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At least 19 records

Developing a Cas9-Based Tool to Engineer Native Plasmids in Synechocystis sp. PCC 6803

The oxygenic photosynthetic bacterium Synechocystis sp. PCC 6803 (S6803) is a model cyanobacterium widely used for fundamental research and biotechnology applications. Due to its polyploidy, existing methods for genome engineering of S6803 require multiple rounds of selection to modify all genome copies, which is time consuming and inefficient. In this study, we engineered the Cas9 tool for onestep, segregationfree genome engineering. We further used our Cas9 tool to delete three of seven S6803 native plasmids. Our results show that all three smallsize native plasmids, but not the largesize native plasmids, can be deleted with this tool. To further facilitate heterologous gene expression in S6803, a shuttle vector based on the native plasmid pCC5.2 was created. The shuttle vector can be introduced into Cas9containing S6803 in one step without requiring segregation and can be stably maintained without antibiotic pressure for at least 30 days. Moreover, genes encoded on the shuttle vector remain functional after 30 days of continuous cultivation without selective pressure. Thus, this study provides a set of new tools for rapid modification of the S6803 genome and for stable expression of heterologous genes, potentially facilitating both fundamental research and biotechnology applications using S6803.

biotechnology

Developing a Genetic Variant Calling Pipeline for Quantifying the Complex Mutagenic Load Accumulated in BioNutrients-1 Production Pack Samples

Microorganisms hold great promise for on demand production of labile nutrients and pharmaceuticals as well recycling and in situ resource utilization. The utilization of microorganisms for such tasks on space missions is hindered by the limited data on how microbes respond to spaceflight. For example, the genetic stability of microorganisms, and the genomic engineered traits added to deliver desired functions, over long-term storage in the spacecraft environment is poorly understood. The BioNutrients-1 (BN-1) mission conducted a 5-year study of desiccated storage in Low Earth Orbit (LEO) to evaluate the suitability of eight synthetic biology chassis organisms for long-duration space missions. We are employing high-depth, whole genome sequencing (WGS) to determine the mutagenic load that accumulated during long-term storage. Mutation analysis pipelines are well established for homogenous culture grown from a single colony, but the mutational landscape of the BN-1 samples present a unique analysis challenge, as every cell in the BN-1 samples had a unique genetic journey of DNA damage and repair. Consequently, sequence variants are expected at low allele frequency within samples. To address this genetic complexity, we apply two distinct computational approaches to identify mutations in pre-existing WGS data collected from populations of Chlamydomonas reinhardtii that were exposed to UV mutagenesis and growth in LEO. For reference genome free mutation detection, we utilized DiscoSNP++, which is a de Bruijn graph approach. For reference genome-based mutation detection we utilize GATK for Microbes, which is a Bayesian probabilistic approach. We will benchmark these approaches against the mutations originally identified using CRISP, a method optimized for pooled samples. Ultimately, quantifying the mutation load imposed by storage or growth on the ISS will help identify chassis organisms with both high levels of genome stability and viability, which are desirable traits for implementation of bioproduction in long-duration missions.

SNP

Selection Factors for Space Crops

NASA is actively researching space crop production to determine its potential to contribute to food system security on long duration missions beyond Low Earth Orbit. Our near-term focus is on nutrient and variety supplementation of prepackaged food with fresh produce that requires little or no processing. The longer-term goal is caloric replacement to become less dependent on Earth, and this will require cultivation of staple crops, processing and cooking equipment, integration with spacecraft air, water, and power systems, and automation. There are numerous technology and knowledge gaps remaining for sustainable space crop production systems, but one high-impact area is in the development of crops specifically customized to meet the needs of controlled environment crop production, astronaut health and well-being, and space-unique environments. Modern crop breeding and genome engineering tools are allowing for rapid development of new genotypes with incredible specificity. Targeted aspects to optimize crops for space have been identified and characterized into five categories: plant growth and development, plant physiology, produce nutrition, produce organoleptic acceptability, and postharvest characteristics. Within each category there are several targets that further the development of crop production systems for spaceflight, such as crop size and harvest index, tolerance to specific environmental stresses, optimizing target nutrients that are low or degrade in the packaged diet, maintenance time requirements, and less indigestible structural material. NASA-funded PIs are already beginning to develop candidate crops, and spaceflight testing and validation of novel space crops is on the horizon. Crops developed for space also have the potential to benefit terrestrial controlled environment agriculture crop production systems. This research was supported by NASA’s Space Biology and Human Research Programs.

