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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 181 records · Page 10
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Quantification of Metabolic Products from Microbial Hosts in Complex Media Using Optically Diffracting Hydrogels

In this work, we herein describe a highly versatile platform approach for the in situ and real-time screening of microbial biocatalysts for enhanced production of bioproducts using photonic crystal hydrogels. This approach was demonstrated by preparing optically diffracting films based on polymerized N-isopropylacrylamide that contracted in the presence of alcohols and organic acids. The hydrogel films were prepared in a microwell plate format, which allows for high-throughput screening, and characterized optically using a microwell plate reader. While demonstrating the ability to detect a broad range of relevant alcohols and organic acids, we showed that the response of the films correlated strongly with the octanol-water partition coefficient (log P) of the analyte. Differences in the secretion of ethanol and succinic acid from strains of Zymomonas mobilis and Actinobacillus succinogenes, respectively, were further detected via optical characterization of the films. These differences, which in some cases were as low as ~3 g/L, were confirmed by high-performance liquid chromatography, thereby demonstrating the sensitivity of this approach. Our findings highlight the potential utility of this multiplexed approach for the detection of small organic analytes in complex biological media, which overcomes a major challenge in conventional optical sensing methods.

09 BIOMASS FUELS↗
DOE OSTI2023

Quantifying the local adaptive landscape of a nascent bacterial community

Abstract The fitness effects of all possible mutations available to an organism largely shape the dynamics of evolutionary adaptation. Yet, whether and how this adaptive landscape changes over evolutionary times, especially upon ecological diversification and changes in community composition, remains poorly understood. We sought to fill this gap by analyzing a stable community of two closely related ecotypes (“L” and “S”) shortly after they emerged within the E. coli Long-Term Evolution Experiment (LTEE). We engineered genome-wide barcoded transposon libraries to measure the invasion fitness effects of all possible gene knockouts in the coexisting strains as well as their ancestor, for many different, ecologically relevant conditions. We find consistent statistical patterns of fitness effect variation across both genetic background and community composition, despite the idiosyncratic behavior of individual knockouts. Additionally, fitness effects are correlated with evolutionary outcomes for a number of conditions, possibly revealing shifting patterns of adaptation. Together, our results reveal how ecological and epistatic effects combine to shape the adaptive landscape in a nascent ecological community.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Author Correction: Engineering self-deliverable ribonucleoproteins for genome editing in the brain

Correction to: Nature Communicationshttps://doi.org/10.1038/s41467-024-45998-2, published online 26 February 2024. In the Acknowledgements section of this article, the grant number relating to National Institutes of Health funding to J.A.D. was incorrectly given as RM1HG009490 and should have been U19NS132303. The grant number 2334028 relating to the National Science Foundation funding to J.A.D. was omitted. Funding from Hampton University Summer Undergraduate Research Program, Mr. Li Ka Shing, Emerson Collective and the Innovative Genomics Institute (IGI) were omitted. The original article has been corrected.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2019

Nontoxic nanopore electroporation for effective intracellular delivery of biological macromolecules

We present a simple nanopore-electroporation (NanoEP) platform for delivery of nucleic acids, functional protein, and Cas9 single-guide RNA ribonucleoproteins into both adherent and suspension cells with up to 80% delivery efficiency and >95% cell viability. Low-voltage electric pulses permeabilize a small area of cell membrane as a cell comes into close contact with the nanopores. The biomolecule cargo is then electrophoretically drawn into the cells through the nanopores. In addition to high-performance delivery with low cell toxicity, the NanoEP system does not require specialized buffers, expensive materials, complicated fabrication processes, or cell manipulation; it simply consists of a generic nanopore-embedded water-filter membrane and a low-voltage square-wave generator. Ultimately, the NanoEP platform offers an effective and flexible method for universal intracellular delivery.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Pan-Coronavirus CRISPR-CasRx Effector System Significantly Reduces Viable Titer in HCoV-OC43, HCoV-229E, and SARS-CoV-2

CRISPR-based technology has become widely used as an antiviral strategy, including as a broad-spectrum human coronavirus (HCoV) therapeutic. In this work, we have designed a CRISPR-CasRx effector system with guide RNAs (gRNAs) that are cross-reactive among several HCoV species. We tested the efficacy of this pan-coronavirus effector system by evaluating the reduction in viral viability associated with different CRISPR targets in HCoV-OC43, HCoV-229E, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We determined that several CRISPR targets significantly reduce viral titer, despite the presence of single nucleotide polymorphisms in the gRNA when compared with a non-targeting, negative control gRNA. CRISPR targets reduced viral titer between 85% and >99% in HCoV-OC43, between 78% and >99% in HCoV-229E, and between 70% and 94% in SARS-CoV-2 when compared with an untreated virus control. These data establish a proof-of-concept for a pan-coronavirus CRISPR effector system that is capable of reducing viable virus in both Risk Group 2 and Risk Group 3 HCoV pathogens.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

