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DOE OSTI2026

Optimizing resource allocation in Miscanthus breeding via sparse testing designs for genomic prediction

Phenotyping high-biomass perennial crops is laborious and the rate of genetic gain in conventional perennial crop breeding programs is typically low. So, it is especially important to identify methods that produce efficiency gains in the breeding process. Miscanthus is a C4 perennial grass with favorable characteristics for producing biomass as a feedstock for biofuels and diverse bio-based products. Increasing biomass yield will increase profitability and environmental benefits, so it is a key target for Miscanthus breeding. In addition, the identification of well-adapted genotypes across a wide range of environmental conditions requires the establishment of multi-environment trials (METs). Sparse testing is a genomic prediction-based strategy that reduces the phenotyping costs in METs by selecting a subset of genotypes to evaluate in a subset of environments and then predicts the performance of the unobserved genotype-environment combinations. A Miscanthus sacchariflorus (MSA) population comprising 336 genotypes observed across three environments was analyzed implementing sparse testing designs. Three prediction models considering main effects (environments, genotypes, genomic) and interaction effects (genotype-by-environment; G×E interaction) were implemented for forecasting dry biomass yield (YDY), total culm (TCM), average internode length (AIL), and culm node number (CNN). Multiple calibration sets based on different compositions and sizes were considered to evaluate performance in terms of the predictive ability (PA) and the mean square error (MSE) for a fixed testing set size. The training set size ranged from 52 to 112 to predict a fixed set of 224 unobserved genotypes across all three environments. The results showed that the model accounting for G×E interaction consistently presented the highest PA and the lowest MSE: for CNN (PA: ~0.77, MSE: ~0.5) and YDY (PA: ~0.70, MSE: ~1.3) while for TCM and AIL these ranged from ~0.28 to 0.41 and ~1.3 to 4.3, respectively. Overall, varying training sets and allocation strategies did not affect PA and MSE, with 52 non-overlapping and 0 overlapping genotypes per environment as the optimal cost-effective allocation framework. This suggests that implementing sparse testing designs could significantly reduce phenotyping costs by fivefold, without compromising PA in breeding programs for perennial crops such as Miscanthus.

Miscanthus sacchariflorus (MSA)↗

DOE OSTI2024

Altering translation allows E. coli to overcome chemically stabilized G-quadruplexes

Genomic DNA from each sample was prepared using the Wizard Genomic DNA Purification Kit (Promega) and after, DNA was quantified using the QuantiFluor ONE dsDNA System (Promega). Genomic DNA underwent shearing to ~200 bp fragments via sonication and the gDNA fragments were prepared for sequencing using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB). Bead-based size selection was used to select ~200 bp fragments and the fragments then underwent a splinkerette PCR using a Tn5-enriching forward primer and custom reverse primers for multiplexing. A final bead-based size selection was used to select for the correct length DNA. DNA was sequenced at the University of Michigan Advanced Genomics Core using Illumina sequencing with a custom read primer reading the last 10 nt of the transposon. PhiX174 DNA spike was added to the run to ensure sufficient sequence diversity on the flow cell. Then, a custom index read primer and standard Illumina primer were used to sequence the index reads and PhiX174, respectively.

