USA. One milliliter of each culture was transferred to a microcentrifuge tube and centrifuged at 15,000 rpm at room temperature for 1 min. Then, 900 μL of supernatant were removed from each tube and replaced with 900 μL of 100% methanol. Each tube was vortexed to break up the pellet, allowed to sit for 5 min at room temperature in the dark, and then vortexed again. Tubes were again centrifuged at 15,000 rpm at room temperature for 1 min. The supernatant of each tube was transferred to a cuvette, and absorbance was read at 665 nm against a 90% methanol blank. The absorbance value was multiplied by 12.7 to obtain μg Chl a/mL (based on the extinction coefficient, ref (38)) and cell density (1 μg/mL Chl a ≈ 106 cells). Our target cell density was 10 μg of Chl a/mL for a 50 mL culture to prepare electrocompetent cells.

Transformation Conditions

Plasmid pSCR11933 (Figure 3b) was obtained from stocks kept in the Summers laboratory. This shuttle plasmid is compatible with both E. coli and N. punctiforme and confers kanamycin/neomycin (kan/neo) resistance. The plasmid DNA was transformed into E. coli following a standard heat shock protocol and then isolated by miniprep (cat. no. 27104, QIAprep Spin Miniprep Kit, Qiagen, Germantown, MD, USA) following the manufacturer’s instructions and eluted in molecular grade water to minimize the salt concentration for electroporation into N. punctiforme.

Transformations by electroporation were performed following a protocol by Summers et al.33 with some modifications (Table S-2). Each 50 mL culture was transferred to a 50 mL falcon tube and centrifuged at 4000 rpm for 5 min. The supernatant was removed so that each culture was concentrated down to 5 mL. The 5 mL concentrated cultures were sonicated to break up the N. punctiforme filaments using a 2 mm microtip, amplitude 1, and the following settings were tested: 10 × 10 s bursts, 5 × 10 s bursts, 4 × 5 s bursts, or 3 × 5 s bursts (Table S-2). After sonication, a droplet of each culture was mounted on a glass slide and observed under a compound microscope to confirm that most N. punctiforme filaments were down to 1–4 cells in length. The 5 mL concentrated cultures were individually transferred to 45 mL of BG11 in 250 mL flasks and allowed to recover overnight at 50% light intensity, 100 rpm, 30 °C. The next morning, the recovered cultures were centrifuged at 4000 rpm for 5 min, and then the supernatant was removed. The pellets were washed with 40 mL of autoclaved room temperature Milli-Q water four times, centrifuging each time. After the fourth wash, the pellets were resuspended to 5 mL in room temperature Milli-Q water. In microcentrifuge tubes, 400 μL of concentrated washed cells and 10 μg of plasmid DNA were mixed by pipetting and allowed to incubate on ice for 1 min. The mixture was transferred to a 2 mm cuvette for electroporation with the following parameters: 600 Ω, 1.6 kEV, and 25 μF. The goal was to obtain a time constant of 11.5–13.5 ms. Immediately after electroporation, 1 mL of BG11 supplemented with MgCl2 (20 mM final concentration) was added to the microcentrifuge tube, and then the content of the tube was transferred to 50 mL of the same media in a 250 mL baffled flask. All the flasks were allowed to recover for 24 h at 25% light intensity, 80 rpm, 30 °C. Cultures were then centrifuged again and transferred to fresh BG11 with either kanamycin or neomycin to reach a final concentration of 1.25 or 2.5 or 5 or 10 μg/mL (different concentrations were tested), and the flasks were incubated at 25% light intensity, 80 rpm, 30 °C for 14 days.

