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At least 145 records · Page 8

Data for Discovery, Characterization, and Application of Chromosomal Integration Sites for Stable Heterologous Gene Expression in Rhodotorula toruloides

Rhodotorula toruloides is a non-model, oleaginous yeast uniquely suited to produce acetyl-CoA-derived chemicals. However, the lack of well-characterized genomic integration sites has impeded the metabolic engineering of this organism. Here we report a set of computationally predicted and experimentally validated chromosomal integration sites in R. toruloides . We first implemented an in silico platform by integrating essential gene information and transcriptomic data to identify candidate sites that meet stringent criteria. We then conducted a full experimental characterization of these sites, assessing integration efficiency, gene expression levels, impact on cell growth, and long-term expression stability. Among the identified sites, 12 exhibited integration efficiencies of 50% or higher, making them sufficient for most metabolic engineering applications. Using selected high-efficiency sites, we achieved simultaneous double and triple integrations and efficiently integrated long functional pathways (up to 14.7 kb). Additionally, we developed a new inducible marker recycling system that allows multiple rounds of integration at our characterized sites. We validated this system by performing five sequential rounds of GFP integration and three sequential rounds of MaFAR integration for fatty alcohol production, demonstrating, for the first time, precise gene copy number tuning in R. toruloides . These characterized integration sites should significantly advance metabolic engineering efforts and future genetic tool development in R. toruloides .

Conversion↗

Discovery, characterization, and application of chromosomal integration sites for stable heterologous gene expression in Rhodotorula toruloides

Rhodotorula toruloides is a non-model, oleaginous yeast uniquely suited to produce acetyl-CoA-derived chemicals. However, the lack of well-characterized genomic integration sites has impeded the metabolic engineering of this organism. Here we report a set of computationally predicted and experimentally validated chromosomal integration sites in R. toruloides. We first implemented an in silico platform by integrating essential gene information and transcriptomic data to identify candidate sites that meet stringent criteria. We then conducted a full experimental characterization of these sites, assessing integration efficiency, gene expression levels, impact on cell growth, and long-term expression stability. Among the identified sites, 12 exhibited integration efficiencies of 50% or higher, making them sufficient for most metabolic engineering applications. Using selected high-efficiency sites, we achieved simultaneous double and triple integrations and efficiently integrated long functional pathways (up to 14.7 kb). Additionally, we developed a new inducible marker recycling system that allows multiple rounds of integration at our characterized sites. Here, we validated this system by performing five sequential rounds of GFP integration and three sequential rounds of MaFAR integration for fatty alcohol production, demonstrating, for the first time, precise gene copy number tuning in R. toruloides. These characterized integration sites should significantly advance metabolic engineering efforts and future genetic tool development in R. toruloides.

59 BASIC BIOLOGICAL SCIENCES↗

Utah FORGE 3-2514: A Strain Sensing Array to Characterize Deformation at the FORGE Site - Workshop Presentation

This is a presentation on the Strain Sensing Array to Characterize Deformation at the FORGE Site project by Clemson University, presented by Lawrence Murdoch. The project's objective was to evaluate the feasibility of measuring and interpreting tensor strain data to improve the performance of EGS. This presentation was featured in the Utah FORGE R&D Annual Workshop on September 8, 2023. The workshop provided a valuable opportunity to explore the progress made in each of the 17 Research and Development projects funded under Solicitation 2020-1 which aim to enhance our understanding of the crucial factors influencing the development of Enhanced Geothermal Systems (EGS) reservoirs and resources.

15 GEOTHERMAL ENERGY↗

Evaluation of the Radiological Characterization for Off-Site Source Recovery Program Waste Streams LA-OS-00-01.001, LA-OS-00-03, and LA-OS-00-04

The purpose of this memorandum is to satisfy the requirements of Section 4.4 of the CCP Acceptable Knowledge Documentation procedure CCP-TP-005 (Ref. 1). This evaluation is updating the previously issued memo NEN3:24-045 issued in August 2024. CCP-TP-005 requires an AK Expert and the OSRP group to evaluate the radionuclide characterization of a waste stream and prepare the NDA Memorandum (letter to CCP Records). This NDA memorandum was written with input from the OSRP group, as required by CCP-TP-005. This memo includes a discussion of the limitations for the radiological characterization and a description of the characterization method.