Keck, James L.↗

NASA NTRS2011

Stress-Driven Selection of Novel Phenotypes

A process has been developed that can confer novel properties, such as metal resistance, to a host bacterium. This same process can also be used to produce RNAs and peptides that have novel properties, such as the ability to bind particular compounds. It is inherent in the method that the peptide or RNA will behave as expected in the target organism. Plasmid-born mini-gene libraries coding for either a population of combinatorial peptides or stable, artificial RNAs carrying random inserts are produced. These libraries, which have no bias towards any biological function, are used to transform the organism of interest and to serve as an initial source of genetic variation for stress-driven evolution. The transformed bacteria are propagated under selective pressure in order to obtain variants with the desired properties. The process is highly distinct from in vitro methods because the variants are selected in the context of the cell while it is experiencing stress. Hence, the selected peptide or RNA will, by definition, work as expected in the target cell as the cell adapts to its presence during the selection process. Once the novel gene, which produces the sought phenotype, is obtained, it can be transferred to the main genome to increase the genetic stability in the organism. Alternatively, the cell line can be used to produce novel RNAs or peptides with selectable properties in large quantity for separate purposes. The system allows for easy, large-scale purification of the RNAs or peptide products. The process has been reduced to practice by imposing sub-inhibitory concentrations of NiCl2 on cells of the bacterium Escherichia coli that were transformed separately with the peptide library and RNA library. The evolved resistant clones were isolated, and sequences of the selected mini-gene variants were established. Clones resistant to NiCl2 were found to carry identical plasmid variants with a functional mini-gene that specifically conferred significant nickel tolerance on the host cells. Sequencing of the selected mini-gene revealed a propensity of the encoded peptide to bind transient metal ions. Expression of the mini-gene markedly improved growth parameters of the evolved clones at sub-inhibitory concentrations of NiCl2 while being slightly detrimental in the absence of stress. Similar results have been obtained with the RNA libraries. Overall, the results demonstrate a very natural outcome of the selection experiments in which the mini-genes were expected to be either successfully integrated into bacterial genetic networks, or rejected depending upon their effect on host fitness. This described approach can be useful as a laboratory model to study the dynamics of bacterial adaptive evolution on the molecular level. It can also provide a strategy for screening expressed DNA libraries in search of novel genes with desirable properties.

Fox, George E.↗

NASA NTRS2016

Genomic and Phenotypic Characterization of Yeast Biosensor for Deep-space Radiation

The BioSentinel mission was selected to launch as a secondary payload onboard NASA Exploration Mission 1 (EM-1) in 2018. In BioSentinel, the budding yeast Saccharomyces cerevisiae will be used as a biosensor to measure the long-term impact of deep-space radiation to living organisms. In the 4U-payload, desiccated yeast cells from different strains will be stored inside microfluidic cards equipped with 3-color LED optical detection system to monitor cell growth and metabolic activity. At different times throughout the 12-month mission, these cards will be filled with liquid yeast growth media to rehydrate and grow the desiccated cells. The growth and metabolic rates of wild-type and radiation-sensitive strains in deep-space radiation environment will be compared to the rates measured in the ground- and microgravity-control units. These rates will also be correlated with measurements obtained from onboard physical dosimeters. In our preliminary long-term desiccation study, we found that air-drying yeast cells in 10% trehalose is the best method of cell preservation in order to survive the entire 18-month mission duration (6-month pre-launch plus 12-month full-mission periods). However, our study also revealed that desiccated yeast cells have decreasing viability over time when stored in payload-like environment. This suggests that the yeast biosensor will have different population of cells at different time points during the long-term mission. In this study, we are characterizing genomic and phenotypic changes in our yeast biosensor due to long-term storage and desiccation. For each yeast strain that will be part of the biosensor, several clones were reisolated after long-term storage by desiccation. These clones were compared to their respective original isolate in terms of genomic composition, desiccation tolerance and radiation sensitivity. Interestingly, clones from a radiation-sensitive mutant have better desiccation tolerance compared to their original isolate without losing radiation sensitivity. We employed Next-Generation Sequencing technology to better understand this phenotypic variation. Current effort is focusing on the analysis of high-throughput sequencing data to look for genomic changes in these reisolated clones compared to their original isolate.

yeast↗

DOE OSTI2025

Amino acid substrate specificities and tissue expression profiles of the nine CYP79A encoding genes in Sorghum bicolor

Cytochrome P450s of the CYP79 family catalyze two N-hydroxylation reactions, converting a selected number of amino acids into the corresponding oximes. The sorghum genome (Sorghum bicolor) harbours nine CYP79A encoding genes, and here sequence comparisons of the CYP79As along with their substrate recognition sites (SRSs) are provided. The substrate specificity of previously uncharacterized CYP79As was investigated by transient expression in Nicotiana benthamiana and subsequent transformation of the oximes formed into the corresponding stable oxime glucosides catalyzed by endogenous UDPG-glucosyltransferases (UGTs). CYP79A61 uses phenylalanine as a substrate, whereas CYP79A91, CYP79A93, and CYP79A95 use valine and isoleucine as substrates, with CYP79A93 showing the ability also to use phenylalanine. CYP79A94 uses isoleucine as a substrate. Analysis of 249 sorghum transcriptomes from two different sorghum cultivars showed the expression levels and tissue-specific expression of the CYP79As. CYP79A1 is the committed gene in dhurrin formation and was the highest expressed gene in most tissues/organs. CYP79A61 was primarily expressed in fully developed leaf blades and leaf sheaths. CYP79A91 and CYP79A92 were expressed mainly in roots >200 cm below ground, while CYP79A93 and CYP79A94 were most highly expressed in the leaf collar and leaf sheath, respectively. Here, the possible signalling effects of the oximes and their metabolites produced in different sorghum tissues are discussed.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2026

