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DOE OSTI2025

RT-EZ: A Golden Gate Assembly Toolkit for Streamlined Genetic Engineering of Rhodotorula toruloides

For economic and sustainable biomanufacturing, the oleaginous yeast Rhodotorula toruloides has emerged as a promising platform for producing biofuels, pharmaceuticals, and other valuable chemicals. However, genetic manipulation of R. toruloides has been limited by its high GC content and the lack of a replicating plasmid, necessitating gene integration into the genome of the yeast. To address these challenges, we developed the RT-EZ (R. toruloides Efficient Zipper) toolkit, a versatile tool based on Golden Gate assembly, designed to streamline R. toruloides engineering with improved efficiency and flexibility. The RT-EZ toolkit simplifies vector construction by incorporating new features such as bidirectional promoters and 2A peptides, color-based screening using RFP, and sequences optimized for both Agrobacterium tumefaciens-mediated transformation (ATMT) and easy linearization, enabling straightforward selection and transformation. Notably, the RT-EZ kit can be used to construct an expression cassette with four different genes in one assembly reaction, significantly improving vector construction speed and efficiency. The utility of the RT-EZ toolkit was demonstrated through the successful synthesis of arachidonic acid in R. toruloides by coexpressing fatty acid elongases and desaturases. Furthermore, this result underscores the potential of the RT-EZ toolkit to advance synthetic biology in R. toruloides, providing a streamlined method for addressing genetic engineering challenges in the yeast.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2020

RCSB Protein Data Bank 1D tools and services

Abstract Motivation Interoperability between polymer sequences and structural data is essential for providing a complete picture of protein and gene features and helping to understand biomolecular function. Results Herein, we present two resources designed to improve interoperability between the RCSB Protein Data Bank, the NCBI and the UniProtKB data resources and visualize integrated data therefrom. The underlying tools provide a flexible means of mapping between the different coordinate spaces and an interactive tool allows convenient visualization of the 1-dimensional data over the web. Availabilityand implementation https://1d-coordinates.rcsb.org and https://rcsb.github.io/rcsb-saguaro. Supplementary information Supplementary data are available at Bioinformatics online.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

The mole genome reveals regulatory rearrangements associated with adaptive intersexuality

Linking genomic variation to phenotypical traits remains a major challenge in evolutionary genetics. In this study, we use phylogenomic strategies to investigate a distinctive trait among mammals: the development of masculinizing ovotestes in female moles. By combining a chromosome-scale genome assembly of the Iberian mole, Talpa occidentalis, with transcriptomic, epigenetic, and chromatin interaction datasets, we identify rearrangements altering the regulatory landscape of genes with distinct gonadal expression patterns. These include a tandem triplication involving CYP17A1, a gene controlling androgen synthesis, and an intrachromosomal inversion involving the pro-testicular growth factor gene FGF9, which is heterochronically expressed in mole ovotestes. Transgenic mice with a knock-in mole CYP17A1 enhancer or overexpressing FGF9 showed phenotypes recapitulating mole sexual features. Our results highlight how integrative genomic approaches can reveal the phenotypic impact of noncoding sequence changes.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Data for "RT-EZ: A Golden Gate Assembly Toolkit for Streamlined Genetic Engineering of Rhodotorula toruloides"

For economic and sustainable biomanufacturing, the oleaginous yeast Rhodotorula toruloides has emerged as a promising platform for producing biofuels, pharmaceuticals, and other valuable chemicals. However, genetic manipulation of R. toruloides has been limited by its high GC content and the lack of a replicating plasmid, necessitating gene integration into the genome of the yeast. To address these challenges, we developed the RT-EZ ( R. toruloides Efficient Zipper) toolkit, a versatile tool based on Golden Gate assembly, designed to streamline R. toruloides engineering with improved efficiency and flexibility. The RT-EZ toolkit simplifies vector construction by incorporating new features such as bidirectional promoters and 2A peptides, color-based screening using RFP, and sequences optimized for both Agrobacterium tumefaciens-mediated transformation (ATMT) and easy linearization, enabling straightforward selection and transformation. Notably, the RT-EZ kit can be used to construct an expression cassette with four different genes in one assembly reaction, significantly improving vector construction speed and efficiency. The utility of the RT-EZ toolkit was demonstrated through the successful synthesis of arachidonic acid in R. toruloides by coexpressing fatty acid elongases and desaturases. This result underscores the potential of the RT-EZ toolkit to advance synthetic biology in R. toruloides , providing a streamlined method for addressing genetic engineering challenges in the yeast.