07 ISOTOPE AND RADIATION SOURCES↗

NRAP-open-IAM: A flexible open-source integrated-assessment-model for geologic carbon storage risk assessment and management

Large-scale implementation of geologic carbon storage (GCS) to help reduce atmospheric greenhouse gas emissions requires stakeholder confidence that injected CO2 will remain contained and that potential subsurface environmental risks are acceptably small and manageable. The U.S. Department of Energy’s National Risk Assessment Partnership (NRAP) has developed an open-source integrated assessment model (NRAP-Open-IAM) to help address questions about a potential GCS site’s ability to effectively contain injected CO 2 and protect groundwater and other overlying environmentally sensitive receptors. NRAP-Open-IAM allows a user to: (1) incorporate relevant site geologic and injection scenario data; (2) characterize important site features and events;(3) couple fast prediction models of various system components of the engineered geologic system; and (4) execute stochastic, dynamic simulation of whole GCS system performance, leakage risk assessment, and uncertainty quantification. NRAP-Open-IAM is available on GitLab (https://gitlab.com/NRAP/OpenIAM), and is accompanied by multiple application examples and detailed user and developer guides.

54 ENVIRONMENTAL SCIENCES↗

Lessons Learned in the Development of Industry Guidance - 20279

The availability of industry guidance on different aspects of waste management forms a valuable reference for waste practitioners. The guidance typically details the principles, processes and practices that should be consulted during each stage of the relevant activities. It also includes relevant regulatory and international requirements, appropriate case studies and examples of best practices to illustrate technical concepts and make the information available to the reader in an accessible manner. Over recent years Wood have developed (or are developing) a number of industry guidance documents on a range of topics including Solid Waste Characterization, Routine Water Quality Monitoring on Nuclear Sites: Pipeline Characterization: Determining Background Radioactivity for Land Quality Assessment: Interim Storage and Aqueous Waste Management. This talk provides an overview of lessons learned along the way including: - the importance of stakeholder engagement through the process: - how to identify 'good practice'; - the importance of iterative development; and - the selection of illustrative and informative case studies. (authors)

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗

Characterizing Integrase-Attachment Site Pairs: A Machine Learning Approach

Genomic islands (GIs) are mobile genetic elements that integrate into host genomes via self-encoded integrases at specific DNA sequences known as attachment (att) sites. The ability to predict the target att site from an integrase's protein sequence is a central challenge in genomics due to the sequence diversity of integrases and the subtlety of their DNA recognition motifs.

59 BASIC BIOLOGICAL SCIENCES↗

GPS-Based Gamma Survey for Characterizing and Decommissioning NORM Sites - 20389

Gamma survey techniques are an especially powerful decommissioning tool at naturally occurring radioactive material (NORM) sites due to both the low cost to obtain data over a large spatial scale and the abundance of gamma emitters in the uranium and thorium decay series. Gamma surveys are executed by coupling a detector - most often a sodium iodide crystal - to a global positioning system (GPS), then reporting a location and gross gamma reading coincidentally to a data logger. Systems may be carried by workers or mounted to a car, all-terrain vehicle, or unmanned aerial system (UAS). The resulting data set provides a high-resolution but low precision map of the gamma radiation field over the area surveyed. Frequently this map is also correlated to soil concentrations of NORM radionuclides (most often, Ra-226) and/or exposure rate. Gamma survey parameters such as movement speed, transect spacing, and data logging frequency define the spatial resolution of the resulting surface, and can be optimized depending on the desired survey sensitivity. This paper examines gamma survey as a tool for decommissioning NORM sites and provides an overview of current gamma survey technology designed to improve the efficiency and effectiveness of the decommissioning process. Topics to be discussed in the paper include: - An overview of gamma survey systems, and the utility of different delivery vehicles depending on desired cost, desired spatial resolution, and site topography. - The influence of physical detector characteristics on detection sensitivity and survey planning. - The tradeoff between high-resolution and large spatial extent, but inherently uncertain data, and low-resolution, low spatial extent, but highly certain data, as well as the specific utility of each of these types of data during NORM facility decommissioning. - Confounding variables that may limit the utility of gamma survey at some sites (e.g., radon gas and spatial heterogeneity / hot spots), and methods to plan for and control these conditions. Results show that the confounding variables, such as radon and data output can greatly influence the overall data quality associated with the decommissioning process. In addition, the use of real-time and aerial survey platforms provides a method for ensuring proper spatial extent of the data. When applied thoughtfully, gamma survey is a powerful tool for detecting NORM radionuclides in the environment and a cost-effective technique for identifying areas requiring remediation. However, entities performing or using gamma survey as a decommissioning tool must be aware of both its advantages and its limitations before basing remediation or regulatory action on gamma survey results. (authors)

