Results for “KB” — Page 7 | Engineering Papers

DOE OSTI2024

KBase Narrative - Porphyromonadaceae sp. W3.11 genome

Narratives for The phenotype and genotype of fermentative prokaryotes This is the Narrative for Porphyromonadaceae sp. W3.11. A complementary Narrative for Lachnospiraceae sp. C1.1 is available here. This is the Narrative for Lachnospiraceae sp. C1.1. A complementary Narrative for Porphyromonadaceae sp. W3.11 is available here. Background and Isolation This Narrative and its complementary Narrative contain assembly and annotation of two bacterial isolates that were isolated by our laboratory from the rumen of a Holstein heifer. All procedures with animals have been approved by University of California Davis’s Institutional Animal Care and Use Committee. Rumen contents were collected through a rumen fistula and strained through two layers of cheesecloth into a bottle. The bottle was sealed to exclude air and maintained at 39°C. Contents were brought to the laboratory and bubbled under O2-free CO2 within 15 min. At the laboratory, serial dilutions were made with anaerobic dilution solution for Lachnospiraceae sp. C1.1 and propionibacterium diluent for Porphyromonadaceae sp. W3.11 (table S2). Aliquots (0.1 ml) of each dilution were injected into anaerobic bottle plates (1) containing 9 ml of LH medium (table S2). After incubation at 37°C for 7 days, isolated colonies were picked. Lachnospiraceae sp. C1.1 was picked from a bottle inoculated with a 104 dilution of rumen contents, and Porphyromonadaceae sp. W3.11 was picked from a bottle inoculated with a 103 dilution. After initial isolation, these organisms were purified by growing on anaerobic roll tubes (2) and picking isolated colonies. We performed de novo sequencing of Lachnospiraceae sp. C1.1 and Porphyromonadaceae sp. W3.11. Aliquots of liquid culture (9 and 1.5 ml, respectively) were collected by syringe and centrifuged (21,000g for 10 min at 4°C). Cell pellets were submitted to Molecular Research LP for DNA extraction, library preparation, and sequencing. After resuspending pellets in 180 µl of ATL buffer (Qiagen), DNA was extracted using the MagAttract HMW DNA Kit (Qiagen). DNA was eluted in 100 µl of AE buffer (Qiagen) and then cleaned using the DNEasy PowerClean Pro Cleanup Kit (Qiagen). DNA was then sheared using the Covaris g-TUBE (Covaris). Sequencing libraries were prepared using the SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences) and 1500 ng of the sheared and purified DNA. The SMRTbell libraries were size-selected (>6 Kb) using a BluePippin instrument (Sage Science) and 0.75% agarose gel. Libraries were then sequenced using the PacBio Sequel II (Pacific Biosciences) platform and a 30-hour movie time. Narrative Summary In these Narratives, we filtered low-quality reads using Trimmomatic (v0.36), assembled filtered reads with SPAdes (v3.15.3), and then checked completeness and contamination of the assembled genomes with CheckM (v1.0.18). Statistics for sequencing and assembly are in table S3. Using the assembled contigs (genomes), we called genes and annotated them. Protein-coding genes were called using Prodigal (v2.6.3) (3) locally or using KBase via RASTtk (v1.073), with identical results. Genes were annotated with KO IDs using KAAS (4). They were further annotated with pfam and TIGRFAM IDs using KBase and the Annotate Domains in a Genome app. We classified putative genes for hydrogenases using HydDB. Genes for 16S ribosomal RNA (rRNA) were called using RASTtk (v1.073) in KBase. The contigs (genomes) were analyzed to determine whether they belonged to new species. Taxonomy was assigned using GTDB-Tk (v1.7.0) in KBase. The identity of 16S rRNA genes to other organisms was found using EzBioCloud (5). Values of digital DNA-DNA hybridization (dDDH) were found with Type (Strain) Genome Server (6). These analyses suggest that Lachnospiraceae sp. C1.1 and Porphyromonadaceae sp. W3.11 represent novel species or genera. GTDB-Tk assigned Lachnospiracae sp. C1.1 to family Lachnospiraceae and genus NK4A144, which contains no type strains. It assigned Porphyromonadaceae sp. W3.11 to Porphyromonadaceae and genus Porphyromonas_A. Values of 16S rRNA identity and dDDH with respect to type strains were low (table S4). Although more phenotypic data are needed, available evidence supports assignment of genomes to new species or genera. Related publication Hackmann TJ, Zhang B. The phenotype and genotype of fermentative prokaryotes. Sci Adv. 2023 Sep 29;9(39):eadg8687. doi: 10.1126/sciadv.adg8687. Epub 2023 Sep 27. PMID: 37756392; PMCID: PMC10530074.

