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At least 91 records · Page 5
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Species-specific ribosomal RNA-FISH identifies interspecies cellular-material exchange, active-cell population dynamics and cellular localization of translation machinery in clostridial cultures and co-cultures

ABSTRACT The development of synthetic microbial consortia in recent years has revealed that complex interspecies interactions, notably the exchange of cytoplasmic material, exist even among organisms that originate from different ecological niches. Although morphogenetic characteristics, viable RNA and protein dyes, and fluorescent reporter proteins have played an essential role in exploring such interactions, we hypothesized that ribosomal RNA-fluorescence in situ hybridization (rRNA-FISH) could be adapted and applied to further investigate interactions in synthetic or semisynthetic consortia. Despite its maturity, several challenges exist in using rRNA-FISH as a tool to quantify individual species population dynamics and interspecies interactions using high-throughput instrumentation such as flow cytometry. In this work, we resolve such challenges and apply rRNA-FISH to double and triple co-cultures of Clostridium acetobutylicum, Clostridium ljungdahlii, and Clostridium kluyveri . In pursuing our goal to capture each organism’s population dynamics, we demonstrate dynamic rRNA, and thus ribosome, exchange between the three species leading to the formation of hybrid cells. We also characterize the localization patterns of the translation machinery in the three species, identifying distinct, dynamic localization patterns among them. Our data also support the use of rRNA-FISH to assess the culture’s health and expansion potential, and, here again, our data find surprising differences among the three species examined. Taken together, our study argues for rRNA-FISH as a valuable and accessible tool for quantitative exploration of interspecies interactions, especially in organisms which cannot be genetically engineered or in consortia where selective pressures to maintain recombinant species cannot be used. IMPORTANCE Though dyes and fluorescent reporter proteins have played an essential role in identifying microbial species in co-cultures, we hypothesized that ribosomal RNA-fluorescence in situ hybridization (rRNA-FISH) could be adapted and applied to quantitatively probe complex interactions between organisms in synthetic consortia. Despite its maturity, several challenges existed before rRNA-FISH could be used to study Clostridium co-cultures of interest. First, species-specific probes for Clostridium acetobutylicum and Clostridium ljungdahlii had not been developed. Second, “state-of-the-art” labeling protocols were tedious and often resulted in sample loss. Third, it was unclear if FISH was compatible with existing fluorescent reporter proteins. We resolved these key challenges and applied the technique to co-cultures of C. acetobutylicum , C. ljungdahlii , and Clostridium kluyveri . We demonstrate that rRNA-FISH is capable of identifying rRNA/ribosome exchange between the three organisms and characterized rRNA localization patterns in each. In combination with flow cytometry, rRNA-FISH can capture sub-population dynamics in co-cultures.

Hill, John D.↗
NASA NTRS1983

Complexes of polyadenylic acid and the methyl esters of amino acids

A study of amino acid methyl esters binding to polyadenylic acid supports the theory that the genetic code originated through weak but selective affinities between amino acids and nucleotides. NMR, insoluble complex analysis, and ultraviolet spectroscopy are used to illustrate a correlation between the hydrophybicities of A amino acids and their binding constants, which, beginning with the largest, are in the order of Phe (having nominally a hydrophobic AAA anticodon), Ile, Leu, Val and Gly (having a hydrophilic anticodon with no A). In general, the binding constants are twice the values by Reuben and Polk (1980) for monomeric AMP, which suggests that polymer amino acids are interacting with only one base. No real differences are found betwen poly A binding for free Phe, Phe methyl ester or Phe amide, except that the amide value is slightly lower.

Khaled, M. A.↗
NASA NTRS1984

Evolutionary constraints and the neutral theory

The neutral theory of molecular evolution postulates that nucleotide substitutions inherently take place in DNA as a result of point mutations followed by random genetic drift. In the absence of selective constraints, the substitution rate reaches the maximum value set by the mutation rate. The rate in globin pseudogenes is about 5 x 10 to the -9th substitutions per site per year in mammals. Rates slower than this indicate the presence of constraints imposed by negative (natural) selection, which rejects and discards deleterious mutations.

Jukes, T. H.↗
NASA NTRS1998

Automatic Evolution of Molecular Nanotechnology Designs

This paper describes strategies for automatically generating designs for analog circuits at the molecular level. Software maps out the edges and vertices of potential nanotechnology systems on graphs, then selects appropriate ones through evolutionary or genetic paradigms.