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗

Insights From Dayflow: A Historical Streamflow Reanalysis Dataset for the Conterminous United States

Abstract Reconstructed historical streamflow time series can supplement limited streamflow gauge observations. However, there are common challenges of typical modeling approaches: process‐based hydrologic models can be data/computation‐intensive, and statistics‐based models can be region/stream‐specific. Here we present a nationally scalable modeling framework integrating the simulated runoff from the Variable Infiltration Capacity (VIC) model with the Routing Application for Parallel computatIon of Discharge (RAPID) routing model leveraging high‐performance computing. We demonstrate an efficient method of assimilating streamflow at US Geological Survey (USGS) streamflow monitoring sites using a simple hierarchical approach in the VIC‐RAPID framework. The result is a reconstructed 36‐year (1980–2015) daily and monthly streamflow dataset (Dayflow) at ∼2.7 million NHDPlusV2 stream reaches in the conterminous US (CONUS). We perform a comprehensive evaluation at 7,526 USGS sites and characterize their error statistics. The results demonstrate that 49% of the USGS sites demonstrate Kling–Gupta Efficiency (KGE) > 0.5 and 58% of the sites show percentage bias within ±20% for the daily naturalized streamflow. Streamflow data assimilation across CONUS shows an overall improvement over naturalized streamflow, notably in the western semiarid‐to‐arid regions. Comparison to other national and global streamflow reanalysis datasets such as the National Water Model and Global Reach‐scale A priori Discharge Estimates for SWOT demonstrates improved KGE, reduced bias, and directions for Dayflow improvements. Investigations of error statistics with key hydrologic, hydroclimatic, and geomorphologic basin characteristics reveal region‐specific patterns which may help improve future framework applications. Overall, Dayflow may enable a better understanding of hydrologic conditions in a changing environment, especially in locations currently not represented by streamflow monitoring networks.

54 ENVIRONMENTAL SCIENCES↗

Molecular Catalyst Synthesis Strategies to Prepare Atomically Dispersed Fe-N-C Heterogeneous Catalysts

We report a strategy to integrate atomically dispersed iron within a heterogeneous nitrogen-doped carbon (N-C) support, inspired by routes for metalation of molecular macrocyclic iron complexes. The N-C support, derived from pyrolysis of a ZIF-8 metal–organic framework, is metalated via solution-phase reaction with FeCl 2 and tributyl amine, as a Brønsted base, at 150 °C. Fe active sites are characterized by 57 Fe Mössbauer spectroscopy and aberration-corrected scanning transmission electron microscopy. The site density can be increased by selective removal of Zn 2+ ions from the N-C support prior to metalation, resembling the transmetalation strategy commonly employed for the preparation of molecular Fe-macrocycles. As a result, the utility of this approach is validated by the higher catalytic rates (per total Fe) of these materials relative to established Fe-N-C catalysts, benchmarked using an aerobic oxidation reaction.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Isolating the Contributions of Specific Network Sites to the Diffuse Vibrational Spectrum of Interfacial Water with Isotopomer-Selective Spectroscopy of Cold Clusters

Decoding the structural information contained in the interfacial vibrational spectrum of water requires understanding how the spectral signatures of individual water molecules respond to their local hydrogen bonding environments. In this study, we isolated the contributions for the five classes of sites that differ according to the number of donor (D) and acceptor (A) hydrogen bonds that characterize each site. These patterns were measured by exploiting the unique properties of the water cluster cage structures formed in the gas phase upon hydration of a series of cations M+·(H2O)n (M = Li, Na, Cs, NH4, CH3NH3, H3O, and n = 5, 20-22). This selection of ions was chosen to systematically express the A, AD, AAD, ADD, and AADD hydrogen bonding motifs. The spectral signatures of each site were measured using two-color, IR-IR isotopomer-selective photofragmentation vibrational spectroscopy of the cryogenically cooled, mass selected cluster ions in which a single intact H2O is introduced without isotopic scrambling, an important advantage afforded by the cluster regime. The resulting patterns provide an unprecedented picture of the intrinsic line shapes and spectral complexities associated with excitation of the individual OH groups, as well as the correlation between the frequencies of the two OH groups on the same water molecule, as a function of network site. The properties of the surrounding water network that govern this frequency map are evaluated by dissecting electronic structure calculations that explore how changes in the nearby network structures, both within and beyond the first hydration shell, affect the local frequency of an OH oscillator. The qualitative trends are recovered with a simple model that correlates the OH frequency with the network-modulated local electron density in the center of the OH bond.