Hackmann, Timothy↗

DOE OSTI2024

KBase Narrative - Lachnospiraceae sp. C1.1 genome

Narratives for The phenotype and genotype of fermentative prokaryotes This is the Narrative for Porphyromonadaceae sp. W3.11. A complementary Narrative for Lachnospiraceae sp. C1.1 is available here. This is the Narrative for Lachnospiraceae sp. C1.1. A complementary Narrative for Porphyromonadaceae sp. W3.11 is available here. Background and Isolation This Narrative and its complementary Narrative contain assembly and annotation of two bacterial isolates that were isolated by our laboratory from the rumen of a Holstein heifer. All procedures with animals have been approved by University of California Davis’s Institutional Animal Care and Use Committee. Rumen contents were collected through a rumen fistula and strained through two layers of cheesecloth into a bottle. The bottle was sealed to exclude air and maintained at 39°C. Contents were brought to the laboratory and bubbled under O2-free CO2 within 15 min. At the laboratory, serial dilutions were made with anaerobic dilution solution for Lachnospiraceae sp. C1.1 and propionibacterium diluent for Porphyromonadaceae sp. W3.11 (table S2). Aliquots (0.1 ml) of each dilution were injected into anaerobic bottle plates (1) containing 9 ml of LH medium (table S2). After incubation at 37°C for 7 days, isolated colonies were picked. Lachnospiraceae sp. C1.1 was picked from a bottle inoculated with a 104 dilution of rumen contents, and Porphyromonadaceae sp. W3.11 was picked from a bottle inoculated with a 103 dilution. After initial isolation, these organisms were purified by growing on anaerobic roll tubes (2) and picking isolated colonies. We performed de novo sequencing of Lachnospiraceae sp. C1.1 and Porphyromonadaceae sp. W3.11. Aliquots of liquid culture (9 and 1.5 ml, respectively) were collected by syringe and centrifuged (21,000g for 10 min at 4°C). Cell pellets were submitted to Molecular Research LP for DNA extraction, library preparation, and sequencing. After resuspending pellets in 180 µl of ATL buffer (Qiagen), DNA was extracted using the MagAttract HMW DNA Kit (Qiagen). DNA was eluted in 100 µl of AE buffer (Qiagen) and then cleaned using the DNEasy PowerClean Pro Cleanup Kit (Qiagen). DNA was then sheared using the Covaris g-TUBE (Covaris). Sequencing libraries were prepared using the SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences) and 1500 ng of the sheared and purified DNA. The SMRTbell libraries were size-selected (>6 Kb) using a BluePippin instrument (Sage Science) and 0.75% agarose gel. Libraries were then sequenced using the PacBio Sequel II (Pacific Biosciences) platform and a 30-hour movie time. Narrative Summary In these Narratives, we filtered low-quality reads using Trimmomatic (v0.36), assembled filtered reads with SPAdes (v3.15.3), and then checked completeness and contamination of the assembled genomes with CheckM (v1.0.18). Statistics for sequencing and assembly are in table S3. Using the assembled contigs (genomes), we called genes and annotated them. Protein-coding genes were called using Prodigal (v2.6.3) (3) locally or using KBase via RASTtk (v1.073), with identical results. Genes were annotated with KO IDs using KAAS (4). They were further annotated with pfam and TIGRFAM IDs using KBase and the Annotate Domains in a Genome app. We classified putative genes for hydrogenases using HydDB. Genes for 16S ribosomal RNA (rRNA) were called using RASTtk (v1.073) in KBase. The contigs (genomes) were analyzed to determine whether they belonged to new species. Taxonomy was assigned using GTDB-Tk (v1.7.0) in KBase. The identity of 16S rRNA genes to other organisms was found using EzBioCloud (5). Values of digital DNA-DNA hybridization (dDDH) were found with Type (Strain) Genome Server (6). These analyses suggest that Lachnospiraceae sp. C1.1 and Porphyromonadaceae sp. W3.11 represent novel species or genera. GTDB-Tk assigned Lachnospiracae sp. C1.1 to family Lachnospiraceae and genus NK4A144, which contains no type strains. It assigned Porphyromonadaceae sp. W3.11 to Porphyromonadaceae and genus Porphyromonas_A. Values of 16S rRNA identity and dDDH with respect to type strains were low (table S4). Although more phenotypic data are needed, available evidence supports assignment of genomes to new species or genera. Related publication Hackmann TJ, Zhang B. The phenotype and genotype of fermentative prokaryotes. Sci Adv. 2023 Sep 29;9(39):eadg8687. doi: 10.1126/sciadv.adg8687. Epub 2023 Sep 27. PMID: 37756392; PMCID: PMC10530074.