A High-efficacy CRISPRi System for Gene Function Discovery in Zymomonas mobilis

Zymomonas mobilis is a promising biofuel producer due to its high alcohol tolerance and streamlined metabolism that efficiently converts sugar to ethanol. Z. mobilis genes are poorly characterized relative to those of model bacteria, hampering our ability to rationally engineer the genome with pathways capable of converting sugars from plant hydrolysates into valuable biofuels and bioproducts. Many of the unique properties that make Z. mobilis an attractive biofuel producer are controlled by essential genes; however, these genes cannot be manipulated using traditional genetic approaches (e.g., deletion or transposon insertion) because they are required for viability. CRISPR interference (CRISPRi) is a programmable gene knockdown system that can precisely control the timing and extent of gene repression, thus enabling targeting of essential genes. Here, we establish a stable, high-efficacy CRISPRi system in Z. mobilis that is capable of perturbing all genes—including essential genes. We show that Z. mobilis CRISPRi causes either strong knockdowns (>100-fold) using single guide RNA (sgRNA) spacers that perfectly match target genes or partial knockdowns using spacers with mismatches. We demonstrate the efficacy of Z. mobilis CRISPRi by targeting essential genes that are universally conserved in bacteria, are key to the efficient metabolism of Z. mobilis, or underlie alcohol tolerance. Our Z. mobilis CRISPRi system will enable comprehensive gene function discovery, opening a path to rational design of biofuel production strains with improved yields.

lignocellulosic hydrolysate↗
DOE OSTI2025

Data for "Efficient Mutagenesis and Genotyping of Maize Inbreds Using Biolistics, Multiplex CRISPR/Cas9 Editing, and Indel-Selective PCR"

CRISPR/Cas9 based genome editing has advanced our understanding of a myriad of important biological phenomena. Important challenges to multiplex genome editing in maize include assembly of large complex DNA constructs, few genotypes with efficient transformation systems, and costly/labor-intensive genotyping methods. Here we present an approach for multiplex CRISPR/Cas9 genome editing system that delivers a single compact DNA construct via biolistics to Type I embryogenic calli, followed by a novel efficient genotyping assay to identify desirable editing outcomes. We first demonstrate the creation of heritable mutations at multiple target sites within the same gene. Next, we successfully created individual and stacked mutations for multiple members of a gene family. Genome sequencing found off-target mutations are rare. Multiplex genome editing was achieved for both the highly transformable inbred line H99 and Illinois Low Protein1 (ILP1), a genotype where transformation has not previously been reported. In addition to screening transformation events for deletion alleles by PCR, we also designed PCR assays that selectively amplify deletion or insertion of a single nucleotide, the most common outcome from DNA repair of CRISPR/Cas9 breaks by non-homologous end-joining. The Indel-Selective PCR (IS-PCR) method enabled rapid tracking of multiple edited alleles in progeny populations. The ‘end to end’ pipeline presented here for multiplexed CRISPR/Cas9 mutagenesis can be applied to accelerate maize functional genomics in a broader diversity of genetic backgrounds.

gene editing↗
DOE OSTI2023

Data for Construction of a Compact Array of Microplasma Jet Devices and Its Application for Random Mutagenesis of Rhodosporidium toruloides

A small and efficient DNA mutation-inducing machine was constructed with an array of microplasma jet devices (7 × 1) that can be operated at atmospheric pressure for microbial mutagenesis. Using this machine, we report disruption of a plasmid DNA and generation of mutants of an oleaginous yeast Rhodosporidium toruloides . Specifically, a compact-sized microplasma channel (25 × 20 × 2 mm3) capable of generating an electron density of greater than 1013 cm–3 was constructed to produce reactive species (N2*, N2+, O, OH, and Hα) under helium atmospheric conditions to induce DNA mutagenesis. The length of microplasma channels in the device played a critical role in augmenting both the volume of plasma and the concentration of reactive species. First, we confirmed that microplasma treatment can linearize a plasmid by creating nicks in vitro. Second, we treated R. toruloides cells with a jet device containing 7 microchannels for 5 min; 94.8% of the treated cells were killed, and 0.44% of surviving cells showed different colony colors as compared to their parental colony. Microplasma-based DNA mutation is energy-efficient and can be a safe alternative for inducing mutations compared to conventional methods using toxic mutagens. This compact and scalable device is amenable for industrial strain improvement involving large-scale mutagenesis.