Yang, Nan↗

Discovery, characterization, and application of chromosomal integration sites in the hyperthermophilic archaeon Sulfolobus islandicus

Sulfolobus islandicus , an emerging archaeal model organism, offers unique advantages for metabolic engineering and synthetic biology applications owing to its ability to thrive in extreme environments. Although several genetic tools have been established for this organism, the lack of well-characterized chromosomal integration sites has limited its potential as a cellular factory. Here, in this work, we systematically identified and characterized 13 artificial CRISPR RNAs targeting eight integration sites in S. islandicus using the CRISPR-COPIES pipeline and a multi-omics-informed computational workflow. We leveraged the endogenous CRISPR-Cas system to integrate the reporter gene lacS and validated heterologous expression through a β-galactosidase assay, revealing significant positional effects. As a proof of concept, we utilized these sites to genetically manipulate lipid ether composition by overexpressing glycerol dibiphytanyl glycerol tetraether (GDGT) ring synthase B (GrsB). This study expands the genetic toolbox for S. islandicus and advances its potential as a robust platform for archaeal synthetic biology and industrial biotechnology.

59 BASIC BIOLOGICAL SCIENCES↗

Wabash CarbonSAFE. Final Report

This document summarizes work detailed in separate Wabash CarbonSAFE reports; the report describes the data collection efforts of the project and consolidates the geologic characterization, well testing, and storage complex modeling results for the Mt. Simon Sandstone and Potosi Dolomite, two distinct reservoirs characterized at the Wabash CarbonSAFE project site. Also presented are summaries of work to characterize the CO 2 source and infrastructure network, as well as summaries of reports analyzing stakeholder engagement options, policy, regulatory and legal considerations, and risk assessment associated with the Wabash CarbonSAFE project site. The report then presents recommendations for the next steps for site characterization, identifies data gaps for future activities, and provides an overall assessment of site potential.

29 ENERGY PLANNING, POLICY, AND ECONOMY↗

CRISPR-COPIES: Web Tool

CRISPR/Cas system has emerged as a powerful genome-editing tool for metabolic engineering and human gene therapy. However, the conundrum of where to integrate heterologous genes on the chromosome using the CRISPR/Cas system remains an open question. Selecting a site for gene integration requires incorporation of complex criteria such as factors involved in CRISPR/Cas-mediated integration, genetic stability, and gene expression and therefore, usually requires strenuous characterization of sites on particular or different chromosomal locations. To address these issues, we developed CRISPR-COPIES, a COmputational Pipeline for the Identification of CRISPR/Cas-facilitated intEgration Sites. The tool applies ScaNN, a state-of-the-art model on the embedding-based nearest neighbor search for fast and accurate off-target search and can identify genome-wide intergenic sites for most bacterial and fungal genomes within minutes. This submission contains the code we developed to create a user-friendly web interface for CRISPR-COPIES (https://biofoundry.web.illinois.edu/copies/). We anticipate CRISPR-COPIES will serve as a useful tool for targeted DNA integration and aid in the characterization of synthetic biology toolkits, rapid strain construction to produce valuable biochemicals, and human gene and cell therapy.

Bioinformatics↗

Physical and Hydraulic Properties of RCRA Borehole Samples from the Hanford Site : Final Report, Fiscal Years 2023-2024

Sediment from 44 core samples collected from Resource Conservation and Recovery Act (RCRA) boreholes drilled in the 200 East and 200 West areas of the Hanford Site were characterized for physical and hydraulic properties (Table S.1). Characterization data included gravimetric water contents and matric potentials, grain-size distributions, saturated hydraulic conductivity, water retention characteristics, and unsaturated hydraulic conductivity. These properties provide site-specific data and parameters that can be used in subsurface flow and contaminant transport models to assess the transport and fate of contaminants in the vadose zone and underlying aquifer systems. The analyzed core samples come from specific areas and depth intervals at the Hanford Site that were targeted for sampling to address data gaps identified by site contractors (Khaleel 2020). X-ray computed tomography (XCT) was used to evaluate the general textural characteristics of the samples and to determine which samples to use for further physical and hydraulic property characterization. Subsequent sample selection was determined by consensus after review of the XCT images by Pacific Northwest National Laboratory, Central Plateau Cleanup Company, and INTERA staff.