Globus, Al↗
NASA NTRS2002

Development of Biocompatibility Procedures for Assessment of Plant Growth in Ground Test Hardware for the EMCS

The European Modular Cultivation System (EMCS) is an European Space Agency-developed facility designed to support plant research in microgravity on the IS NASA is responsible for providing US specific hardware to use within the EMCS. In preparation for flight, research will be developed and tested at Ames Research Center in the EMCS ground test hardware, the Experiment Reference Module (ERM) In order to determine the acceptability of the ERM for such purposes, biocompatibility tests will be performed to determine that the hardware functions as intended and successfully supports the' growth of plants. In this report, we describe the development of procedures and the collection of baseline data against which to compare ERM function, e.g. biocompatibility testing. A simple and robust system was developed to grow whole Arabidopsis thaliana plants within the confined volumes characteristic of spaceflight hardware. Our system for growing plants eliminated the necessity of a water/nutrient delivery system and allowed for quantifiable assessment of individual plants, as well as entire population dynamics. To insure uniform germination, seeds were started in small straw segments and transplanted into modified scintillation vials. Seedlings were selected prior to transplantation to decrease genetic variability. Plants were grown for a total of 24 days in standard laboratory plant growth chambers under controlled conditions. Sequential digital still images were taken on a daily basis. Analysis of these images allowed for the quantification of even minute environmental effect, on growth dynamics whole plants. The data collected provide reliable growth curves against which to compare plants grown in the ERM.

Bowman, R. N.↗
NASA NTRS2006

Orbit design and optimization based on global telecommunication performance metrics

The orbit selection of telecommunications orbiters is one of the critical design processes and should be guided by global telecom performance metrics and mission-specific constraints. In order to aid the orbit selection, we have coupled the Telecom Orbit Analysis and Simulation Tool (TOAST) with genetic optimization algorithms. As a demonstration, we have applied the developed tool to select an optimal orbit for general Mars telecommunications orbiters with the constraint of being a frozen orbit. While a typical optimization goal is to minimize tele-communications down time, several relevant performance metrics are examined: 1) area-weighted average gap time, 2) global maximum of local maximum gap time, 3) global maximum of local minimum gap time. Optimal solutions are found with each of the metrics. Common and different features among the optimal solutions as well as the advantage and disadvantage of each metric are presented. The optimal solutions are compared with several candidate orbits that were considered during the development of Mars Telecommunications Orbiter.

genetic algorithms↗
DOE OSTI2025

Inducible flippase-mediated metabolic engineering of Rhodosporidium toruloides for enhanced 3-hydroxypropionic acid production from corn stover hydrolysate

Rhodosporidium toruloides has gained increasing interests as a promising non-model host organism to produce a wide range of bioproducts from lignocellulosic biomass. Increasing the bioproduct titers, rates, and yields remains a challenge, largely due to a lack of robust and well-characterized genetic tools in this host. Here we developed an inducible flippase (FLP) and flippase recognition target (FRT) system that enables genetic manipulations without the need for additional selection markers. Synthetic inducible promoters were established, enabling regulation of FLP expression and efficient antibiotic marker removal. Leveraging this system, we engineered a strain to optimize 3-hydroxypropionic acid (3HP) production. Over four rounds of iterative genomic editing to resolve pathway bottlenecks, we achieved a 3HP titer of 69.4 g/L in fed-batch fermentation - the highest level reported in yeast from lignocellulosic hydrolysates. The engineered high 3HP producing strain offers a robust platform for sustainable bio-based chemical production from lignocellulosic feedstocks.

3-hydroxypropionic acid↗
NASA NTRS1985

Wideband Disturbance Accomodation in Precision Flexible Space Structures

Numerous spacecraft missions currently being formulated exhibit several basic and sharply conflicting features: selected components (e.g., an optical train) of inherently flexible structures are to maintain precision pointing performance in a wideband disturbance environment. Within the overall process of synthesizing an active controller to deal with this difficult problem, the aspects of reduced-order modelling and of actuator and sensor selection are crucial to the successful implementation of any controller feedback strategy. The principal focus in this paper is on the influence of actuator and sensor selection upon the effectiveness of a specified controller strategy for wideband disturbance accommodation. A genetic optical support structure is used for a number of design examples. Active transducer selections are made systematically based upon their direct contributuion to optical pointing error. Controller designs incorporating the various transducer selections exhibit stability in the presence of unmodeled modes over a frequency range substantially beyond the bandwidth of the disturbance.

Hegg, D. R.↗
NASA NTRS2017

Towards a Universal Biology: Is the Origin and Evolution of Life Predictable?