From Reads to Function Workshop - Milano 2026

The Bicocca Sampling Days (BSDs) model offers a reproducible “citizen science” framework integrating research, education, and public engagement through large-scale microbiome sampling, followed by a workshop of data analysis on select samples. We identified 9 bacterial and archaeal metagenome-assembled genomes from six soil samples across three separate sampling days in two approaches with indidivual sample and replicate co-assembly spanning three unique classes, providing genomic insights into microbial nutrient cycling in these systems.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

An essential and highly selective protein import pathway encoded by nucleus-forming phage

Targeting proteins to specific subcellular destinations is essential in prokaryotes, eukaryotes, and the viruses that infect them. Chimalliviridae phages encapsulate their genomes in a nucleus-like replication compartment composed of the protein chimallin (ChmA) that excludes ribosomes and decouples transcription from translation. These phages selectively partition proteins between the phage nucleus and the bacterial cytoplasm. Currently, the genes and signals that govern selective protein import into the phage nucleus are unknown. Here, we identify two components of this protein import pathway: a species-specific surface-exposed region of a phage intranuclear protein required for nuclear entry and a conserved protein, PicA (Protein importer of chimalliviruses A), that facilitates cargo protein trafficking across the phage nuclear shell. We also identify a defective cargo protein that is targeted to PicA on the nuclear periphery but fails to enter the nucleus, providing insight into the mechanism of nuclear protein trafficking. Using CRISPRi-ART protein expression knockdown of PicA, we show that PicA is essential early in the chimallivirus replication cycle. Together, our results allow us to propose a multistep model for the Protein Import Chimallivirus pathway, where proteins are targeted to PicA by amino acids on their surface and then licensed by PicA for nuclear entry. The divergence in the selectivity of this pathway between closely related chimalliviruses implicates its role as a key player in the evolutionary arms race between competing phages and their hosts.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Genome-resolved analysis of Serratia marcescens strain SMTT infers niche specialization as a hydrocarbon-degrader

Abstract Bacteria that are chronically exposed to high levels of pollutants demonstrate genomic and corresponding metabolic diversity that complement their strategies for adaptation to hydrocarbon-rich environments. Whole genome sequencing was carried out to infer functional traits of Serratia marcescens strain SMTT recovered from soil contaminated with crude oil. The genome size (Mb) was 5,013,981 with a total gene count of 4,842. Comparative analyses with carefully selected S. marcescens strains, 2 of which are associated with contaminated soil, show conservation of central metabolic pathways in addition to intra-specific genetic diversity and metabolic flexibility. Genome comparisons also indicated an enrichment of genes associated with multidrug resistance and efflux pumps for SMTT. The SMTT genome contained genes that enable the catabolism of aromatic compounds via the protocatechuate para-degradation pathway, in addition to meta-cleavage of catechol (meta-cleavage pathway II); gene enrichment for aromatic compound degradation was markedly higher for SMTT compared to the other S. marcescens strains analysed. Our data presents a valuable genetic inventory for future studies on strains of S. marcescens and provides insights into those genomic features of SMTT with industrial potential.

Genetics & Heredity↗

DOE OSTI2025

Orthogonal replication with optogenetic selection evolves yeast JEN1 into a mevalonate transporter

Abstract The in vivo continuous evolution system OrthoRep (orthogonal replication) is a powerful strategy for rapid enzyme evolution inSaccharomyces cerevisiaethat diversifies genes at a rate exceeding the endogenous genome mutagenesis rate by several orders of magnitude. However, it is difficult to neofunctionalize genes using OrthoRep partly because of the way selection pressures are applied. Here we combine OrthoRep with optogenetics in a selection strategy we call OptoRep, which allows fine-tuning of selection pressure with light. With this capability, we evolved a truncated form of the endogenous monocarboxylate transporterJEN1 (JEN1t)into a de novo mevalonate importer. We demonstrate the functionality of the evolvedJEN1t(JEN1t Y180C/G ) in the production of farnesene, a renewable aviation biofuel, from mevalonate fed to fermentation media or produced by microbial consortia. This study shows that the light-induced complementation of OptoRep may improve the ability to evolve functions not currently accessible for selection, while its fine tunability of selection pressure may allow the continuous evolution of genes whose desired function has a restrictive range between providing effective selection and cellular viability.

