Genetic stability of Hydrogenomonas eutropha
Genetic stability and phage ineffectivity of Hydrogenomonas eutropha
Decicco, B. T.↗
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Synthetic overlapping genes stabilize genetic systems
Overlapping genes—wherein two different proteins are translated from alternative reading frames of the same DNA sequence—provide a means to stabilize an engineered gene by directly linking its evolutionary fate with that of an overlapping gene. However, creating overlapping gene pairs is challenging, as it requires redesigning both protein products to accommodate overlap constraints. Here, we present a new “overlapping, alternate-frame insertion” (OAFI) method for creating synthetic overlapping genes by inserting an “inner” gene, encoded in an alternate frame, into a flexible region of an “outer” gene. Using OAFI, we create new overlapping gene pairs of genetic reporters and bacterial toxins within an antibiotic resistance gene. We show that both the inner and outer genes retain function despite redesign, with translation of the inner gene influenced by its overlap position in the outer gene. Importantly, we show that, despite these inner gene sequences not contributing to outer gene function, selection for the outer gene alters the permitted inactivating mutations in the inner gene, and that overlapping toxins can restrict horizontal gene transfer of the antibiotic resistance gene. Overall, OAFI offers a versatile tool for synthetic biology, expanding the applications of overlapping genes in gene stabilization and biocontainment.
Biological and medical sciences↗
Space Algae-2 Ground and Lunar Analog Studies in Preparation for Long-Duration Propagation of Cyanobacteria in Spaceflight
There are numerous applications for microalgae in spaceflight missions and on Earth, such as oxygen production, carbon dioxide removal, nutrition, wastewater processing, and biofuel production. Space Algae-2 aims to test the genetic stability of Arthrospira platensis, commonly known as spirulina, during six-months of continuous culture on the International Space Station. Long-duration exposure to ionizing radiation and microgravity may impact growth, nutrient composition, and genetic stability. The high protein, vitamin, antioxidant content, and radiation resistance make spirulina a promising candidate for bioregenerative life support systems. A concept of operations was developed to grow and harvest algal biomass in space. Preflight testing experiments optimized conditions for an extended growth period in a gas permeable bioreactor bag. Preflight and post-harvest storage methods were developed in addition to a novel cryopreservation method. After sample return, multi-omics analysis will be conducted to determine the mutation rate, gene expression, and the protein and metabolite profile. The concept of operations for Space Algae-2 was tested during a lunar mission simulation within a semi-controlled environment. During a six-day lunar analog mission at the Hawai’i Space Exploration Analog and Simulation (HI-SEAS) A. platensis was successfully grown using flight-like hardware. The cyanobacteria were harvested and used to supplement bread as an example of spirulina biomass utilization. Overall, the data collected from Space Algae-2 will inform potential bioengineering of spirulina for space and terrestrial applications.
Algae↗
Space Algae-2 Ground and Lunar Analog Studies in Preparation for Long-Duration Propagation of Cyanobacteria in Spaceflight
There are numerous applications for microalgae in spaceflight missions and on Earth, such as oxygen production, carbon dioxide removal, nutrition, wastewater processing, and biofuel production. Space Algae-2 aims to test the genetic stability of Arthrospira platensis, commonly known as spirulina, during six-months of continuous culture on the International Space Station. Long-duration exposure to ionizing radiation and microgravity may impact growth, nutrient composition, and genetic stability. The high protein, vitamin, antioxidant content, and radiation resistance make spirulina a promising candidate for bioregenerative life support systems. A concept of operations was developed to grow and harvest algal biomass in space. Preflight testing experiments optimized conditions for an extended growth period in a gas permeable bioreactor bag. Preflight and post-harvest storage methods were developed in addition to a novel cryopreservation method. After sample return, multi-omics analysis will be conducted to determine the mutation rate, gene expression, and the protein and metabolite profile. The concept of operations for Space Algae-2 was tested during a lunar mission simulation within a semi-controlled environment. During a six-day lunar analog mission at the Hawai’i Space Exploration Analog and Simulation (HI-SEAS) A. platensis was successfully grown using flight-like hardware. The cyanobacteria were harvested and used to supplement bread as an example of spirulina biomass utilization. Overall, the data collected from Space Algae-2 will inform potential bioengineering of spirulina for space and terrestrial applications.