Hackmann, Timothy↗

DOE OSTI2026

Genome_shuffling_enables_quantitative_trait_locus_mapping_in_Bacillus_subtilis

Genetic mapping is a powerful tool for eukaryotic genetics that has only been applied to bacteria in limited circumstances. Quantitative trait locus (QTL) mapping generally relies on sexual recombination to break linkages between genes, yet bacteria rarely undergo sufficient homologous recombination to generate suitable mapping populations. In this work, we used iterative biparental genome shuffling by protoplast fusion inBacillus subtilisto generate a population of bacteria with substantial random recombination throughout their genomes. Individual shuffled progeny were arrayed in well plates, resequenced, and characterized for a range of complex phenotypes including spore germination and swarming motility. Genetic mapping of the resulting phenotypes identified high-confidence QTLs of moderate size (∼10 kb), and these associations were validated through targeted genetic swaps. ThisB. subtilisQTL population can easily be used to map additional phenotypes, and the general approach for QTL mapping is applicable in a wide range of bacteria.

Bacillus subtilis↗

DOE OSTI2022

Clostridium autoethanogenum isopropanol production via native plasmid pCA replicon

Clostridium autoethanogenum is a model gas-fermenting acetogen for commercial ethanol production. It is also a platform organism being developed for the carbon-negative production of acetone and isopropanol by gas fermentation. We have assembled a 5.5 kb pCA plasmid for type strain DSM10061 (JA1-1) using three genome sequence datasets. pCA is predicted to encode seven open-reading frames and estimated to be a low-copy number plasmid present at approximately 12 copies per chromosome. RNA-seq analyses indicate that pCA genes are transcribed at low levels and two proteins, CAETHG_05090 (putative replication protein) and CAETHG_05115 (hypothetical, a possible Mob protein), were detected at low levels during batch gas fermentations. Thiolase (thlA), CoA-transferase (ctfAB), and acetoacetate decarboxylase (adc) genes were introduced into a vector for isopropanol production in C. autoethanogenum using the native plasmid origin of replication. The availability of the pCA sequence will facilitate studies into its physiological role and could form the basis for genetic tool optimization.

09 BIOMASS FUELS↗

DOE OSTI2021

Hybridization History and Repetitive Element Content in the Genome of a Homoploid Hybrid, Yucca gloriosa (Asparagaceae)