Conversion↗
DOE OSTI2025

Data for "Constitutive Down-Regulation of Liguleless Alleles in Sorghum Drives Increased Productivity and Water Use Efficiency"

Plant architecture influences the microenvironment throughout the canopy layer. Plants with a more erect leaf architecture allow for an increase in planting densities and allow more light to reach lower canopy leaves. This is predicted to increase crop carbon assimilation. Frictional resistance to wind reduces air movement in the lower canopy, resulting in higher humidity. By increasing the proportion of canopy photosynthesis in the more humid lower canopy, gains in the efficiency of water use might be expected, although this may be slightly offset by the more open erectophile form canopy. An anatomical feature in members of the Poaceae family that impacts leaf angle is the articulated junction of the sheath and blade, which also bares the ligule and auricles. Mutants, which lack ligules and auricles, show no articulation at this junction, resulting in leaves that are near vertical. In maize, these phenotypes termed liguleless result from null mutations of genes: ZmLG1 (Zm00001eb67740) and ZmLG2 (Zm00001eb147220). In sorghum, SbiRTx430.06G264300 (SbLG1) and SbiRTx430.03G392300 (SbLG2) are annotated as the respective maize homologues. A hair-pin element designed to down-regulate both SbLG1 and SbLG2 was introduced into the grain sorghum genotype RTx430. Derived transgenic events harbouring the hair-pin failed to develop ligules and displayed reduced leaf angles to the vertical, but less vertical than in null mutations. Under field settings, plots sown with these sorghum events having an erect architecture phenotype displayed an increase in photosynthesis in lower canopy levels, which led to increases in above-ground biomass and seed yield, without an increase in water use.

Genome Engineering↗
DOE OSTI2017

Next-Gen 3 : Sequencing, Modeling, and Advanced Biofuels - Final Technical Report

Successful, scalable implementation of biofuels is dependent on the efficient and near complete utilization of diverse biomass sources. One approach is to utilize the large recalcitrant biomass fraction (or any organic waste stream) through the thermochemical conversion of organic compounds to syngas, a mixture of carbon monoxide (CO), carbon dioxide (CO 2 ), and hydrogen (H 2 ), which can subsequently be metabolized by acetogenic microorganisms to produce next-gen biofuels. The goal of this proposal was to advance the development of the acetogen Clostridium ljungdahlii as a chassis organism for next-gen biofuel production from cheap, renewable sources and to detail the interconnectivity of metabolism, energy conservation, and regulation of acetogens using next-gen sequencing and next-gen modeling. To achieve this goal we determined optimization of carbon and energy utilization through differential translational efficiency in C. ljungdahlii. Furthermore, we reconstructed a next-generation model of all major cellular processes, such as macromolecular synthesis and transcriptional regulation and deployed this model to predicting proteome allocation, overflow metabolism, and metal requirements in this model acetogen. In addition we explored the evolutionary significance of tRNA operon structure using the next-gen model and determined the optimal operon structure for bioproduction. Our study substantially enhanced the knowledgebaase for chemolithoautotrophs and their potential for advanced biofuel production. It provides next-generation modeling capability, offer innovative tools for genome-scale engineering, and provide novel methods to utilize next-generation models for the design of tunable systems that produce commodity chemicals from inexpensive sources.

09 BIOMASS FUELS↗
DOE OSTI2022

PARA: A New Platform for the Rapid Assembly of gRNA Arrays for Multiplexed CRISPR Technologies

Multiplexed CRISPR technologies have great potential for pathway engineering and genome editing. However, their applications are constrained by complex, laborious and time-consuming cloning steps. In this research, we developed a novel method, PARA, which allows for the one-step assembly of multiple guide RNAs (gRNAs) into a CRISPR vector with up to 18 gRNAs. Here, we demonstrate that PARA is capable of the efficient assembly of transfer RNA/Csy4/ribozyme-based gRNA arrays. To aid in this process and to streamline vector construction, we developed a user-friendly PARAweb tool for designing PCR primers and component DNA parts and simulating assembled gRNA arrays and vector sequences.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

BioEnergy Science Center

The BioEnergy Science Center, led by Oak Ridge National Laboratory, has been making advances in biofuels for over a decade. These achievements in plant genomics, microbial engineering, biochemistry, and plant physiology will carry over into the Center for Bioenergy Innovation, a new Department of Energy bioenergy research center.