To check for successful transformation, the cell density of transformants and control (no plasmid added) was measured every few days by Chl a extraction as described previously to confirm that transformants were growing more than wild type (WT) cultures in the presence of kan/neo (Figure 3c). In addition, the presence of the kan/neo resistance genes in transformant cultures was checked every few days by PCR, gel electrophoresis, and Sanger sequencing. To do so, 1 mL of each culture was pelleted by centrifugation at 8000 rpm for 3 min, and then the supernatant was removed. The pellets were resuspended in 50 μL of BG11 and 1 μL was then used as the DNA template for PCR. PCR was conducted following the manufacturer’s instructions for the Q5 High-Fidelity 2X Master Mix (cat. no. M0492S, New England BioLabs, Inc., Ipswich, MA, USA), using the following conditions: 98 °C for 30 s and (98 °C for 10 s, 71 °C for 5 s, 72 °C for 30 s) × 35 cycles. The following primers were used: forward 5′-CTGCAATGATACCGCGAGACCC-3′ and reverse 5′-CCAGTCCGCAGAAACGGTGC-3′ with an expected PCR product size of 1288 bp for gene encoding the kan/neo resistance marker gene.

Sample Preparation for Transportation to the International Space Station

Successful transformants and WT N. punctiforme cultures were sterile filtered onto 0.22 μm cellulose acetate membranes in a Corning 150 mL Bottle Top Filter (cat. no. 430626, Corning Inc., Corning, NY, USA). The filters were cut out of the bottle top using autoclaved metal scissors and tweezers and placed into Petri dishes and then allowed to dry overnight in the dark at room temperature (Figure 4a). The following day, each filter was cut in half, and each half-filter was placed in a Rhodium Cryotube 0005 (NASA part no.: RhCT-0005) (Figure 4b). These Rhodium proprietary tubes provide a 4 mL working volume for samples and have been NASA flight safety certified for operation on the ISS. Filters were loaded into the RhCT-0005s using autoclaved metal scissors and tweezers.

Figure 4 (a) Images of filters with a layer of wild type or transformed N. punctiforme. The Petri dishes provide a sterile container to rest cut filters for the duration of the drying period. (b) Image of cells in cryotubes packaged for shipment to and from the Kennedy Space Center and the International Space Station.

Since the limitation of cell quantity may be different for resuscitation and DNA extraction after a time period as cargo in space travel, we prepared several concentrations of cell mass on filter paper from both transformed and WT N. punctiforme (Table 1). The project allowed 36 samples to be included in the space cargo, and samples were planned to span a reasonable range of cell quantities of transformed and WT N. punctiforme. With this strategy, we produced filters carrying approximately 3,302,000 to 14,986,000 cells for transformed N. punctiforme and 1,240,000 to 445,770,000 for WT N. punctiforme (Figure 4 and Table 1).

Table 1 Flight and Ground Sample Lista

a Corresponding flight and ground samples (e.g., 1 and 37) are two halves of the same filter. Wild-type samples were used to test different DNA extraction and revival methods upon samples returning to the laboratory.

All samples were air-couriered from Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA, to the Kennedy Space Center (KSC), Merritt Island, FL, USA on December 14, 2021. Upon arrival, Rhodium Scientific packed the RhCT-0005s containing samples into two sets of four Rhodium Science Chamber 05CT (NASA part no.: RhSC-05CT) facilities. Each facility contained nine samples contained within two layers of Bitran(R) bags, labeled following the NASA approved labeling plan, then grouped in sets of four into gallon-size plastic storage bags. This configuration was flight safety certified by Rhodium for integration and operation on the ISS. One set of samples, hereafter referred to as the “ground” samples, remained at KSC, while the other identical set of samples, hereafter referred to as the “flight” samples, was launched to the International Space Station (ISS) on the SpaceX Crew Dragon Commercial Resupply Service Mission 24 (CRS-24) on December 21, 2021. Rhodium monitored astronaut on orbit stowage and retrieval activities from Rhodium Mission Control in Houston, a facility with video and audio links routed from the ISS through NASA Marshall Space Flight Center. The flight samples remained on the ISS undisturbed within the Japanese Experiment Module for 34 days before splashdown and return to KSC on January 24, 2022. Flight and ground samples were then shipped to LBNL. The timeline of the project and the scheduled space flights to ISS provided the constraints for the planning the necessary steps of this project. As such, pre- and post-flight steps were organized and completed to utilize project times available to us before and after scheduled space flight operations at the ISS that occurred during the timeline of this project.