Hybridization in plants results in phenotypic and genotypic perturbations that can have dramatic effects on hybrid physiology, ecology, and overall fitness. Hybridization can also perturb epigenetic control of transposable elements, resulting in their proliferation. Understanding the mechanisms that maintain genomic integrity after hybridization is often confounded by changes in ploidy that occur in hybrid plant species. Homoploid hybrid species, which have no change in chromosome number relative to their parents, offer an opportunity to study the genomic consequences of hybridization in the absence of change in ploidy. Yucca gloriosa (Asparagaceae) is a young homoploid hybrid species, resulting from a cross between Yucca aloifolia and Yucca filamentosa. Previous analyses of ~11 kb of the chloroplast genome and nuclear-encoded microsatellites implicated a single Y. aloifolia genotype as the maternal parent of Y. gloriosa. Using whole genome resequencing, we assembled chloroplast genomes from 41 accessions of all three species to re-assess the hybrid origins of Y. gloriosa. We further used re-sequencing data to annotate transposon abundance in the three species and mRNA-seq to analyze transcription of transposons. The chloroplast phylogeny and haplotype analysis suggest multiple hybridization events contributing to the origin of Y. gloriosa, with both parental species acting as the maternal donor. Transposon abundance at the superfamily level was significantly different between the three species; the hybrid was frequently intermediate to the parental species in TE superfamily abundance or appeared more similar to one or the other parent. In only one case—Copia LTR transposons—did Y. gloriosa have a significantly higher abundance relative to either parent. Expression patterns across the three species showed little increased transcriptional activity of transposons, suggesting that either no transposon release occurred in Y. gloriosa upon hybridization, or that any transposons that were activated via hybridization were rapidly silenced. The identification and quantification of transposon families paired with expression evidence paves the way for additional work seeking to link epigenetics with the important trait variation seen in this homoploid hybrid system.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Dependence of Solar Wind Proton Temperature on the Polarization Properties of Alfvénic Fluctuations at Ion-kinetic Scales

In this work, we use fluctuating magnetic helicity to investigate the polarization properties of Alfvénic fluctuations at ion-kinetic scales in the solar wind as a function of β p , the ratio of proton thermal pressure to magnetic pressure, and θ vB , the angle between the proton flow and local mean magnetic field, B 0 . Using almost 15 yr of Wind observations, we separate the contributions to helicity from fluctuations with wavevectors, k, quasi-parallel and oblique to B 0 , finding that the helicity of Alfvénic fluctuations is consistent with predictions from linear Vlasov theory. This result suggests that the nonlinear turbulent fluctuations at these scales share at least some polarization properties with Alfvén waves. Furthermore, we also investigate the dependence of proton temperature in the β p –θ vB plane to probe for possible signatures of turbulent dissipation, finding that it correlates with θ vB . The proton temperature parallel to B 0 is higher in the parameter space where we measure the helicity of right-handed Alfvénic fluctuations, and the temperature perpendicular to B 0 is higher where we measure left-handed fluctuations. This finding is inconsistent with the general assumption that by sampling different θ vB in the solar wind we can analyze the dependence of the turbulence distribution on θ kB , the angle between k and B 0 . After ruling out both instrumental and expansion effects, we conclude that our results provide new evidence for the importance of local kinetic processes that depend on θ vB in determining proton temperature in the solar wind.

79 ASTRONOMY AND ASTROPHYSICS↗

DOE OSTI2021

PlasmidMaker: a Versatile, Automated, and High Throughput End-to-End Platform for Plasmid Construction

Plasmids are used extensively in basic and applied biology. However, design and construction of plasmids, specifically the ones carrying complex genetic information, remains one of the most time-consuming, labor-intensive, and rate-limiting steps in performing sophisticated biological experiments. Here, we report the development of a versatile, robust, automated end-to-end platform named PlasmidMaker that allows error-free construction of plasmids with virtually any sequences in a high-throughput manner. This platform consists of a most versatile DNA assembly method using Pyrococcus furiosus Argonaute ( Pf Ago)-based artificial restriction enzymes, a user-friendly frontend for plasmid design, and a backend that streamlines the workflow and integration with a robotic system. As a proof of concept, we used this platform to generate ~100 plasmids from six different species ranging from 5 to 18 kb in size from up to 11 DNA fragments within 3 days. PlasmidMaker should greatly expand the potential of synthetic biology.