The origin and evolution of life seems an unpredictable oddity, based on the quirks of contingency. Celebrated by the late Stephen Jay Gould in several books, "evolution by contingency" has all the adventure of a thriller, but lacks the predictive power of the physical sciences. Not necessarily so, replied Simon Conway Morris, for convergence reassures us that certain evolutionary responses are replicable. The outcome of this debate is critical to Astrobiology. How can we understand where we came from on Earth without prophesy? Further, we cannot design a rational strategy for the search for life elsewhere - or to understand what the future will hold for life on Earth and beyond - without extrapolating from pre-biotic chemistry and evolution. There are several indirect approaches to understanding, and thus describing, what life must be. These include philosophical approaches to defining life (is there even a satisfactory definition of life?), using what we know of physics, chemistry and life to imagine alternate scenarios, using different approaches that life takes as pseudoreplicates (e.g., ribosomal vs non-ribosomal protein synthesis), and experimental approaches to understand the art of the possible. Given that: (1) Life is a process based on physical components rather than simply an object; (2). Life is likely based on organic carbon and needs a solvent for chemistry, most likely water, and (3) Looking for convergence in terrestrial evolution we can predict certain tendencies, if not quite "laws", that provide predictive power. Biological history must obey the laws of physics and chemistry, the principles of natural selection, the constraints of an evolutionary past, genetics, and developmental biology. This amalgam creates a surprising amount of predictive power in the broad outline. Critical is the apparent prevalence of organic chemistry, and uniformity in the universe of the laws of chemistry and physics. Instructive is the widespread occurrence of convergent or parallel evolution, which suggests that under certain conditions similar solutions are arrived at independently.

Universal biology; life everywhere↗
DOE OSTI2024

Robust wind farm layout optimization

Wake interactions in wind farms cause losses in annual energy production (AEP) on the order of 10%. Wind farm designers optimize the layout of the farm to mitigate wake losses, especially in the dominant site-specific wind directions. As wind turbines and wind farms grow in scale, optimization becomes more complex. Offshore wind farms regularly comprise more than 100 wind turbines and are characterized by complex boundaries due to shipping lanes, neighboring wind farms, and other constraints. Layout optimization methods are broadly split between gradient-based and gradient-free approaches. Gradient-based approaches can converge quickly and perform well for smaller, academic problems but are often sensitive to initial conditions and tuning parameters and require expert knowledge to use. On the other hand, gradient-free approaches can be more robust to problem complexities. We present a robust layout optimization approach based on a random search algorithm. The algorithm is intended for those who are not optimization experts and has few tuning parameters that need specification to achieve satisfactory results. Unlike off-the-shelf methods, which use generally available, non-domain-specific optimization routines that accept as inputs an optimization function and constraint definitions, this approach takes advantage of the relative computational costs of the different evaluations by evaluating cheaper computations first (boundary and minimum distance constraints) and running expensive AEP evaluations only if all other checks pass. Moreover, an outer genetic algorithm allows multiple solutions to evolve in parallel, enabling rapid solution development on high-performance computers. We discuss the relative ease of selecting necessary tuning parameters and demonstrate the efficacy of the genetic random search on a complex layout problem consisting of placing 70 turbines in a nonconvex and unconnected boundary region.

17 WIND ENERGY↗
NASA NTRS2000

Constructing high complexity synthetic libraries of long ORFs using in vitro selection

We present a method that can significantly increase the complexity of protein libraries used for in vitro or in vivo protein selection experiments. Protein libraries are often encoded by chemically synthesized DNA, in which part of the open reading frame is randomized. There are, however, major obstacles associated with the chemical synthesis of long open reading frames, especially those containing random segments. Insertions and deletions that occur during chemical synthesis cause frameshifts, and stop codons in the random region will cause premature termination. These problems can together greatly reduce the number of full-length synthetic genes in the library. We describe a strategy in which smaller segments of the synthetic open reading frame are selected in vitro using mRNA display for the absence of frameshifts and stop codons. These smaller segments are then ligated together to form combinatorial libraries of long uninterrupted open reading frames. This process can increase the number of full-length open reading frames in libraries by up to two orders of magnitude, resulting in protein libraries with complexities of greater than 10(13). We have used this methodology to generate three types of displayed protein library: a completely random sequence library, a library of concatemerized oligopeptide cassettes with a propensity for forming amphipathic alpha-helical or beta-strand structures, and a library based on one of the most common enzymatic scaffolds, the alpha/beta (TIM) barrel. Copyright 2000 Academic Press.