gene editing↗

DOE OSTI2022

Preliminary Sensitivity Analysis for Sensors Impacts on Building Control Performance

This report describes the preliminary sensitivity analysis for sensor impacts on building control performance through the US Department of Energy’s Oak Ridge National Laboratory’s Flexible Research Platform (FRP-2) building. The rooftop unit system provides cooling and heating to the building. The main heating coil is a gas heating coil. Each zone is served by a variable air volume box with an electricity reheat coil. The rooftop unit and variable air volume box controls adopted the practical control sequences from ASHRAE Guideline 36-2018: High-Performance Sequences of Operation. For sensors, the incipient (time-changing) sensor errors, including bias sensor error and precision sensor error, are the inputs of interest. The outputs are energy consumption and thermal comfort (e.g., the predicted percentage of dissatisfied occupants). The large-scale simulation (3,600 cases) was conducted on a cloud platform by integrating sensor errors and ASHRAE Guideline 36 control sequences into an emulator based on the EnergyPlus simulation program with Python energy management system feature. The surrogate models were developed based on cloud simulation results. The uncertainty analysis showed that the sensor errors substantially affect building energy consumption and thermal comfort. The sensitivity analysis shows a ranking of sensor error impacts for each interested output item (e.g., cooling energy, reheat coil heating energy, predicted percentage of dissatisfied occupants). In FY 2022, sensor locations, types, and costs will be evaluated. The field test in Oak Ridge National Laboratory’s Flexible Research Platform building regarding sensor impacts will also be performed. Finally, a comparative analysis will be conducted based on the field test results and emulator results.

32 ENERGY CONSERVATION, CONSUMPTION, AND UTILIZATI↗

NASA NTRS1972

Computational efficiency achieved by time splitting of finite difference operators.

A technique is presented for computing multidimensional time-dependent flow fields that avoids much of the inefficiency typically found in finite difference calculations. The technique initially divides the flow field into regions, each containing a mesh of general quadrilateral cells chosen to provide spatial resolution of the local features of the flow. A finite difference operator of second order accuracy, consisting of a sequence of one-dimensional operators (each operating at near maximum Courant-Friedrich-Lewy number) is then constructed for each region. Numerical results illustrating the technique for inviscid flows about simple bodies that generate shock waves, embedded shock waves, and expansion fans are presented and compared with exact theory.

Maccormack, R. W.↗

NASA NTRS1972

A TDM link with channel coding and digital voice.

The features of a TDM (time-division multiplexed) link model are described. A PCM telemetry sequence was coded for error correction and multiplexed with a digitized voice channel. An all-digital implementation of a variable-slope delta modulation algorithm was used to digitize the voice channel. The results of extensive testing are reported. The measured coding gain and the system performance over a Gaussian channel are compared with theoretical predictions and computer simulations. Word intelligibility scores are reported as a measure of voice channel performance.

Jones, M. W.↗

NASA NTRS1975

Hydrodynamic models of a Cepheid atmosphere. I - Deep envelope models

The implicit hydrodynamic code of Kutter and Sparks has been modified to include radiative transfer effects. This modified code has been used to compute deep envelope models of a classical Cepheid with a period of 12 days. It is shown that in this particular model the hydrogen ionization region plays only a small role in producing the observed phase lag between the light and velocity curves. The cause of the bumps on the model's light curve is examined, and a mechanism is presented to explain those Cepheids with two secondary features on their light curves. This mechanism is shown to be consistent with the Hertzsprung sequence only if the evolutionary mass-luminosity law is used.

Karp, A. H.↗

NASA NTRS1981

A Module Experimental Process System Development Unit (MEPSDU)

The technical readiness of a cost effective process sequence that has the potential for the production of flat plate photovoltaic modules which met the price goal in 1986 of $.70 or less per Watt peak was demonstrated. The proposed process sequence was reviewed and laboratory verification experiments were conducted. The preliminary process includes the following features: semicrystalline silicon (10 cm by 10 cm) as the silicon input material; spray on dopant diffusion source; Al paste BSF formation; spray on AR coating; electroless Ni plate solder dip metallization; laser scribe edges; K & S tabbing and stringing machine; and laminated EVA modules.