Enghiad, Behnam↗

DOE OSTI2022

Design of a neutral thermal scattering (NeTS) module for hydrogen in light water

The accurate representation of thermal scattering law (TSL) data is integral to the design and characterization of many modern nuclear systems, particularly those using light water as a moderator/coolant. As a material-dependent distribution over energy-momentum phase space, the TSL may exhibit a variety of unique and relevant conditional (e.g., temperature, pressure) and compositional (e.g., porosity, stoichiometry, radiation damage) dependencies. Currently, there are various approaches to incorporate temperature dependence, which is especially important in coupled neutronic-thermal hydraulic simulations. In each approach, there is an inherent tradeoff between memory consumption and accuracy. Some techniques require tens to hundreds of MBs or more, while others fail to reproduce the underlying data to within 10% error over the considered input domain, despite having a reduced storage burden. This work aims to address both sides of the tradeoff simultaneously by implementing a novel deep learning (DL) approach to TSL representation. The neural thermal scattering (NeTS) concept, which is amenable to an arbitrary number of dependencies, is demonstrated via the inclusion of temperature dependence into a highly compact, highly accurate functional form of the multi- variate TSL (i.e., S(α, β, T)) for hydrogen in light water. Resulting storage requirements are on the order of 100 kB, and median and maximum percent deviations are on the order of 0.1% and 1%, respectively. These measures represent a step improvement over previous techniques. Notably, the developed neural network and feature methodology build on those employed in prior work on beryllium oxide. (authors)

22 GENERAL STUDIES OF NUCLEAR REACTORS↗

DOE OSTI2020

An acidic polysaccharide (AGC3) isolated from North American ginseng ( Panax quinquefolius ) suspension culture as a potential immunomodulatory nutraceutical

Polysaccharides isolated from Panax quinquefolius roots are widely used as nutraceuticals due to their immunomodulatory properties. Despite their popularity, several challenges exist in isolating ginseng root polysaccharides such as batch-to-batch structural inconsistencies and bacterial endotoxin contamination. A plant tissue culture-based platform offers a potential solution to isolate natural polysaccharide fractions with consistent chemical characteristics and reduced endotoxin content. In this study, an acidic polysaccharide fraction (AGC3) with immunomodulatory properties was isolated from Panax quinquefolius suspension cultures. The heterogeneous fraction (molecular weight: 4.81 and 32.14 kDa), purified by anion exchange chromatography, was predominantly composed of galactose (>60%) along with the presence of rhamnose, arabinose, glucose, glucuronic acid and galacturonic acid. The major glycosidic linkages were found to be t-Galp (47.7%), 4-Galp (15.6%), 2,4-Rhap (8.1%), 6-Galp (8.1%) and 4-GalAp (6.8%). Structural analyses indicated the presence of a pectic rhamnogalacturonan I polysaccharide in AGC3. AGC3 significantly (p < 0.05) stimulated RAW 264.7 murine macrophage cells and primary murine splenocytes by enhancing the production of several immunomodulatory mediators such as IL-6, TNF-α, GM-CSF and MCP-1. The results also indicated the putative roles of NF-κB (p65/RelA) and MAPK (p38) signaling pathways in the immunostimulatory response. Additionally, AGC3 induced murine splenocyte proliferation, another major indicator of immunostimulation. Overall, AGC3 has the potential to be used as an immunostimulatory nutraceutical.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Multidimensional perspectives of geo-epidemiology: from interdisciplinary learning and research to cost–benefit oriented decision-making

Research typically promotes two types of outcomes (inventions and discoveries), which induce a virtuous cycle: something suspected or desired (not previously demonstrated) may become known or feasible once a new tool or procedure is invented and, later, the use of this invention may discover new knowledge. Research also promotes the opposite sequence—from new knowledge to new inventions. This bidirectional process is observed in geo-referenced epidemiology—a field that relates to but may also differ from spatial epidemiology. Geo-epidemiology encompasses several theories and technologies that promote inter/transdisciplinary knowledge integration, education, and research in population health. Based on visual examples derived from geo-referenced studies on epidemics and epizootics, this report demonstrates that this field may extract more (geographically related) information than simple spatial analyses, which then supports more effective and/or less costly interventions. Actual (not simulated) bio-geo-temporal interactions (never captured before the emergence of technologies that analyze geo-referenced data, such as geographical information systems) can now address research questions that relate to several fields, such as Network Theory. Thus, a new opportunity arises before us, which exceeds research: it also demands knowledge integration across disciplines as well as novel educational programs which, to be biomedically and socially justified, should demonstrate cost-effectiveness. Grounded on many bio-temporal-georeferenced examples, this report reviews the literature that supports this hypothesis: novel educational programs that focus on geo-referenced epidemic data may help generate cost-effective policies that prevent or control disease dissemination.

59 BASIC BIOLOGICAL SCIENCES↗