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗

Characterizing Performance of Freshwater Wetland Methane Models Across Time Scales at FLUXNET-CH 4 Sites Using Wavelet Analyses

Process-based land surface models are important tools for estimating global wetland methane (CH 4 ) emissions and projecting their behavior across space and time. So far there are no performance assessments of model responses to drivers at multiple time scales. In this study, we apply wavelet analysis to identify the dominant time scales contributing to model uncertainty in the frequency domain. We evaluate seven wetland models at 23 eddy covariance tower sites. Our study first characterizes site-level patterns of freshwater wetland CH 4 fluxes (FCH 4 ) at different time scales. A Monte Carlo approach was developed to incorporate flux observation error to avoid misidentification of the time scales that dominate model error. Our results suggest that (a) significant model-observation disagreements are mainly at multi-day time scales (<15 days); (b) most of the models can capture the CH 4 variability at monthly and seasonal time scales (>32 days) for the boreal and Arctic tundra wetland sites but have significant bias in variability at seasonal time scales for temperate and tropical/subtropical sites; (c) model errors exhibit increasing power spectrum as time scale increases, indicating that biases at time scales <5 days could contribute to persistent systematic biases on longer time scales; and (d) differences in error pattern are related to model structure (e.g., proxy of CH 4 production). Our evaluation suggests the need to accurately replicate FCH 4 variability, especially at short time scales, in future wetland CH 4 model developments.

58 GEOSCIENCES↗

Subsurface Characterization of Hydraulic Fracture Test Site-2 (HFTS-2), Delaware Basin

Hydraulic Fracturing Test Site-2 (HFTS-2) is a field-based research experiment performed in the Wolfcamp Formation of the Permian (Delaware) Basin. This paper focuses on integration, advanced geological characterization, and 3D subsurface modeling of the comprehensive HFTS-2 dataset. The study showcases a multidisciplinary reservoir characterization approach that incorporates geology, petrophysics, geochemistry, geomechanics, microseismic, and subsurface engineering analysis. Subsurface characterization of organic-rich mudstone formations requires understanding complex hydraulic fracture network growth in relation to inherent lithology, geomechanical properties, and interaction with pre-existing natural fractures. This paper presents a characterization workflow incorporating pre- and post-stimulation subsurface data, unique to the HFTS-2 dataset. The study integrated: (1) rock properties from logs, cores, and thin sections; (2) natural and hydraulic fracture descriptions from cores and image logs; (3) local and regional stresses; (4) geomechanics; (5) microseismic; (6) fiber optic (FO) and bottomhole pressure gauge (BHPG) response; and (7) produced fluids analysis. During a stimulation treatment, creation of the stimulated rock volume (SRV) is influenced by several subsurface factors. Key contributing factors include structural context, stress conditions, lithology, facies architecture, pre-existing natural fractures, and geomechanical properties. The HFTS-2 subsurface data integration indicates that the SRV is comprised of a complex juxtaposition of hydraulic fracture swarms, as evidenced by image logs analysis, core description, and microseismic monitoring. The HFTS-2 microseismic event density was used to generate 3D heat maps that serve as a representative SRV footprint, corroborated by secondary datasets. These maps were further integrated with petrophysical and geomechanical characteristics, as well as responses from FO and BHPG, to estimate the lateral and vertical dimensions of the effective fractures. The geological characterization for the HFTS-2 dataset combined with 3D modeling for petrophysical and geomechanical properties provides a strong foundation for subsurface simulation and optimization studies. Downloaded from http://onepetro.org/URTECONF/proceedings-pdf/21URTC/1-21URTC/D011S005R001/2477501/urtec-2021-5243-ms.pdf/1 by Carol Worster on 28 February 2022 URTeC 5243 The workflow improved our understanding of HFTS-2 hydraulic fracture propagation and characteristics in relation to offset pressure depletion and interaction with pre-existing natural fractures. Analysis showed that fracture geometry varies by stage and by well, and a complex fracture network is generated with varying fracture density. The multidisciplinary workflow presented herein for integration and characterization serves as a foundation to evaluate completion efficiency and estimate areal and vertical stimulation and depletion extent for the project. Furthermore, the workflow and learnings can also be transferred to other unconventional plays.

58 GEOSCIENCES↗

AmeriFlux FLUXNET-1F US-Akn Savannah River Site

This is the AmeriFlux Management Project (AMP) created FLUXNET-1F version of the carbon flux data for the site US-Akn Savannah River Site. This is the FLUXNET version of the carbon flux data for the site US-Akn Savannah River Site produced by applying the standard ONEFlux (1F) software. Site Description - The site is characterized by a mix of pine and deciduous hardwood forests with a general canopy height of 25m which was established in the 1950s. Management of the forest around the site is performed by the USFS-Savannah River and consists of prescribed burning of the understory on a 3-4 year cycle.

Leclerc, Monique↗