Biochemistry & Molecular Biology↗

DOE OSTI2024

Genomic prediction of regional-scale performance in switchgrass ( Panicum virgatum ) by accounting for genotype-by-environment variation and yield surrogate traits

Switchgrass is a potential crop for bioenergy or carbon capture schemes, but further yield improvements through selective breeding are needed to encourage commercialization. To identify promising switchgrass germplasm for future breeding efforts, we conducted multisite and multitrait genomic prediction with a diversity panel of 630 genotypes from 4 switchgrass subpopulations (Gulf, Midwest, Coastal, and Texas), which were measured for spaced plant biomass yield across 10 sites. Our study focused on the use of genomic prediction to share information among traits and environments. Specifically, we evaluated the predictive ability of cross-validation (CV) schemes using only genetic data and the training set (cross-validation 1: CV1), a subset of the sites (cross-validation 2: CV2), and/or with 2 yield surrogates (flowering time and fall plant height). We found that genotype-by-environment interactions were largely due to the north–south distribution of sites. The genetic correlations between the yield surrogates and the biomass yield were generally positive (mean height r = 0.85; mean flowering time r = 0.45) and did not vary due to subpopulation or growing region (North, Middle, or South). Genomic prediction models had CV predictive abilities of –0.02 for individuals using only genetic data (CV1), but 0.55, 0.69, 0.76, 0.81, and 0.84 for individuals with biomass performance data from 1, 2, 3, 4, and 5 sites included in the training data (CV2), respectively. To simulate a resource-limited breeding program, we determined the predictive ability of models provided with the following: 1 site observation of flowering time (0.39); 1 site observation of flowering time and fall height (0.51); 1 site observation of fall height (0.52); 1 site observation of biomass (0.55); and 5 site observations of biomass yield (0.84). The ability to share information at a regional scale is very encouraging, but further research is required to accurately translate spaced plant biomass to commercial-scale sward biomass performance.

09 BIOMASS FUELS↗

DOE OSTI2025

Leveraging CRISPR Cas9 RNPs and Cre- loxP in Picochlorum celeri for generation of field deployable strains and selection marker recycling

As new highly productive strains of algae are discovered and developed to meet the energy, chemical, and food requirements of the future, genetic engineering of those strains in a manner that yields deployable transformants is paramount. This study introduces the novel CRoxP ($\underline{\textrm{C}}$$\textrm{as9}$ $\underline{\textrm{R}}$$\textrm{NPs}$ coupled with an inducible $\underline{\textrm{CR}}$$\textrm{e}$-$\textrm{l}\underline{\textrm{oxP}}$) system for rapid generation of marker- and transgene-free strains of Picochlorum celeri. The CRoxP system allows reuse of selection markers without Cas9 expression in vivo, eliminating many of the bottlenecks associated with conventional CRISPR Cas9 use for precise genome editing. In P. celeri, transformants were generated with a turnaround time as short as 21 days between transformation and being ready for another round of transformation with the same selection marker by using the CRoxP system. As a use-case for CRoxP, depigmented strains of P. celeri were generated by multiplexed Cas9 disruption of major LHCII genes followed by either a second round of LHCII targeting, or knockout of an LHCI gene. One transformant tested in flask culture (R6) exhibited similar biomass production to the wild type with 46% less Chl a + b on a biomass basis. In photobioreactors and under diel light simulating a solar day, a transformant (LhcBM31) exhibited 34 g AFDW m –2 d –1 with 54% less Chl a + b on a biomass basis vs. wild type.