Space Crop Production

Selection Factors for Space Crops

NASA is actively researching space crop production to determine its potential to contribute to food system security on long duration missions beyond Low Earth Orbit. Our near-term focus is on nutrient and variety supplementation of prepackaged food with fresh produce that requires little or no processing. The longer-term goal is caloric replacement to become less dependent on Earth, and this will require cultivation of staple crops, processing and cooking equipment, integration with spacecraft air, water, and power systems, and automation. There are numerous technology and knowledge gaps remaining for sustainable space crop production systems, but one high-impact area is in the development of crops specifically customized to meet the needs of controlled environment crop production, astronaut health and well-being, and space-unique environments. Modern crop breeding and genome engineering tools are allowing for rapid development of new genotypes with incredible specificity. Targeted aspects to optimize crops for space have been identified and characterized into five categories: plant growth and development, plant physiology, produce nutrition, produce organoleptic acceptability, and postharvest characteristics. Within each category there are several targets that further the development of crop production systems for spaceflight, such as crop size and harvest index, tolerance to specific environmental stresses, optimizing target nutrients that are low or degrade in the packaged diet, maintenance time requirements, and less indigestible structural material. NASA-funded PIs are already beginning to develop candidate crops, and spaceflight testing and validation of novel space crops is on the horizon. Crops developed for space also have the potential to benefit terrestrial controlled environment agriculture crop production systems. This research was supported by NASA’s Space Biology and Human Research Programs.

Space Crop Production

Three-Dimensional Transgenic Cell Models to Quantify Space Genotoxic Effects

In this paper we describe a three-dimensional, multicellular tissue-equivalent model, produced in NASA-designed, rotating wall bioreactors using mammalian cells engineered for genomic containment of mUltiple copies of defined target genes for genotoxic assessment. The Rat 2(lambda) fibroblasts (Stratagene, Inc.) were genetically engineered to contain high-density target genes for mutagenesis. Stable three-dimensional, multicellular spheroids were formed when human mammary epithelial cells and Rat 2(lambda) fibroblasts were cocultured on Cytodex 3 Beads in a rotating wall bioreactor. The utility of this spheroidal model for genotoxic assessment was indicated by a linear dose response curve and by results of gene sequence analysis of mutant clones from 400micron diameter spheroids following low-dose, high-energy, neon radiation exposure

Gonda, S.

Plant adaptation to low atmospheric pressures: potential molecular responses

There is an increasing realization that it may be impossible to attain Earth normal atmospheric pressures in orbital, lunar, or Martian greenhouses, simply because the construction materials do not exist to meet the extraordinary constraints imposed by balancing high engineering requirements against high lift costs. This equation essentially dictates that NASA have in place the capability to grow plants at reduced atmospheric pressure. Yet current understanding of plant growth at low pressures is limited to just a few experiments and relatively rudimentary assessments of plant vigor and growth. The tools now exist, however, to make rapid progress toward understanding the fundamental nature of plant responses and adaptations to low pressures, and to develop strategies for mitigating detrimental effects by engineering the growth conditions or by engineering the plants themselves. The genomes of rice and the model plant Arabidopsis thaliana have recently been sequenced in their entirety, and public sector and commercial DNA chips are becoming available such that thousands of genes can be assayed at once. A fundamental understanding of plant responses and adaptation to low pressures can now be approached and translated into procedures and engineering considerations to enhance plant growth at low atmospheric pressures. In anticipation of such studies, we present here the background arguments supporting these contentions, as well as informed speculation about the kinds of molecular physiological responses that might be expected of plants in low-pressure environments.

