Search NASA⌕ Search

SEARCH · Search NASA

Results for “Genetic engineering”

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.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 289 records · Page 16
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Digital Droplet PCR and Mesocosm-Based Methods to Evaluate Biocontainment Strategies in a Native Soil Ecosystem

Genetically modified industrial production microbes and their associated bioproducts have emerged as an integral component of a sustainable bioeconomy. However, the rapid development of these innovative technologies raises biosecurity concerns, namely, the risk of environmental escape. Thus, the realization of a bioeconomy hinges not only on the development and deployment of microbial production hosts, but also on the development of secure biosystems and biocontainment designs. Current laboratory-based biocontainment testing systems do not accurately reflect the complexities found in natural environments, necessitating an environmentally relevant analysis pipeline that allows for the detection of rare escapees within a complex soil microbiome and differentiation between closely related strains. To this end, we have developed an approach that utilizes soil mesocosms and integrated digital droplet PCR (ddPCR) system to evaluate the efficacy of novel biocontainment strategies. We demonstrate the utility of this approach by modeling contamination with industrial microbial chasses versus their biocontained counterparts. Here we demonstrate the broad utility of this system by highlighting findings from strains of Saccharomyces cerevisiae that are contained with an inducible toxin anti-toxin system, strains of Synechocystis sp. PCC 6803 contained via gene knockout or toxin anti-toxin system, and strains of Escherichia coli that are contained via genomic recoding. We also show that ddPCR can be used to detect gene copies from E. coli equal to those counted by traditional spot plating assays. The resultant data demonstrates that this system has broad utility across diverse microbial chassis and biocontainment strategies and enables researchers to track the fate of our contaminating microbe with high sensitivity in the soil. The findings presented here support the use of this mesocosm-based approach to assess the environmental impact of industrial microbes and to validate biocontainment strategies.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

XA21-mediated resistance to Xanthomonas oryzae pv. oryzae is dose dependent

The rice receptor kinase XA21 confers broad-spectrum resistance to Xanthomonas oryzae pv. oryzae ( Xoo ), the causal agent of rice bacterial blight disease. To investigate the relationship between the expression level of XA21 and resulting resistance, we generated independent HA-XA21 transgenic rice lines accumulating the XA21 immune receptor fused with an HA epitope tag. Whole-genome sequence analysis identified the T-DNA insertion sites in sixteen independent T0 events. Further, through quantification of the HA-XA21 protein and assessment of the resistance to Xoo strain PXO99 in six independent transgenic lines, we observed that XA21-mediated resistance is dose dependent. In contrast, based on the four agronomic traits quantified in these experiments, yield is unlikely to be affected by the expression level of HA-XA21 . These findings extend our knowledge of XA21-mediated defense and contribute to the growing number of well-defined genomic landing pads in the rice genome that can be targeted for gene insertion without compromising yield.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

BETO 2021 Peer Review: Algae Biotechnology Partnership - Technology Area Session: Advanced Algae Systems

Development of advanced genetic and genomic tools for targeted algal metabolic engineering pursuits will be integral to achieving target biomass productivity and, ultimately, the BETO goal of cost-competitive biofuels derived from algal biomass by 2022. However, at present, broad-host-range tool development is currently hindered by strain-specific negative regulatory mechanisms. Indeed, the biological processes controlling algal transcription and translation are subject to complex host regulation, which often presents hurdles for targeted genetic engineering strategies. Advanced genetic approaches offer a means to rewire these regulatory systems and/or introduce novel functionality into algal biocatalysts. Synthetic systems biology approaches also present a means to construct novel genetic regulatory networks and rewire natural biological systems to establish an “orthogonal central dogma,” wherein non-native control elements are introduced into or evolved in host microbes for bypass of host control. To this end, the Algae Biotechnology Partnership aims to develop advanced genetic editing tools and synthetic and orthogonal genetic regulatory systems to enable universal metabolic engineering strategies in top-candidate deployment algal strains. Successful development will ultimately open the door for targeted strain-engineering strategies, aimed at maximizing algal outdoor biomass production, composition, and strain robustness.