09 BIOMASS FUELS↗

NASA NTRSDate not supplied

Cleanroom Microbes Survive Drying, Vacuum, and Proton Irradiation

Introduction : The goal of planetary protection at NASA is to mitigate the risk of contaminating sensitive target bodies with biological life. While many cleaning procedures have been put in place to reduce bioburden on spacecraft, microbes are experts at evolving to survive harsh conditions. Specifically, the dry, low-nutrient environment of a cleanroom (commonly used for assembly of spacecraft) can represent an environment where extremophiles can survive. Methods : Scientists at NASA MSFC wished to gather a snapshot of the microbial population within a variety of cleanrooms on site. A study was undertaken to collect air, surface, and floor samples from clean-rooms and isolate unique morphologies. From this study, 95 isolates were collected and saved in a microbial library. About 86% of these were identified at least to a genus level. Following identification, 24 microbes were selected, based on a literature review, as potential extremophiles. These were grown in liquid cultures, diluted to a set optical density, washed with water, and then applied to a sterilized Kapton coupon. Droplets were allowed to dry overnight in a biosafety cabinet. Coupons were then installed in a pelletron and pumped down to high vacuum (~1E-6 Torr). Samples were then subjected 100 keV protons at a fluence of 2x10 15 p+/cm 2 up to 4x10 15 p+/cm 2 . Following exposure, samples were returned to the microbiology lab where they were pro-cessed by submerging in water, vortexing, and then plating either droplets or spread plates. Recovery data collected was qualitative with a ranking or +, minor, or – for growth. Some selected radiotolerant strains were sequenced using the Illumina sequencing platform. The resulting genomes were annotated with the Rapid Annotations using Subsystems Technology (RAST) server and analyzed for conserved and unique stress response relevant genomic signatures to identify clues related to specific tolerances. Results and Discussion : After five rounds of proton radiation, we narrowed our isolates to five, non-spore forming bacteria that demonstrated survival: Arthrobacter koreensis, Paenarthrobacter nitroguajacolicus, Mycetocola manganoxydans , and an Erwinia sp. Furthermore, we exposed these four microbes to 254 nm wavelength light at an intensity of 80 W/m 2 at a distance of ~18 cm for 10 minutes. Only A. koreensis demonstrated survival following UV exposure. Finally, we performed whole genome sequencing on the four strains to look for genetic markers of stress resistance. When we compared the genomes of the four strains, we found that genes coding for GGDEF and EAL domains with PAS/PAC sensors were only found in A. koreensis . These domains, modulated by PAS/PAC sensors, are hypothesized to facilitate survival under drying, desiccation, and proton irradiation. Drying and Desiccation : PAS domains sense hydration changes and modulate GGDEF and EAL domain activity to adjust c-di-GMP levels, enhancing resistance to desiccation. For instance, in Pseudomonas aeruginosa , the PAS domain of RbdA modulates activity under varying hydration conditions, affecting stress responses [1]. Proton Irradiation : Proton irradiation causes oxidative stress, leading to ROS generation. PAS domains detect this stress and modulate GGDEF and EAL domains to manage oxidative stress responses. In Shewanella , EAL domain proteins modulated by PAS sensors help bacteria adapt to extreme conditions [2]. These genes upregulate other stress response genes, protecting membrane function, protein stability, DNA repair, and antioxidant defenses. The modulation of c-di-GMP by PAS domains is crucial for bacterial adaptation to stress conditions, enabling dynamic physio-logical adjustments [3]. Understanding these mechanisms provides insights into bacterial stress responses and strategies for controlling bacterial growth [4]. Conclusions : These findings indicate that clean-rooms harbor extremophile microbes that may be able to survive conditions in deep space. Furthermore, while we identified certain stress-response genes that may be at least partly responsible for the phenotypes observed in this study, there are likely unidentified genes or characteristics about A. koreensis , and other bacteria, that may allow them to survive in harsh environments. Future studies will focus on identifying these unknown genes and characteristics, further elucidating the mechanisms of extremophile survival and potentially informing the development of new biotechnologies for space exploration and other extreme environments.