Sample Rehydration and DNA Extraction and Sequencing

Flight and ground transformant and WT samples were taken out of the tubes and placed in Petri dishes by using autoclaved tools. To attempt reviving the samples, 2 mL of BG11 were pipetted onto the filters and the samples were incubated overnight at room temperature in the dark. The cells were then gently scraped off the filter and resuspended in BG11 by using sterile inoculation loops. The BG11 containing resuspended cells were transferred to a glass tube, and 3 mL of additional BG11 was added. The tubes were placed at 25% light, 80 rpm, 30 °C. Another way the samples were attempted to be revived was by placing filters directly into tubes containing 5 mL of BG11 and placing them on a shaker at 25% light, 80 rpm, and 30 °C.

To directly extract DNA from the desiccated flight and ground transformants and WT samples, we experimented with different methods (Table S-3). First, we placed filters in 2 mL tubes with beads and 1 mL of TE buffer (Qiagen Plasmid Isolation Kit, Qiagen Germantown, MD, USA) and ran them in the tissue lyser at either 1/2 speed or max speed for 30 s or 1 min. Then, we followed a standard phenol:chloroform DNA extraction.39,40 Another way we attempted to extract DNA from the desiccated samples was by rehydrating filters in BG11 for 15 min, 2 h, 12 h, 24 h, and 48 h, gently scraping the cells of the filters with inoculation loops and placing the resuspended cells into the first tube of the DNeasy PowerSoil Pro Kit (cat. no. 47016, Qiagen, Germantown, MD, USA) containing beads, and then following the manufacturer’s instructions. We also placed filters whole or cut into different sized pieces (2 × 2, 4 × 4, 6 × 6, and 8 × 8 mm) in the first tube of the DNeasy PowerSoil Pro Kit using autoclaved scissors and tweezers. Then, 500 μL of buffer (10 mM Tris-Cl and 0.5 mM EDTA; pH 8.0) was added and samples were allowed to incubate for 15 min, 2 h, 12 h, 24 h, and 48 h in the dark at room temperature. Then, samples were vortexed at different speeds and lengths of time: 10 min at full speed, 10 min at half speed, and 15 min at half speed. Of the methods used (Table S-3), the approach that provided the best samples was placing filters cut into 4 × 4 mm pieces in the first tube of the DNeasy PowerSoil Pro Kit with 500 μL of buffer (10 mM Tris-Cl and 0.5 mM EDTA; pH 8.0) and incubating for 24 h in the dark at room temperature and then vortexing the tubes for 15 min at half speed. The rest of the protocol followed the manufacturer’s instructions.

The quantity and quality of the extracted raw DNA were measured by a NanoDrop spectrophotometer (Thermo Fisher, USA) per the manufacturer’s instructions. Additionally, 100 ng of DNA was transformed into NEB 10-β competent E. coli (high efficiency) (cat. no. C3019H, New England BioLabs, Inc., Ipswich, MA, USA) following the manufacturer’s instructions with a recovery time of either 1 h (standard time) or increased to 2 or 3 h. For each sample, the entire reaction mixture was plated onto selective LB plates. Plates were incubated overnight at 37 °C (Figure S-1).

For each plate, colonies were counted (Table S-4) and then scraped off the plate and pooled in 1 mL of LB Kan media. The pooled colonies were resuspended and mixed by pipetting, then 500 μL were used for DNA isolation by miniprep (cat. no. 27104, QIAprep Spin Miniprep Kit, Qiagen, Germantown, MD, USA). The extracted DNA was quantified using the Qubit dsDNA BR Assay Kit (cat. no. Q32850, ThermoFisher Scientific, Waltham, MA, USA), and its purity was determined with a NanoDrop spectrophotometer (Table S-5). Raw plasmid DNA and isolated plasmid DNA were sent for Illumina sequencing at QB3 Genomics (UC Berkeley, Berkeley, CA).