Algae↗
Space Algae-2 Ground and Lunar Analog Studies in Preparation for Long-Duration Propagation of Cyanobacteria in Spaceflight
There are numerous applications for microalgae in spaceflight missions and on Earth, such as, oxygen production, carbon dioxide removal, nutrition, wastewater processing, and biofuel production. Space Algae-2 aims to test the genetic stability of Arthrospira platensis, commonly known as spirulina, during six-months of continuous culture in spaceflight on the International Space Station. Long-duration exposure to ionizing radiation and microgravity may impact growth, nutrient composition, and genetic stability. The high protein, vitamin, antioxidant content, and radiation resistance make spirulina a promising candidate for bioregenerative life support systems during long-duration missions. A concept of operations was developed to grow and harvest algal biomass in space. Preflight testing experiments were conducted to optimize conditions for an extended growth period in a gas permeable bioreactor bag. Preflight and post-harvest storage methods were developed in addition to a novel cryopreservation method. After sample return, multi-omics analysis will be conducted to determine the mutation rate, gene expression, and protein and metabolite profile. The concept of operations for Space Algae-2 was tested at HI-SEAS (Hawai’i Space Exploration Analog and Simulation) during a six-day lunar analog mission (EMMIHS23, EuroMoonMars, International MoonBase Alliance, HI-SEAS, 2023). A. platensis was grown in the semi-controlled environment using flight-like hardware and solar powered LED lights. Then, the biomass was harvested and used to supplement bread as an example of A. platensis utilization. Overall, the data collected from Space Algae-2 will inform potential bioengineering of spirulina for space and terrestrial applications.
Algae↗
Some properties of the hydrogen bonds in biochemistry with particular reference to the stability of the genetic code
Importance of H-bonding on stability of genetic code of DNA as expressed by Watson-Crick model and template idea
Loewdin, P.-O.↗
The MELISSA pilot plant facility as as integration test-bed for advanced life support systems
The different advances in the Micro Ecological Life Support System Alternative project (MELISSA), fostered and coordinated by the European Space Agency, as well as in other associated technologies, are integrated and demonstrated in the MELISSA Pilot Plant laboratory. During the first period of operation, the definition of the different compartments at an individual basis has been achieved, and the complete facility is being re-designed to face a new period of integration of all these compartments. The final objective is to demonstrate the potentiality of biological systems such as MELISSA as life support systems. The facility will also serve as a test bed to study the robustness and stability of the continuous operation of a complex biological system. This includes testing of the associated instrumentation and control for a safe operation, characterization of the chemical and microbial safety of the system, as well as tracking the genetic stability of the microbial strains used. The new period is envisaged as a contribution to the further development of more complete biological life support systems for long-term manned missions, that should be better defined from the knowledge to be gained from this integration phase. This contribution summarizes the current status of the Pilot Plant and the planned steps for the new period. c2004 COSPAR. Published by Elsevier Ltd. All rights reserved.
Waste Management/instrumentation/methods↗
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↗
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.↗
ECUT: Energy Conversion and Utilization Technologies program - Biocatalysis research activity
The activities of the Biocatalysis Research Activity are organized into the Biocatalysis and Molecular Modeling work elements and a supporting planning and analysis function. In the Biocatalysis work element, progress is made in developing a method for stabilizing genetically engineered traits in microorganisms, refining a technique for monitoring cells that are genetically engineered, and identifying strains of fungi for highly efficient preprocessing of biomass for optimizing the efficiency of bioreactors. In the Molecular Modeling work element, a preliminary model of the behavior of enzymes is developed. A preliminary investigation of the potential for synthesizing enzymes for use in electrochemical processes is completed. Contact with industry and universities is made to define key biocatalysis technical issues and to broaden the range of potential participants in the activity. Analyses are conducted to identify and evaluate potential concepts for future research funding.