Chelsi Cassilly↗

DOE OSTI2026

Continental-scale integration of soil metagenomes and organic matter chemistry reveals ubiquitous microbial capacity for chemically-recalcitrant carbon decomposition

Soil organic matter (SOM) decomposition by microorganisms is a major uncertainty in predicting terrestrial carbon–atmosphere feedbacks, partly because we lack understanding of the microbial diversity involved in depolymerizing different carbon pools across environmental gradients. We address this gap using a continental-scale dataset pairing shotgun metagenomes with high-resolution SOM chemistry, assembling 0.76 Tbp of prokaryotic MAGs (828 genomes) and identifying 66,727 SOM molecules from 47 standardized U.S. soil cores selected using respiration rates from 106 soils. Integrating these datasets reveals widespread microbial potential for depolymerizing chemically-recalcitrant SOM previously considered stable. We uncover complementary metabolic specialization between genera affiliated with two abundant bacterial orders, Rhizobiales and Chthoniobacterales, and an archaeal order, Nitrososphaerales. This metabolic partitioning is consistent across soil depths and activity levels, suggesting coordinated decomposition of complex SOM through distinct but complementary biochemical strategies. The metabolic potential for depolymerization of chemically-recalcitrant compounds is supported by the abundance of these molecules across the soils, as indicated by Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry (FTICR-MS), and by flux balance analysis of metabolic models. Our results show that a substantial portion of ostensibly stable SOM remains vulnerable to microbial decomposition, a mechanism not captured in current Earth System Models.

Song, Young C. [Pacific Northwest National Laborat↗

DOE OSTI2026

Vicennial metagenomic time series unveils evolutionary dynamics of giant viruses in a freshwater ecosystem

Giant viruses play crucial ecological roles in aquatic ecosystems, yet their evolutionary dynamics in response to environmental changes, particularly in freshwater environments, are not well understood. We analyzed a 20-year time series (2000-2019) of 471 co-assembled metagenomes from Lake Mendota (USA) to reconstruct 1512 giant virus metagenome-assembled genomes, providing insights into viral genome evolution. Viruses in the order Imitervirales dominate the virome, remaining consistent across seasons and years. Our findings reveal gene duplication (23% of genes) and horizontal gene transfer (29% of genes) as key drivers of genomic innovation. A co-occurrence network analysis indicates increased virus-host interactions following the introduction of an invasive predatory zooplankton in 2009, highlighting potential hosts in Bigyra, Perkinsea, and Euglenozoa. While single nucleotide polymorphism analysis shows predominantly purifying selection in viral genes, there is a significant increase in positively selected genes post-invasion, particularly those related to infection. Comparative evolutionary analyses reveal that giant viruses exhibit genome-wide substitution rates similar to co-occurring bacteria but significantly slower than smaller dsDNA phages, suggesting both stability and adaptability. Our study demonstrates that freshwater giant viruses employ various evolutionary strategies to respond to environmental change. These results underscore their significant yet often underappreciated role in freshwater ecosystem dynamics.

Vasquez, Yumary M↗

NASA NTRS2015

Karyotyping of Chromosomes in Human Bronchial Epithelial Cells Transformed by High Energy Fe Ions

Lung cancer induced from exposures to space radiation is one of the most significant health risks for long-term space travels. Evidences show that low- and high- Linear energy transfer (LET)-induced transformation of normal human bronchial epithelial cells (HBEC) that are immortalized through the expression of Cdk4 and hTERT. The cells were exposed to gamma rays and high-energy Fe ions for the selection of transformed clones. Transformed HBEC are identified and analyzed chromosome aberrations (i.e. genomic instability) using the multi-color fluorescent in situ hybridization (mFISH), as well as the multi-banding in situ hybridization (mBAND) techniques. Our results show chromosomal translocations between different chromosomes and several of the breaks occurred in the q-arm of chromosome 3. We also identified copy number variations between the transformed and the parental HBEC regardless of the exposure conditions. We observed chromosomal aberrations in the lowand high-LET radiation-induced transformed clones and they are imperfectly different from clones obtain in spontaneous soft agar growth.