DNA Sequence Analysis

Illumina adapter sequences and phiX sequences were removed from raw reads using BBduk (https://sourceforge.net/projects/bbmap/) with default parameters. Reads were subsequently quality filtered using Sickle (https://github.com/najoshi/sickle) with default parameters. Trimmed and quality filtered reads from each sample (mean depth ∼98 M, 150 bp reads per sample) were mapped back to a combined reference sequence file containing the N. punctiforme genome (taxonomy ID, 63737) and the pSCR119 plasmid (taxonomy ID, 282192) using bowtie2 with default parameters.41 Mapped read files from bowtie2 were converted into sorted indexed BAM format using Samtools.42 Reference sequence coverage and divergent nucleotide sites were quantified from BAM files using the inStrain profile within the inStrain package.18 Divergent nucleotide sites are the sum of single nucleotide substitutions (SNS), a fixed nucleotide change supported by all reads relative to the reference sequence, and single nucleotide variants (SNV), a nucleotide change relative to the reference sequence supported by a significant subfraction of the mapped reads. inStrain was run with default parameters which required ≥95% alignment identity to a reference for a read to be used in the analysis, the minimum coverage to call a variant to be ≥5X, and the minimum fraction of reads with a variant to call an SNV to be ≥5%.

Outputs from inStrain were combined and analyzed by using a custom R script (Supplementary Data S-1). We first assessed the coverage and divergent site count estimated by InStrain for the N. punctiforme genome and pSCR119 plasmid in each sample and each pair of samples retained on the ground (ground) or sent to space (flight). We observed very low coverage, likely due to inefficient genomic DNA extraction from lyophilized samples, for the N. punctiforme genome, and thus limited our analysis to divergent sites in the pSCR119 plasmid sequence. For analysis of divergent sites in the pSCR119 plasmid sequence, we only retained samples for analysis that had a complete ground–flight pair and where the difference in coverage between samples in a pair was ≤10-fold (Figure S-5). All plotting was performed using ggplot2 in R,43 and statistical testing to evaluate significant differences in divergent sites between ground and flight sample pairs was performed in R using both the Student’s t test and the Wilcoxon rank sum test.

Data Availability Statement

The plasmid sequences used in this study are available through NCBI BioProject ID, PRJNA1034103.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssynbio.3c00672.Supplementary figures, including images of plates and assessment of coverage for extracted plasmid DNA sequences, and supplementary tables, including all media, transformation protocols, DNA extraction, and transformation methods tested (PDF)

Snapgene sequence for pSCR119 used and R script for data analysis (ZIP)

Supplementary Material

sb3c00672_si_001.pdf

sb3c00672_si_002.zip

Author Contributions

A.K., A.M., H.J.M., and O.G.H. developed the research plan. A.K. conducted the experiments, prepared samples, and collected the data. A.K. and S.D. analyzed the data. H.J.M. and O.G.H. handled the samples at the KSC. F.G.P., M.C.S., and T.E. provided guidance and resources. A.K., S.D., and A.M. drafted the manuscript. All authors provided input, made revisions, and helped finalize the manuscript.

This study was supported by funding from Rhodium Scientific, LLC, (Sponsor Ref CWMD1918-008, Award AWD00005599). A.M. and A.K. are supported by the ENIGMA, Ecosystems and Networks Integrated with Genes and Molecular Assemblies (enigma.lbl.gov), a Scientific Focus Area Program.A.M. and T.E. are supported by the Joint BioEnergy Institute (jbei.org) at Lawrence Berkeley National Laboratory funded by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under Contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the U.S. Department of Energy.

The authors declare no competing financial interest.

Acknowledgments

We thank Kevin Klicki for helping us obtain the Nostoc strains and plasmids, Alex Codik for archiving strains and plasmids, and Juliana Artier and Andreja Kust for their thoughtful advice on cyanobacteria culturing. We also thank R.P. Oats for the initial work in drafting the proposal for this study and Lucas Waldburger for help with uploading data to public repositories.
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