Source record↗

NASA NTRS1982

Morphology and movements of polarization features on Venus

Ground and spacecraft based polarization observations of Venus detected the signature of the so called 1-micron radius cloud particles that constitute the main cloud deck on Venus, as well as that of sub-micron sized particles that form a haze above this main cloud layer. Whether such observations showed any local clustering or organization in the polarimetry data remained uncertain until the relatively high resolution observations from the Pioneer Venus Orbiter Cloud Photopolarimeter (OCPP) became available. The available polarization data from this instrument was studied to see whether local organization was present, to study the morphology of such features or polarization clouds, and finally, to see if they can be followed in a sequence of observations to study their motions.

Limaye, S. S.↗

NASA NTRS1987

Space telerobotics technology demonstration program

The paper reports the ongoing development of a telerobot demonstrator. The demonstrator is implemented as a laboratory-based research testbed, and will show proof-of-concept for supervised automation of space assembly, servicing, and repair operations. The demonstrator system features a hierarchically layered intelligent control architecture which enables automated planning and run-time sequencing of complex tasks by a supervisory human operator. The demonstrator also provides a full bilateral force-reflecting hand control teleroperations capability. The operator may switch smoothly between the automated and teleroperated tasking modes in run-time, either on a preplanned or operator-designated basis.

Szirmay, S. Z.↗

NASA NTRS1987

The NASA telerobot technology demonstrator

The ongoing development of a telerobot technology demonstrator is reported. The demonstrator is implemented as a laboratory-based research testbed, and will show proof-of-concept for supervised automation of space assembly, servicing, and repair operations. The demonstrator system features a hierarchically layered intelligent control architecture which enables automated planning and run-time sequencing of complex tasks by a supervisory human operator. The demonstrator also provides a full bilateral force-reflecting hand control teleoperations capability. The operator may switch smoothly between the automated and teleoperated tasking modes in run-time, either on a preplanned or operator-designated basis.

Schenker, P. S.↗

NASA NTRS1989

The interaction of supernovae with circumstellar bubbles

This paper examines the interaction of a massive star supernova with the shell created by the fast wind from a blue supergiant, either in the main-sequence phase or in a late evolutionary phase. Making a number of idealizations, the general features of shell interaction are described by semianalytical solutions. The expected properties of the supernova and its environment are discussed, and the hydrodynamics of the interaction is described. It is found that, typically, the shock traversal occurs before the energy transfer is significant. Applications of the model to observed objects are considered, with special attention given to the interaction of SN 1987A with its circumstellar shell, which is expected to occur within decades.

Chevalier, Roger A.↗

NASA NTRS2005

Moonshine Versus Earthshine: Physics Makes a Difference

Introduction: Recently released, high-resolution images from the Mars Orbiter Camera (MOC) and the Thermal Emission Imaging System (THEMIS) reveal a myriad of intriguing landforms banked along the northern edge of Terby Crater located on the northern rim of Hellas (approx.28degS, 287degW). Landforms within this crater include north-trending troughs and ridges, a remarkable 2.5 km-thick sequence of exposed layers, mantled ramps that extend across and between layered sequences, fan-like structures, sinuous channels, collapse pits, a massive landslide and viscous flow features. The suite of diverse landforms in Terby and its immediate surroundings attest to a diversity of rock types and geologic processes, making this locality ideal for studying landform-climate relationships on Mars. In order to decipher the complicated geologic history of Terby Crater and the nature of the layered deposits, a generalized geomorphic map was created and the slope of the layered deposits was examined.

Wilson, T. L.↗

NASA NTRS1984

Ultraviolet spectral morphology of the O stars - The remarkable luminosity dependence of the Si IV stellar wind effect

An extensive survey of ultraviolet O type spectra, by means of high-resolution data from the International Ultraviolet Explorer archives, shows a strong correlation between both the photospheric and stellar wind features, and the optical spectral types. In particular, the stellar wind effect in the Si IV resonance doublet displays a strong luminosity dependence which has not been completely described before: there is no effect anywhere on the main sequence, but it develops gradually through the intermediate luminosity classes into a full P Cygni profile in the supergiants. This behavior is in sharp contrast to that of the C IV and N V, which have strong P Cygni profiles throughout most of the O type domain, including the main sequence. The Si IV effect simultaneously provides a sensitive classification criterion and poses an outstanding problem for detailed astrophysical interpretation. In addition, a new emission feature at 1574 A, possibly due to N III or to N IV forbidden line, is reported.