biocatalysis↗
DOE OSTI2024

Application of functional genomics for domestication of novel non-model microbes

Abstract With the expansion of domesticated microbes producing biomaterials and chemicals to support a growing circular bioeconomy, the variety of waste and sustainable substrates that can support microbial growth and production will also continue to expand. The diversity of these microbes also requires a range of compatible genetic tools to engineer improved robustness and economic viability. As we still do not fully understand the function of many genes in even highly studied model microbes, engineering improved microbial performance requires introducing genome-scale genetic modifications followed by screening or selecting mutants that enhance growth under prohibitive conditions encountered during production. These approaches include adaptive laboratory evolution, random or directed mutagenesis, transposon-mediated gene disruption, or CRISPR interference (CRISPRi). Although any of these approaches may be applicable for identifying engineering targets, here we focus on using CRISPRi to reduce the time required to engineer more robust microbes for industrial applications. One-Sentence Summary The development of genome scale CRISPR-based libraries in new microbes enables discovery of genetic factors linked to desired traits for engineering more robust microbial systems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Genetic compensation of triacylglycerol biosynthesis in the green microalga Chlamydomonas reinhardtii

Genetic compensation has been proposed to explain phenotypic differences between gene knockouts and knockdowns in several metazoan and plant model systems. With the rapid development of reverse genetic tools such as CRISPR/Cas9 and RNAi in microalgae, it is increasingly important to assess whether genetic compensation affects the phenotype of engineered algal mutants. While exploring triacylglycerol (TAG) biosynthesis pathways in the model alga Chlamydomonas reinhardtii, it was discovered that knockout of certain genes catalyzing rate-limiting steps of TAG biosynthesis, type-2 diacylglycerol acyltransferase genes (DGTTs), triggered genetic compensation under abiotic stress conditions. Genetic compensation of a DGTT1 null mutation by a related PDAT gene was observed regardless of the strain background or mutagenesis approach, for example, CRISPR/Cas 9 or insertional mutagenesis. However, no compensation was found in the PDAT knockout mutant. The effect of PDAT knockout was evaluated in a Δvtc1 mutant, in which PDAT was upregulated under stress, resulting in a 90% increase in TAG content. Knockout of PDAT in the Δvtc1 background induced a 12.8-fold upregulation of DGTT1 and a 272.3% increase in TAG content in Δvtc1/pdat1 cells, while remaining viable. These data suggest that genetic compensation contributes to the genetic robustness of microalgal TAG biosynthetic pathways, maintaining lipid and redox homeostasis in the knockout mutants under abiotic stress. This work demonstrates examples of genetic compensation in microalgae, implies the physiological relevance of genetic compensation in TAG biosynthesis under stress, and provides guidance for future genetic engineering and mutant characterization efforts.

09 BIOMASS FUELS↗
DOE OSTI2024

CRISPR Tools for Engineering Prokaryotic Systems: Recent Advances and New Applications

In the past decades, the broad selection of CRISPR-Cas systems has revolutionized biotechnology by enabling multimodal genetic manipulation in diverse organisms. Rooted in a molecular engineering perspective, we recapitulate the different CRISPR components and how they can be designed for specific genetic engineering applications. We first introduce the repertoire of Cas proteins and tethered effectors used to program new biological functions through gene editing and gene regulation. We review current guide RNA (gRNA) design strategies and computational tools and how CRISPR-based genetic circuits can be constructed through regulated gRNA expression. Then, we present recent advances in CRISPR-based biosensing, bioproduction, and biotherapeutics across in vitro and in vivo prokaryotic systems. Finally, we discuss forthcoming applications in prokaryotic CRISPR technology that will transform synthetic biology principles in the near future.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Natural transformation as a tool in Acinetobacter baylyi : Streamlined engineering and mutational analysis

Natural transformation and homologous recombination in a soil bacterium, Acinetobacter baylyi ADP1, occur with exceptionally high efficiency. These genetic features can be harnessed to address a wide variety of fundamental and applied scientific topics. Recent advances in synthetic biology and laboratory evolution have led to renewed appreciation for the use of A. baylyi as a model organism. To complement several review articles that highlight new tool sets, this chapter focuses on simple protocols and examples of transformation assays that facilitate genetic analysis and engineering. Whole genome sequencing often reveals extensive genetic variation among closely related isolates that can confound the association of genotypic and phenotypic changes. In A. baylyi, such associations can be deciphered in unique ways by directly transforming cells with linear DNA fragments. The resulting allelic replacement, which occurs at high frequency, rapidly generates desired mutants via targeted chromosomal editing. Diverse screening and selection methods can be used to test hypotheses and streamline experimental strategies to reveal the significance of specific DNA sequences. Moreover, large procedural variations are well tolerated, and techniques can be readily adapted for new purposes. Furthermore, one goal of highlighting natural transformation methodology in A. baylyi is to expand the community of researchers using this versatile bacterial host.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Synthetic feed-forward loop circuit boosts transgene expression in sugarcane