NASA Discipline Plant Biology

Evolutionary Flexibility and Rigidity in the Bacterial Methylerythritol Phosphate (Mep) Pathway

Terpenoids are a diverse class of compounds with wide-ranging uses including as industrial solvents, pharmaceuticals, and fragrances. Efforts to produce terpenoids sustainably by engineering microbes for fermentation are ongoing, but industrial production still largely relies on nonrenewable sources. The methylerythritol phosphate (MEP) pathway generates terpenoid precursor molecules and includes the enzyme Dxs and two iron–sulfur cluster enzymes: IspG and IspH. IspG and IspH are rate limiting-enzymes of the MEP pathway but are challenging for metabolic engineering because they require iron–sulfur cluster biogenesis and an ongoing supply of reducing equivalents to function. Therefore, identifying novel alternatives to IspG and IspH has been an on-going effort to aid in metabolic engineering of terpenoid biosynthesis. We report here an analysis of the evolutionary diversity of terpenoid biosynthesis strategies as a resource for exploration of alternative terpenoid biosynthesis pathways. Using comparative genomics, we surveyed a database of 4,400 diverse bacterial species and found that some may have evolved alternatives to the first enzyme in the pathway, Dxs making it evolutionarily flexible. In contrast, we found that IspG and IspH are evolutionarily rigid because we could not identify any species that appear to have enzymatic routes that circumvent these enzymes. The ever-growing repository of sequenced bacterial genomes has great potential to provide metabolic engineers with alternative metabolic pathway solutions. With the current state of knowledge, we found that enzymes IspG and IspH are evolutionarily indispensable which informs both metabolic engineering efforts and our understanding of the evolution of terpenoid biosynthesis pathways.

Bailey Marshall

ICME for NASA Aerospace Applications: Batteries for Electric Aviation

NASA’s approach to computational materials modeling is detailed in the NASA Vision 2040 Roadmap for Multiscale Modeling and Simulation of Materials and Systems. This report is in the spirit of national initiatives such as the Material Genome Initiative (MGI), Integrated Computational Materials Engineering (ICME), and others. We utilize a combination of fundamental modeling, computational high-throughput screening, and data science methods, e.g., machine learning, are used to find innovative solutions to NASA or national technology challenges. Applications of interest are wide ranging from advanced alloys to batteries to coatings, among others. In this talk, we present three examples for recent work related to NASA applications. First, doping advanced sulfur battery cathodes with selenium boosts electrical conductivity important for electric aircraft applications. First principles calculations will be discussed that result in compositional design maps for these materials. Second, development of icephobic coatings is important to mitigate safety hazards associated with icing for aircraft. Molecular dynamics simulations are reported for ice-surface interfaces to understand adhesion mechanisms and help screen optimal ice-phobic coatings. Third, shape memory alloys have numerous applications as actuators, superelastic materials, etc. for aerospace. We report machine learning models that predict martensitic transition temperatures across a broad swath of compositional space.

John Lawson

Robust Informatics Infrastructure Required For ICME: Combining Virtual and Experimental Data

With the increased emphasis on reducing the cost and time to market of new materials, the need for robust automated materials information management system(s) enabling sophisticated data mining tools is increasing, as evidenced by the emphasis on Integrated Computational Materials Engineering (ICME) and the recent establishment of the Materials Genome Initiative (MGI). This need is also fueled by the demands for higher efficiency in material testing; consistency, quality and traceability of data; product design; engineering analysis; as well as control of access to proprietary or sensitive information. Further, the use of increasingly sophisticated nonlinear, anisotropic and or multi-scale models requires both the processing of large volumes of test data and complex materials data necessary to establish processing-microstructure-property-performance relationships. Fortunately, material information management systems have kept pace with the growing user demands and evolved to enable: (i) the capture of both point wise data and full spectra of raw data curves, (ii) data management functions such as access, version, and quality controls;(iii) a wide range of data import, export and analysis capabilities; (iv) data pedigree traceability mechanisms; (v) data searching, reporting and viewing tools; and (vi) access to the information via a wide range of interfaces. This paper discusses key principles for the development of a robust materials information management system to enable the connections at various length scales to be made between experimental data and corresponding multiscale modeling toolsets to enable ICME. In particular, NASA Glenn's efforts towards establishing such a database for capturing constitutive modeling behavior for both monolithic and composites materials

Mutli-scale models

Reveal, A General Reverse Engineering Algorithm for Inference of Genetic Network Architectures