Yeshitla, Samrawit↗

NASA NTRS2016

Automating Microbial Directed Evolution For Bioengineering Applications

From a micro-biology perspective, directed evolution is a technique that uses controlled environmental pressures to select for a desired phenotype. Directed evolution has the distinct advantage over rational design of not needing extensive knowledge of the genome or pathways associated with a microorganism to induce phenotypes. However, there are currently limitations to the applicability of this technique including being time-consuming, error-prone, and dependent on existing assays that may lack selectivity for the given phenotype. The AADEC (Autonomous Adaptive Directed Evolution Chamber) system is a proof-of-concept instrument to automate and improve the technique such that directed evolution can be used more effectively as a general bioengineering tool. A series of tests using the automated system and comparable by-hand survival assay measurements have been carried out using UV-C radiation and Escherichia coli cultures in order to demonstrate the advantages of the AADEC versus traditional implementations of directed evolution such as random mutagenesis. AADEC uses UV-C exposure as both a source of environmental stress and mutagenesis, so in order to evaluate the UV-C tolerance obtained from the cultures, a manual UV-C exposure survival assay was developed alongside the device to compare the survival fractions at a fixed dosage. This survival assay involves exposing E. coli to UV-C radiation using a custom-designed exposure hood to control the flux and dose. Surviving cells are counted then transferred to the next iteration and so on for several iterations to calculate the survival fractions for each exposure iteration.This survival assay primarily serves as a baseline for the AADEC device, allowing quantification of the differences between the AADEC system over the manual approach. The primary data of comparison is survival fractions; this is obtained by optical density and plate counts in the manual assay and by optical density growth curve fits pre- and post-exposure in the automated case. This data can then be compiled to calculate trends over the iterations to characterize increasing UV-C resistance of the E.coli strains. The observed trends are statistically indistinguishable through several iterations from both sources.

Bioengineering↗

NASA NTRS2003

Enhancement of anion-exchange chromatography of DNA using compaction agents

The use of adsorptive chromatography for preparative nucleic acid separations is often limited by low capacity. The possibility that the adsorbent surface area sterically accessible to nucleic acid molecules could be increased by reducing their radius of gyration with compaction agents has been investigated. The equilibrium adsorption capacity of Q Sepharose anion-exchange matrix for plasmid DNA at 600 mM NaCl was enhanced by up to ca. 40% in the presence of 2.5 mM spermine. In addition, compaction agent selectivity has been demonstrated. Spermine, for example, enhances the adsorption of both plasmid and genomic DNA, spermidine enhances binding only of plasmid, and hexamine cobalt enhances only the binding of genomic DNA. Compaction may be generally useful for enhancing adsorptive separations of nucleic acids.

NASA Discipline Environmental Health↗

NASA NTRS2012

Development of Two Color Fluorescent Imager and Integrated Fluidic System for Nanosatellite Biology Applications

Nanosatellites offer frequent, low-cost space access as secondary payloads on launches of larger conventional satellites. We summarize the payload science and technology of the Microsatellite in-situ Space Technologies (MisST) nanosatellite for conducting automated biological experiments. The payload (two fused 10-cm cubes) includes 1) an integrated fluidics system that maintains organism viability and supports growth and 2) a fixed-focus imager with fluorescence and scattered-light imaging capabilities. The payload monitors temperature, pressure and relative humidity, and actively controls temperature. C. elegans (nematode, 50 m diameter x 1 mm long) was selected as a model organism due to previous space science experience, its completely sequenced genome, size, hardiness, and the variety of strains available. Three strains were chosen: two green GFP-tagged strains and one red tdTomato-tagged strain that label intestinal, nerve, and pharyngeal cells, respectively. The integrated fluidics system includes bioanalytical and reservoir modules. The former consists of four 150 L culture wells and a 4x5 mm imaging zone the latter includes two 8 mL fluid reservoirs for reagent and waste storage. The fluidic system is fabricated using multilayer polymer rapid prototyping: laser cutting, precision machining, die cutting, and pressure-sensitive adhesives it also includes eight solenoid-operated valves and one mini peristaltic pump. Young larval-state (L2) nematodes are loaded in C. elegans Maintenance Media (CeMM) in the bioanalytical module during pre-launch assembly. By the time orbit is established, the worms have grown to sufficient density to be imaged and are fed fresh CeMM. The strains are pumped sequentially into the imaging area, imaged, then pumped into waste. Reagent storage utilizes polymer bags under slight pressure to prevent bubble formation in wells or channels. The optical system images green and red fluorescence bands by excitation with blue (473 nm peak) and amber (587 nm peak) LEDs it achieves 8 m lateral resolution using a CMOS imaging chip (as configured for serial data speeds) or 4 m resolution using USB imaging chips. The imager consists of a modified commercial off-the-shelf CMOS chip camera, amber, blue and white LEDs, as well as a relay lens and dual-band filters to obviate moving parts while supporting both fluorescence wavelengths.

Wu, Diana Terri↗