Bio-based products derived from natural plant-derived materials offer a promising alternative to petroleum-based products, which are essential for achieving global sustainability (Gupta et al. 2022). Sugarcane provides 40% of the world’s biofuel (Brant et al. 2025). Recently, metabolic engineering of sugarcane for hyperaccumulation of biomass oil is emerging as a strategy to elevate the crop’s energy content (Cao et al. 2023; Maitra et al. 2024). Synthetic transcription factors offer a powerful tool for modulating entire metabolic pathways by enabling fine-tuned activation or repression of specific genes (Liu and Stewart 2016; Hooghvorst and Altpeter 2023). However, the application of these technologies faces challenges, including the availability of well-characterized genetic building blocks for precise manipulation of gene expression, and the inherently challenging properties of gene expression in highly polyploid crops like sugarcane (Liu and Stewart 2016).

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Towards engineering hybrid incompatibility in plants

The potential for gene flow between genetically modified organisms (GMOs) and non-GMO relatives poses a significant challenge to the development and regulatory approval of GMO crops (Wedger et al., 2024), for example, the spread of herbicide resistance transgenes from crops such as rice or sorghum to cross-pollinating weedy species. Addressing this concern, we developed Engineered Genetic Incompatibility (EGI) (Maselko et al., 2017), a system that establishes species-like barriers to gene flow between otherwise sexually compatible populations. EGI employs Programmable Transcriptional Activators (PTAs) to drive lethal over- and/or ectopic expression of tightly regulated genes following undesired hybridization events (Figure 1a,b). A benign mutation of the target promoter in the EGI organism protects it from ill effects of the PTA, which acts as a sentinel for the wild-type (WT) promoter sequence. Given numerous potential PTA targets, multiple mutually incompatible subpopulations are feasible (Maselko et al., 2020). EGI has been demonstrated in yeast as a proof-of-concept (Maselko et al., 2017) and in insects as a strategy for genetic biocontrol of pests (Maselko et al., 2020; Upadhyay et al., 2022). EGI in plants would provide a strategy to halt gene flow between engineered crops and their domestic and wild relatives without altering normal cultivation or propagation practices. Here, we present promising results towards the demonstration of EGI in plants and highlight technical challenges that still need to be overcome.

CRISPRa↗
DOE OSTI2020

Editorial: Proceedings of ICPSBBB 2018 - 2nd International Conference on Plant Synthetic Biology, Bioengineering and Biotechnology

Plant synthetic biology combines engineering principles with plant biology toward the design, construction, and testing of plants with new traits (Liu and Stewart, 2015). Synthetic biology applies engineering principles, using defined genetic parts with the aim of achieving predictable outcomes. The field aims at providing fuels, pharmaceuticals, bioactive metabolites and increased crop yield. Synthetic biology is increasingly forming a framework in genetic transformation of plants allowing us to improve traits and crop performance. The conference on Plant Synthetic Biology, Bioengineering, and Biotechnology covers broad aspects of plant synthetic biology, ranging from tools to the design of predictable functions and precision genome editing, to re-designed photosynthesis, plant bioengineering techniques, and rational design of novel plant traits. This Frontiers in Plant Science virtual issue on the “Research Topic Proceedings of ICPSBBB 2018 - 2nd International Conference on Plant Synthetic Biology, Bioengineering and Biotechnology” consists of 8 publications, including four reviews and four original research articles, which fall into the following general topics: quantitative and genetic parts, evolution, expression of transgenes and epigenetic changes, engineering of stress tolerance and bioproduction.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Constitutive and inducible oleoresin defenses share genetic architectures and mechanisms in Pinus taeda

The oleoresin defense system of loblolly pine (Pinus taeda) protects trees from insects and pathogens and is an important source of renewable biofuels and chemicals, but the genetic basis of oleoresin production is poorly understood. We characterized the genetic architecture of oleoresin flow, resin canal number, stem wood terpene content, and monoterpene composition in two clonal populations of P. taeda. We used quantitative genetic analyses, genome-wide association studies (GWASs), multiplex network learning, and gene expression profiling to elucidate shared gene networks underlying defense traits and to identify high-quality candidates for breeding and engineering loblolly pine. Genetic analyses revealed polygenic inheritance and trait-to-trait correlations provide strong evidence for shared genes regulating constitutive and induced oleoresin flow. We identified 236 single nucleotide polymorphisms associated with oleoresin flow, resin canal number, and terpene composition and highlight candidate genes likely involved in terpene biosynthesis, cambial meristem reprogramming, and pathogen perception and immune signaling. Fourteen GWAS candidates were methyl jasmonate-responsive in tissues where resin canals initiate and terpene production occurs. Integrating quantitative genetics, GWAS, gene expression, and multiplex network analyses enabled the prioritization of high-quality candidate genes. This work advances the development of more resilient loblolly pine optimized for ecological performance, renewable chemical, and biofuel production.