Given the immanent gene expression mapping covering whole genomes during development, health and disease, we seek computational methods to maximize functional inference from such large data sets. Is it possible, in principle, to completely infer a complex regulatory network architecture from input/output patterns of its variables? We investigated this possibility using binary models of genetic networks. Trajectories, or state transition tables of Boolean nets, resemble time series of gene expression. By systematically analyzing the mutual information between input states and output states, one is able to infer the sets of input elements controlling each element or gene in the network. This process is unequivocal and exact for complete state transition tables. We implemented this REVerse Engineering ALgorithm (REVEAL) in a C program, and found the problem to be tractable within the conditions tested so far. For n = 50 (elements) and k = 3 (inputs per element), the analysis of incomplete state transition tables (100 state transition pairs out of a possible 10(exp 15)) reliably produced the original rule and wiring sets. While this study is limited to synchronous Boolean networks, the algorithm is generalizable to include multi-state models, essentially allowing direct application to realistic biological data sets. The ability to adequately solve the inverse problem may enable in-depth analysis of complex dynamic systems in biology and other fields.

Liang, Shoudan

Final Report - Hypergravity Effects on Chromatin Conformation and Nuclear Structure in Cultured Cells using High-throughput Sequencing

The conformation of the human genome is known to play an important role in transcriptional control of gene expression. Our aim is to assess whether exposure of cultured human cardiomyocytes to hypergravity can induce changes in chromatin organization as assayed by Hi-C proximity ligation technique. Hi-C is used to analyze chromatin interactions by using formaldehyde to crosslink regions of chromatin that are in close proximity. The DNA is then fragmented using a restriction enzyme and ligated under dilute conditions to favor intramolecular ligation of cross-linked fragments. Finally, the DNA is sequenced, allowing reconstruction of genomic structure. iCell cardiomyocyte cultures will be exposed to 40 g via an engineered device (incu-fuge) that allows for the chemical fixation of cells on a spinning centrifuge. Additionally, we plan on using fluorescence microscopy to analyze the nuclear and actin cytoskeletal conformation of cardiomyocytes post hypergravity exposure. We hypothesize that chromatin will rearrange in hypergravity conditions, both rapidly due to direct mechanical forces, as well as over longer time frames due to changes to structures necessary for transcriptional responses such as the de novo formation of promoter-enhancer loops. A negative result–no change between 1 g and hypergravity conditions–would nevertheless be an important data point in our understanding of the rheology of the nucleus, and how cells and cellular structures respond to different gravity fields. The cellular physiology of hypergravity has clinical relevance for spaceflight, and can further inform our understanding of microgravity physiology.

chromatin

Expanding the Frontiers of Knowledge

So space is supremely hostile, but we know this. But when we ask what is the cost of human space missions, we need to consider as many contingencies as possible. This is important because we want to do more than send people on one-way trips, we want to be able to bring astronauts back. So if exploration is what really matters and not just pride of nation, then perhaps we should genetically engineer a version of ourselves that can survive the hostile environments of space. We've got cloning. We're inside the genome. Let s just do it. Well in fact, we ve done that already. Yes, we have emissaries of ourselves that survive the hazards of space; they re called robots. You don t have to feed them or bring them back, and they don t complain if you lose them in space. So my concern is if costs turn out to be what they have historically been and the time to execute programs lasts as long as it historically has, then I am not convinced that economic cycles and political cycles will allow such programs to survive if they do not satisfy one of these three criteria. The record of history tells us this, unless somehow you want to believe that we are different today than 6,000 years of our predecessors.

deGrasse Tyson, Neil

NEEMO 21: Tools, Techniques, Technologies and Training for Science Exploration

The 21st mission of the National Aeronautics and Space Administration (NASA) Extreme Environment Mission Operations (NEEMO) was a highly integrated operational field test and evaluation of tools, techniques, technologies, and training for science driven exploration during extravehicular activity (EVA). The mission was conducted in July 2016 from the Aquarius habitat, an underwater laboratory, off the coast of Key Largo in the Florida Keys National Marine Sanctuary. An international crew of eight (comprised of NASA and ESA astronauts, engineers, medical personnel, and habitat technicians) lived and worked in and around Aquarius and its surrounding reef environment for 16 days. The integrated testing (both interior and exterior objectives) conducted from this unique facility continues to support current and future human space exploration endeavors. Expanding on the scientific and operational evaluations conducted during NEEMO 20, the 21st NEEMO mission further incorporated a diverse Science Team comprised of planetary geoscientists from the Astromaterials Research and Exploration Science (ARES/XI) Division from the Johnson Space Center, marine scientists from the Department of Biological Sciences at Florida International University (FIU) Integrative Marine Genomics and Symbiosis (IMaGeS) Lab, and conservationists from the Coral Restoration Foundation. The Science Team worked in close coordination with the long-standing EVA operations, planning, engineering, and research components of NEEMO in all aspects of mission planning, development, and execution.