Non-NASA Center↗
NASA NTRS2005

Genetic Algorithms Applied to Multi-Objective Aerodynamic Shape Optimization

A genetic algorithm approach suitable for solving multi-objective problems is described and evaluated using a series of aerodynamic shape optimization problems. Several new features including two variations of a binning selection algorithm and a gene-space transformation procedure are included. The genetic algorithm is suitable for finding Pareto optimal solutions in search spaces that are defined by any number of genes and that contain any number of local extrema. A new masking array capability is included allowing any gene or gene subset to be eliminated as decision variables from the design space. This allows determination of the effect of a single gene or gene subset on the Pareto optimal solution. Results indicate that the genetic algorithm optimization approach is flexible in application and reliable. The binning selection algorithms generally provide Pareto front quality enhancements and moderate convergence efficiency improvements for most of the problems solved.

Holst, Terry L.↗
NASA NTRS2004

Genetic Algorithms Applied to Multi-Objective Aerodynamic Shape Optimization

A genetic algorithm approach suitable for solving multi-objective optimization problems is described and evaluated using a series of aerodynamic shape optimization problems. Several new features including two variations of a binning selection algorithm and a gene-space transformation procedure are included. The genetic algorithm is suitable for finding pareto optimal solutions in search spaces that are defined by any number of genes and that contain any number of local extrema. A new masking array capability is included allowing any gene or gene subset to be eliminated as decision variables from the design space. This allows determination of the effect of a single gene or gene subset on the pareto optimal solution. Results indicate that the genetic algorithm optimization approach is flexible in application and reliable. The binning selection algorithms generally provide pareto front quality enhancements and moderate convergence efficiency improvements for most of the problems solved.

Holst, Terry L.↗
NASA NTRS1974

Origins of biological information and the genetic code

Information, defined as the capacity of a molecule or system for selective interactions with other molecules or systems, is followed through its evolution from prebiological information to protoribosomes. Emphasis is on proteins and protein-like polymers, and later on ATP. The research will contribute more to the understanding of the essence of the genetic mechanism.

Fox, S. W.↗
NASA NTRS1973

Ultraviolet selection pressure on the earliest organisms

It had been proposed by Sagan (1957, 1961) that UV light, partially penetrating the primitive reducing atmosphere of the earth, posed a major problem for the earliest evolution of life. This argument is now updated and refined. The picture of a secondary reducing atmosphere is presented. It is assumed that an excess of hydrogen from this atmosphere has already escaped to space. The genetic material surrounded itself as a solution to the UV selection pressure with bases or nucleotides having no function whatever in replication or protein synthesis.

Sagan, C.↗
NASA NTRS2011

Stress-Driven Selection of Novel Phenotypes

A process has been developed that can confer novel properties, such as metal resistance, to a host bacterium. This same process can also be used to produce RNAs and peptides that have novel properties, such as the ability to bind particular compounds. It is inherent in the method that the peptide or RNA will behave as expected in the target organism. Plasmid-born mini-gene libraries coding for either a population of combinatorial peptides or stable, artificial RNAs carrying random inserts are produced. These libraries, which have no bias towards any biological function, are used to transform the organism of interest and to serve as an initial source of genetic variation for stress-driven evolution. The transformed bacteria are propagated under selective pressure in order to obtain variants with the desired properties. The process is highly distinct from in vitro methods because the variants are selected in the context of the cell while it is experiencing stress. Hence, the selected peptide or RNA will, by definition, work as expected in the target cell as the cell adapts to its presence during the selection process. Once the novel gene, which produces the sought phenotype, is obtained, it can be transferred to the main genome to increase the genetic stability in the organism. Alternatively, the cell line can be used to produce novel RNAs or peptides with selectable properties in large quantity for separate purposes. The system allows for easy, large-scale purification of the RNAs or peptide products. The process has been reduced to practice by imposing sub-inhibitory concentrations of NiCl2 on cells of the bacterium Escherichia coli that were transformed separately with the peptide library and RNA library. The evolved resistant clones were isolated, and sequences of the selected mini-gene variants were established. Clones resistant to NiCl2 were found to carry identical plasmid variants with a functional mini-gene that specifically conferred significant nickel tolerance on the host cells. Sequencing of the selected mini-gene revealed a propensity of the encoded peptide to bind transient metal ions. Expression of the mini-gene markedly improved growth parameters of the evolved clones at sub-inhibitory concentrations of NiCl2 while being slightly detrimental in the absence of stress. Similar results have been obtained with the RNA libraries. Overall, the results demonstrate a very natural outcome of the selection experiments in which the mini-genes were expected to be either successfully integrated into bacterial genetic networks, or rejected depending upon their effect on host fitness. This described approach can be useful as a laboratory model to study the dynamics of bacterial adaptive evolution on the molecular level. It can also provide a strategy for screening expressed DNA libraries in search of novel genes with desirable properties.

Fox, George E.↗