genome-wide association study↗
DOE OSTI2023

Genetic algorithm optimization of a chemical kinetic mechanism for propane at engine relevant conditions

Propane has demonstrated significant potential for reductions in greenhouse gas and pollutant emissions in medium- and heavy-duty engine applications, but further improvements require accurate, compact, and scalable chemical kinetic mechanisms to design the next generation of propane fueled engines, particularly at the boosted operating conditions necessary to meet the power density demand of medium- and heavy-duty applications. In this work, six key chemical reactions were identified in a reduced mechanism with 70 species and 352 reactions through a sensitivity analysis performed at conditions typical of thermodynamic trajectories observed in a high compression ratio, long stroke engine operated on propane from throttled to boosted operating conditions. While the original mechanism was validated against rapid compression machine (RCM) data, it was found to overpredict experimental autoignition tendencies in 2-zone, 0-D SI engine simulations performed in Chemkin Pro. Subsequently, a genetic algorithm approach was used to optimize the six reaction rate parameters within established uncertainty bounds by performing RCM simulations and comparing to two independent sets of literature ignition delay times for propane, thus generating two new kinetic mechanisms. The first optimization achieved a mean absolute percent error (MPE) reduction in 2nd stage ignition delay of 61.4% in seven generations, while the second optimization utilized a newer experimental RCM dataset, and achieved MPE reduction of 56.7% in seven generations, and further marginal improvement to 57.8% reduction in 34 generations. Finally, the two mechanisms were then evaluated again in the 2-zone 0-D SI engine model in Chemkin Pro comparing typical mean and knocking cycle trajectories, and it was found that the second optimized mechanism provided better prediction of knock onset at the representative conditions evaluated in this work, particularly for higher load operating conditions.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Rational design of a genetically encoded NMR zinc sensor

Elucidating the biochemical roles of the essential metal ion, Zn 2+ , motivates detection strategies that are sensitive, selective, quantitative, and minimally invasive in living systems. Fluorescent probes have identified Zn 2+ in cells but complementary approaches employing nuclear magnetic resonance (NMR) are lacking. Recent studies of maltose binding protein (MBP) using ultrasensitive 129 Xe NMR spectroscopy identified a switchable salt bridge which causes slow xenon exchange and elicits strong hyperpolarized 129 Xe chemical exchange saturation transfer (hyper-CEST) NMR contrast. To engineer the first genetically encoded, NMR-active sensor for Zn 2+ , we converted the MBP salt bridge into a Zn 2+ binding site, while preserving the specific xenon binding cavity. The zinc sensor (ZS) at only 1 μM achieved ‘turn-on’ detection of Zn 2+ with pronounced hyper-CEST contrast. This made it possible to determine different Zn 2+ levels in a biological fluid via hyper-CEST. ZS was responsive to low-micromolar Zn 2+ , only modestly responsive to Cu 2+ , and nonresponsive to other biologically important metal ions, according to hyper-CEST NMR spectroscopy and isothermal titration calorimetry (ITC). Protein X-ray crystallography confirmed the identity of the bound Zn 2+ ion using anomalous scattering: Zn 2+ was coordinated with two histidine side chains and three water molecules. Penta-coordinate Zn 2+ forms a hydrogen-bond-mediated gate that controls the Xe exchange rate. Metal ion binding affinity, 129 Xe NMR chemical shift, and exchange rate are tunable parameters via protein engineering, which highlights the potential to develop proteins as selective metal ion sensors for NMR spectroscopy and imaging.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Zymomonas mobilis : bringing an ancient human tool into the genomic era

Zymomonas mobilis is an ethanologenic bacterium that has been used for over 1500 years to produce alcoholic beverages. Recently, this microbe has become a top candidate for biofuel production due to its efficient metabolism. Z. mobilis is being developed to utilize lignocellulosic biomass as a feedstock and synthesize a range of valuable chemicals and fuels. Genetic and metabolic engineering strategies are crucial to reach these goals. Recent advances include genome engineering, CRISPR editing, and CRISPRi knockdown of genes. Metabolic engineering has enabled redirection of carbon from the natural product ethanol to chemicals such as 2,3-butanediol and polyhydroxybutyrate. Finally, the approaches summarized here will streamline the development of Z. mobilis as an industrial chassis for sustainable liquid fuels and chemicals.

Boismier, Emma C. [Michigan State Univ., East Lans↗