Wilcox, R.↗
Origins of the plant chloroplasts and mitochondria based on comparisons of 5S ribosomal RNAs
In this paper, we provide macromolecular comparisons utilizing the 5S ribosomal RNA structure to suggest extant bacteria that are the likely descendants of chloroplast and mitochondria endosymbionts. The genetic stability and near universality of the 5S ribosomal gene allows for a useful means to study ancient evolutionary changes by macromolecular comparisons. The value in current and future ribosomal RNA comparisons is in fine tuning the assignment of ancestors to the organelles and in establishing extant species likely to be descendants of bacteria involved in presumed multiple endosymbiotic events.
NASA Discipline Exobiology↗
Space Algae-2: Preflight Testing for A Long-Duration, Multi-Omics Analysis of Arthrospira Platensis
The cyanobacteria Arthrospira platensis NIES-39, commonly known as spirulina, could provide a fresh supply of nutrients for crew on long-duration spaceflight missions. Spirulina is a readily digestible food that is high in protein with all essential amino acids as well as significant levels of B vitamins, antioxidants, and anti-inflammatory metabolites. Spaceflight has multiple abiotic stressors such as increased ionizing radiation and microgravity, which causes a lack of convective mixing. These environmental conditions may impact productivity, nutritional composition, and in long-duration propagation, spaceflight stress may impact the genetic stability of spirulina cultures. We are developing an International Space Station experiment to continuously culture A. platensis for six months. Multi-omics profiling will be used to monitor for changes in the genome, transcriptome, proteome, and metabolome to determine if A. platensis is a suitable nutritional supplement on long-duration missions. During preflight testing we developed a protocol for inoculated liquid cultures to survive a 10-week storage period prior to photo-incubation. The bioreactor bag, temperature, and lighting conditions that support a 14-day growth cycle between passages were also determined. Media testing identified minimal salts supporting robust growth that can be stored in liquid or dry form. A simple filtration method was developed to dewater cultures and harvest biomass for frozen sample return. We optimized a cryopreservation method to enable return of live cells for isolation of individual A. platensis clones. The concept of operations for Space Algae-2 developed from these test results as well as progress on multi-omics analysis methods will be presented.
Algae↗
Space Algae-2: Preflight Testing for A Long-Duration, Multi-Omics Analysis of Arthrospira Platensis
The cyanobacteria Arthrospira platensis NIES-39, commonly known as spirulina, could provide a fresh supply of nutrients for crew on long-duration spaceflight missions. Spirulina is a readily digestible food that is high in protein with all essential amino acids as well as significant levels of B vitamins, antioxidants, and anti-inflammatory metabolites. Spaceflight has multiple abiotic stressors such as increased ionizing radiation and microgravity, which causes a lack of convective mixing. These environmental conditions may impact productivity, nutritional composition, and in long-duration propagation, spaceflight stress may impact the genetic stability of spirulina cultures. We are developing an International Space Station experiment to continuously culture A. platensis for six months. Multi-omics profiling will be used to monitor for changes in the genome, transcriptome, proteome, and metabolome to determine if A. platensis is a suitable nutritional supplement on long-duration missions. During preflight testing we developed a protocol for inoculated liquid cultures to survive a 10-week storage period prior to photo-incubation. The bioreactor bag, temperature, and lighting conditions that support a 14-day growth cycle between passages were also determined. Media testing identified minimal salts supporting robust growth that can be stored in liquid or dry form. A simple filtration method was developed to dewater cultures and harvest biomass for frozen sample return. We optimized a cryopreservation method to enable return of live cells for isolation of individual A. platensis clones. The concept of operations for Space Algae-2 developed from these test results as well as progress on multi-omics analysis methods will be presented.