Walborn, N. R.↗

DOE OSTI2016

Conservation and diversity in the ultralong third heavy-chain complementarity-determining region of bovine antibodies

Antibodies provide a broad defense against a vast array of antigens; however, the structural features that contribute to this diverse antigen recognition vary in different vertebrates. In cows, a subset of antibodies have an exceptionally long third heavy-chain complementarity-determining region (CDR H3) that is highly variable in sequence and includes multiple cysteines. These long CDR H3s (up to 69 residues) fold into a long stalk atop which sits a knob domain that is located far from the antibody surface. We have determined crystal structures of three bovine Fabs to decipher the conserved and variable features of ultralong CDR H3s that lead to diversity in antigen recognition. Despite high sequence variability, the stalks adopt a conserved β-ribbon structure, whereas the knob regions share a conserved β sheet that serves as a scaffold for two connecting loops of variable length and conformation, as well as one conserved disulfide. Variation in patterns and connectivity of the remaining disulfides contribute to the structural diversity of the knob. Finally, the unusual architecture of these ultralong bovine CDR H3s for generating diversity is unique in adaptive immune systems and may inform efforts in antibody engineering.

60 APPLIED LIFE SCIENCES↗

NASA NTRS1979

Stellar model chromospheres. IX - Chromospheric activity in dwarf stars

High-resolution Ca II K line profiles are used to model the upper photospheres and lower chromospheres of eight main-sequence stars ranging in spectral type from F0 to M0 and exhibiting different degrees of chromospheric activity. The model chromospheres are studied as a function of spectral type and activity for stars of similar spectral type in order to obtain evidence of enhanced nonradiative heating in the upper-photospheric models and in the ratio of minimum temperature at the base of the chromosphere to effective temperature, a correlation between activity and temperature in the lower chromospheres, and a correlation of the width at the base of the K-line emission core and at the K2 features with activity. Chromospheric radiative losses are estimated for the modelled stars and other previously analyzed main-sequence stars. The results obtained strengthen the argument that dMe flare stars exhibit fundamentally solar-type activity but on an increased scale.

Kelch, W. L.↗

DOE OSTI2018

Genome-Based Taxonomic Classification of the Phylum Actinobacteria

The application of phylogenetic taxonomic procedures led to improvements in the classification of bacteria assigned to the phylum Actinobacteria but even so there remains a need to further clarify relationships within a taxon that encompasses organisms of agricultural, biotechnological, clinical, and ecological importance. Classification of the morphologically diverse bacteria belonging to this large phylum based on a limited number of features has proved to be difficult, not least when taxonomic decisions rested heavily on interpretation of poorly resolved 16S rRNA gene trees. Here, draft genome sequences of a large collection of actinobacterial type strains were used to infer phylogenetic trees from genome-scale data using principles drawn from phylogenetic systematics. The majority of taxa were found to be monophyletic but several orders, families, and genera, as well as many species and a few subspecies were shown to be in need of revision leading to proposals for the recognition of 2 orders, 10 families, and 17 genera, as well as the transfer of over 100 species to other genera. In addition, emended descriptions are given for many species mainly involving the addition of data on genome size and DNA G+C content, the former can be considered to be a valuable taxonomic marker in actinobacterial systematics. Many of the incongruities detected when the results of the present study were compared with existing classifications had been recognized from 16S rRNA gene trees though whole-genome phylogenies proved to be much better resolved. The few significant incongruities found between 16S/23S rRNA and whole genome trees underline the pitfalls inherent in phylogenies based upon single gene sequences. Similarly good congruence was found between the discontinuous distribution of phenotypic properties and taxa delineated in the phylogenetic trees though diverse non-monophyletic taxa appeared to be based on the use of plesiomorphic character states as diagnostic features.

59 BASIC BIOLOGICAL SCIENCES↗