Graff, T.

NASA Tech Briefs, August 2011

Topics covered include: Miniature, Variable-Speed Control Moment Gyroscope; NBL Pistol Grip Tool for Underwater Training of Astronauts; HEXPANDO Expanding Head for Fastener-Retention Hexagonal Wrench; Diagonal-Axes Stage for Pointing an Optical Communications Transceiver; Improvements in Speed and Functionality of a 670-GHz Imaging Radar; IONAC-Lite; Large Ka-Band Slot Array for Digital Beam-Forming Applications; Development of a 150-GHz MMIC Module Prototype for Large-Scale CMB Radiation; Coupling Between Waveguide-Fed Slot Arrays; PCB-Based Break-Out Box; Multiple-Beam Detection of Fast Transient Radio Sources; Router Agent Technology for Policy-Based Network Management; Remote Asynchronous Message Service Gateway; Automatic Tie Pointer for In-Situ Pointing Correction; Jitter Correction; MSLICE Sequencing; EOS MLS Level 2 Data Processing Software Version 3; DspaceOgre 3D Graphics Visualization Tool; Metallization for Yb14MnSb11-Based Thermoelectric Materials; Solvent/Non-Solvent Sintering To Make Microsphere Scaffolds; Enhanced Fuel-Optimal Trajectory-Generation Algorithm for Planetary Pinpoint Landing; Self-Cleaning Coatings and Materials for Decontaminating Field-Deployable Land and Water-Based Optical Systems; Separation of Single-Walled Carbon Nanotubes with DEP-FFF; Li Anode Technology for Improved Performance; Post-Fragmentation Whole Genome Amplification-Based Method; Microwave Tissue Soldering for Immediate Wound Closure; Principles, Techniques, and Applications of Tissue Microfluidics; Robotic Scaffolds for Tissue Engineering and Organ Growth; Stress-Driven Selection of Novel Phenotypes; Method for Accurately Calibrating a Spectrometer Using Broadband Light; Catalytic Microtube Rocket Igniter; Stage Cylindrical Immersive Display; Vacuum Camera Cooler; Atomic Oxygen Fluence Monitor; Thermal Management Tools for Propulsion System Trade Studies and Analysis; Introduction to Physical Intelligence; Technique for Solving Electrically Small to Large Structures for Broadband Applications; Accelerated Adaptive MGS Phase Retrieval; Large Eddy Simulation Study for Fluid Disintegration and Mixing; Tropospheric Correction for InSAR Using Interpolated ECMWF Data and GPS Zenith Total Delay; Technique for Calculating Solution Derivatives With Respect to Geometry Parameters in a CFD Code; Acute Radiation Risk and BRYNTRN Organ Dose Projection Graphical User Interface; Probabilistic Path Planning of Montgolfier Balloons in Strong, Uncertain Wind Fields; Flight Simulation of ARES in the Mars Environment; Low-Outgassing Photogrammetry Targets for Use in Outer Space; Planning the FUSE Mission Using the SOVA Algorithm; Monitoring Spacecraft Telemetry Via Optical or RF Link; and Robust Thermal Control of Propulsion Lines for Space Missions.

Source record

A Design Framework for Thick and Thin Tow-Steered Composites Using Mechanics of Structure Genome

The design of tow-steered composites is one of the most popular and promising topics under the class of variable stiffness structures. In this work, the authors propose a new design framework and tool for the optimization of tow-steered composites. Mechanics of structure genome (MSG) provides accurate computation of plate/shell section properties including thick and highly curved structures. The computed section properties can be directly used in commercial computer-aided engineering tools. Tow-steered composites are modeled through a design framework wherein tow paths are parameterized in a general way and projected onto a finite element mesh, and MSG calculates the local shell/plate properties. The result is a complete workflow from design parameter input to structural performance evaluation. An open-source optimization software package is used to enable constrained optimization of structural stiffness for the broad tow-steered composite design space. Numerical examples are provided to demonstrate the capabilities of this tool and the promise and potential of tow-steered designs that optimize structural performance while satisfying manufacturing constraints.