Algae↗
Genetic Studies of Hydrogen Bacteria and Their Applications to Biological Life Support Systems Status Report No. 3, May 1 - Oct. 31, 1966
Genetic cultural stabilities of hydrogen bacteria for application to biological life support systems
LIFE SUPPORT SYSTEM↗
Testing A Concept of Operations for the Space Algae-2 Spaceflight Experiment
Algae has abundant potential spaceflight applications to enable future crewed missions and habitation beyond low-Earth orbit, including production of essential nutrients, oxygen, and biofuels. Implementing algae production in space requires understanding how different algae species respond to spaceflight stressors such as microgravity and radiation over a realistic production time course. Space Algae-2 is focusing on Arthrospira platensis, a filamentous cyanobacteria commonly known as Spirulina. A. platensis is a human nutritional supplement on Earth that is a good source of essential amino acids, β-carotene, thiamin, riboflavin, and antioxidants. The Space Algae-2 experiment plans to grow and passage A. platensis continuously aboard the International Space Station for six months. The algae produced will be analyzed with multi-omics profiling to monitor for genetic and phenotype stability in the spaceflight environment. This presentation will report the results from pre-flight science verification tests of the concept of operations for Space Algae-2. The purpose of these tests was to determine if the proposed crew operations for serial passages, sample collection, and sample preservation are feasible to meet science requirements for axenic culturing and preservation of DNA, RNA, protein, and nutritional metabolites.
Microalgae↗
Wheat Crop Traits Conferring High Yield Potential May Also Improve Yield Stability Under Climate Change
Increasing genetic wheat yield potential is considered by many as critical to increasing global wheat yields and production, baring major changes in consumption patterns. Climate change challenges breeding by making target environments less predictable, altering regional productivity and potentially increasing yield variability. Here we used a crop simulation model solution in the SIMPLACE framework to explore yield sensitivity to select trait characteristics (radiation use efficiency [RUE], fruiting efficiency and light extinction coefficient) across 34 locations representing the world’s wheat-producing environments, determining their relationship to increasing yields, yield variability and cultivar performance. The magnitude of the yield increase was trait-dependent and differed between irrigated and rainfed environments. RUE had the most prominent marginal effect on yield, which increased by about 45 % and 33 % in irrigated and rainfed sites, respectively, between the minimum and maximum value of the trait. Altered values of light extinction coefficient had the least effect on yield levels. Higher yields from improved traits were generally associated with increased inter-annual yield variability (measured by standard deviation), but the relative yield variability (as coefficient of variation) remained largely unchanged between base and improved genotypes. This was true under both current and future climate scenarios. In this context, our study suggests higher wheat yields from these traits would not increase climate risk for farmers and the adoption of cultivars with these traits would not be associated with increased yield variability.
Climate change↗
Intraspecific Reaction Norm Variation Controls the Eco-Evolutionary Consequences of Environmental Change
As environmental change accelerates globally, understanding concurrent organismal, species, and community responses is increasingly vital. Here, we examine these collective responses by incorporating genotype-specific thermal reaction norms into an eco-evolutionary predator-prey model, allowing us to track simultaneous phenotypic, ecological, and evolutionary responses to environmental change within ecological communities. We show that the reaction norms expressed by genotypes within a population determine how a community switches between different eco-evolutionary outcomes with changes in temperature. We identify how different components of phenotypic variation in thermal reaction norms—environmental (E), additive environmental and genetic (E + G), and gene-by-environment interactions (G × E)—influence eco-evolutionary dynamics and outcomes as temperature changes. Furthermore, our findings underscore how complex eco-evolutionary responses to environmental change ultimately emerge from variation in reaction norms among genotypes, offering new mechanistic insights into environmental impacts on adaptation, the maintenance of phenotypic and genetic variation, and ecological stability, which is crucial for understanding and predicting eco-evolutionary effects of rapid environmental change in the future.
Eco-evolutionary↗
System and method for embedding emotion in logic systems
A system, method, and computer readable-media for creating a stable synthetic neural system. The method includes training an intellectual choice-driven synthetic neural system (SNS), training an emotional rule-driven SNS by generating emotions from rules, incorporating the rule-driven SNS into the choice-driven SNS through an evolvable interface, and balancing the emotional SNS and the intellectual SNS to achieve stability in a nontrivial autonomous environment with a Stability Algorithm for Neural Entities (SANE). Generating emotions from rules can include coding the rules into the rule-driven SNS in a self-consistent way. Training the emotional rule-driven SNS can occur during a training stage in parallel with training the choice-driven SNS. The training stage can include a self assessment loop which measures performance characteristics of the rule-driven SNS against core genetic code. The method uses a stability threshold to measure stability of the incorporated rule-driven SNS and choice-driven SNS using SANE.
Curtis, Steven A.↗