09 BIOMASS FUELS↗
DOE OSTI2019

A new approach to Cas9-based genome editing in Aspergillus niger that is precise, efficient and selectable

Aspergillus niger and other filamentous fungi are widely used in industry, but efficient genetic engineering of these hosts remains nascent. For example, while molecular genetic tools have been developed, including CRISPR/Cas9, facile genome engineering of A. niger remains challenging. To address these challenges, we have developed a simple Cas9-based gene targeting method that provides selectable, iterative, and ultimately marker-free generation of genomic deletions and insertions. This method leverages locus-specific "popout" recombination to suppress off-target integrations. We demonstrated the effectiveness of this method by targeting the phenotypic marker albA and validated it by targeting the glaA and mstC loci. After two selection steps, we observed 100% gene editing efficiency across all three loci. This method greatly reduces the effort required to engineer the A. niger genome and overcomes low Cas9 transformations efficiency by eliminating the need for extensive screening. This method represents a significant addition to the A. niger genome engineering toolbox and could be adapted for use in other organisms. It is expected that this method will impact several areas of industrial biotechnology, such as the development of new strains for the secretion of heterologous enzymes and the discovery and optimization of metabolic pathways.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Building materials genome from ground‐state configuration to engineering advance

Individual phases are commonly considered as the building blocks of materials. However, the accurate theoretical prediction of properties of individual phases remains elusive. The top-down approach by decoding genomic building blocks of individual phases from experimental observations is nonunique. The density functional theory (DFT), as a state-of-the-art solution of quantum mechanics, prescribes the existence of a ground-state configuration at 0 K for a given system. It is self-evident that the ground-state configuration alone is insufficient to describe a phase at finite temperatures as symmetry-breaking non-ground-state configurations are excited statistically at temperatures above 0 K. Our multiscale entropy approach (recently terms as Zentropy theory) postulates that the entropy of a phase is composed of the sum of the entropy of each configuration weighted by its probability plus the configurational entropy among all configurations. Consequently, the partition function of each configuration in statistical mechanics needs to be evaluated by its free energy rather than total energy. The combination of the ground-state and symmetry-breaking non-ground-state configurations represents the building blocks of materials and can be used to quantitatively predict free energy of individual phases with the free energy of each configuration predicted from DFT as well as all properties derived from free energy of individual phases.

CALPHAD↗
DOE OSTI2016

Chloroplast genomes: diversity, evolution, and applications in genetic engineering

Chloroplasts play a crucial role in sustaining life on earth. The availability of over 800 sequenced chloroplast genomes from a variety of land plants has enhanced our understanding of chloroplast biology, intracellular gene transfer, conservation, diversity, and the genetic basis by which chloroplast transgenes can be engineered to enhance plant agronomic traits or to produce high-value agricultural or biomedical products. In this review, we discuss the impact of chloroplast genome sequences on understanding the origins of economically important cultivated species and changes that have taken place during domestication. Here, we also discuss the potential biotechnological applications of chloroplast genomes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Mechanism-guided engineering of a minimal biological particle for genome editing

The widespread application of genome editing to treat and cure disease requires the delivery of genome editors into the nucleus of target cells. Enveloped delivery vehicles (EDVs) are engineered virally derived particles capable of packaging and delivering CRISPR-Cas9 ribonucleoproteins (RNPs). However, the presence of lentiviral genome encapsulation and replication proteins in EDVs has obscured the underlying delivery mechanism and precluded particle optimization. Here, we show that Cas9 RNP nuclear delivery is independent of the native lentiviral capsid structure. Instead, EDV-mediated genome editing activity corresponds directly to the number of nuclear localization sequences on the Cas9 enzyme. EDV structural analysis using cryo-electron tomography and small molecule inhibitors guided the removal of ~80% of viral residues, creating a minimal EDV (miniEDV) that retains full RNP delivery capability. MiniEDVs are 25% smaller yet package equivalent amounts of Cas9 RNPs relative to the original EDVs and demonstrated increased editing in cell lines and therapeutically relevant primary human T cells. These results show that virally derived particles can be streamlined to create efficacious genome editing delivery vehicles with simpler production and manufacturing.

59 BASIC BIOLOGICAL SCIENCES↗