Su Tian

A Design Framework for Thick and Thin Tow-Steered Composites Using Mechanics of Structure Genome

The design of tow-steered composites is one of the most popular and promising topics under the class of variable stiffness structures. In this work, the authors propose a new design framework and tool for the optimization of tow-steered composites. Mechanics of structure genome (MSG) provides accurate computation of plate/shell section properties including thick and highly curved structures. The computed section properties can be directly used in commercial computer-aided engineering tools. Tow-steered composites are modeled through a design framework wherein tow paths are parameterized in a general way and projected onto a finite element mesh, and MSG calculates the local shell/plate properties. The result is a complete workflow from design parameter input to structural performance evaluation. An open-source optimization software package is used to enable constrained optimization of structural stiffness for the broad tow-steered composite design space. Numerical examples are provided to demonstrate the capabilities of this tool and the promise and potential of tow-steered designs that optimize structural performance while satisfying manufacturing constraints.

Su Tian

Identification of Novel Desiccation-Tolerant S. cerevisiae Strains for Deep Space Biosensors

NASA's BioSentinel mission, a secondary payload that will fly on the Space Launch System's first Exploration Mission (EM-1), utilizes the budding yeast S. cerevisiae to study the biological response to the deep space radiation environment. Yeast samples are desiccated prior to launch to suspend growth and metabolism while the spacecraft travels to its target heliocentric orbit beyond Low Earth Orbit. Each sample is then rehydrated at the desired time points to reactivate the cells. A major risk in this mission is the loss of cell viability that occurs in the recovery period following the desiccation and rehydration process. Cell survival is essential for the detection of the biological response to features in the deep space environment, including ionizing radiation. The aim of this study is to mitigate viable cell loss in future biosensors by identifying mutations and genes that confer tolerance to desiccation stress in rad51, a radiation-sensitive yeast strain. We initiated a screen for desiccation-tolerance after rehydrating cells that were desiccated for three years, and selected various clones exhibiting robust growth. To verify retention of radiation sensitivity in the isolated clones - a crucial feature for a successful biosensor - we exposed them to ionizing radiation. Finally, to elucidate the genetic and molecular bases for observed desiccation-tolerance, we will perform whole-genome sequencing of those rad51 clones that exhibit both robust growth and radiation sensitivity following desiccation. The identification and characterization of desiccation-tolerant strains will allow us to engineer a biological model that will be resilient in face of the challenges of the deep space environment, and will thus ensure the experimental success of future biosensor missions.

S. cerevisiae

Identification of Novel Desiccation-Tolerant S. cerevisiae Strains for Deep Space Biosensors

NASA's BioSentinel mission, a secondary payload that will fly on the Space Launch Systems first Exploration Mission (EM-1), utilizes the budding yeast S. cerevisiae to study the biological response to the deep space radiation environment. Yeast samples are desiccated prior to launch to suspend growth and metabolism while the spacecraft travels to its target heliocentric orbit beyond Low Earth Orbit. Each sample is then rehydrated at the desired time points to reactivate the cells. A major risk in this mission is the loss of cell viability that occurs in the recovery period following the desiccation and rehydration process. Cell survival is essential for the detection of the biological response to features in the deep space environment, including ionizing radiation.The aim of this study is to mitigate viable cell loss in future biosensors by identifying mutations and genes that confer tolerance to desiccation stress in rad51, a radiation-sensitive yeast strain. We initiated a screen for desiccation-tolerance after rehydrating cells that were desiccated for three years, and selected various clones exhibiting robust growth. To verify retention of radiation sensitivity in the isolated clonesa crucial feature for a successful biosensorwe exposed them to ionizing radiation. Finally, to elucidate the genetic and molecular bases for observed desiccation-tolerance, we will perform whole-genome sequencing of those rad51 clones that exhibit both robust growth and radiation sensitivity following desiccation. The identification and characterization of desiccation-tolerant strains will allow us to engineer a biological model that will be resilient in face of the challenges of the deep space environment, and will thus ensure the experimental success of future biosensor missions.

S. cerevisiae