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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Optimizing Selection Pressures and Pest Management to Maximize Cultivation Yield (OSPREY) (Final Technical Report)

This project was proposed in response to AOI 1, Cultivation Intensification Processes for Algae, within the FY19 Bioenergy Technologies Office Multi-Topic Funding Opportunity Announcement (FOA Number: DE-FOA-0002029). The work was designed to address a critical industry need to improve annualized productivity, stability, and quality of algal production strains for biofuels and bioproducts. The overall project goals were to generate process innovations rooted in established outdoor systems for strain selection, improvement, maintenance, and cultivation as well as pest detection and tracking. Planned advances included a 50% improvement in harvest yield based on AFDW (g m 2 d -1 ), 50% improvement in robustness based on stability metrics (e.g., high-productivity cultivation days, pond uptime), and 20% improvement in conversion yield. Individually, each of our planned process improvements (e.g., pest tracking) had the potential to increase productivity. However, to realize increases in yield at the system level, improvements to one unit’s process must be balanced against potential effects on other processes. For example, changes to strains, cultivation, and pest management developed in isolation may hurt other unit operations. Therefore, a critical success factor of the project was the integration of the pipeline components, achieved through iterative field-to- (short term) lab testing. In addition, through sustainability models, we evaluated how improvements would alter industry scenarios.

09 BIOMASS FUELS↗
DOE OSTI2024

Effects of SARS-CoV-2 Main Protease Mutations at Positions L50, E166, and L167 Rendering Resistance to Covalent and Noncovalent Inhibitors

SARS-CoV-2 propagation under nirmatrelvir and ensitrelvir pressure selects for main protease (MPro) drug-resistant mutations E166V (DRM2), L50F/E166V (DRM3), E166A/L167F (DRM4), and L50F/E166A/L167F (DRM5). DRM2-DRM5 undergoes N-terminal autoprocessing to produce mature MPro with dimer dissociation constants (K dimer ) 2–3 times larger than that of the wildtype. Co-selection of L50F restores catalytic activity of DRM2 and DRM4 from ~10 to 30%, relative to that of the wild-type enzyme, without altering K dimer . Binding affinities and thermodynamic profiles that parallel the drug selection pressure, exhibiting significant decreases in affinity through entropy/enthalpy compensation, were compared with GC373. Reorganization of the active sites due to mutations observed in the inhibitor-free DRM3 and DRM4 structures as compared to MPro WT may account for the reduced binding affinities, although DRM2 and DRM3 complexes with ensitrelvir are almost identical to MPro WT -ensitrelvir. In conclusion, chemical reactivity changes of the mutant active sites due to differences in electrostatic and protein dynamics effects likely contribute to losses in binding affinities.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Comparison of Real-Time Pressure Rail Selection Algorithms for the Hybrid Hydraulic Electric Architecture: Case Study on a Track Loader

Abstract The hybrid hydraulic electric architecture (HHEA) seeks to combine the high power/torque/force density of hydraulics with the efficiency of electric machines. A set of common pressure rails is used to provide a majority of the power and this power is modulated by small electric machines to provide precise control for the operator. The HHEA has been studied in previous work using off-line dynamic programming optimization to determine energy efficient pressure rail selections, but this approach requires drive cycle information apriori. A Lagrange multiplier method has also been investigated where a set of gains (Lagrange multipliers) are optimized off-line with the idea the these gains, once determined, could be used for real-time operation. In this work, three new real-time pressure rail selection algorithms that do not require future drive cycle information are investigated; greedy, torque minimizing, and thresholding. The greedy control is found to only use 1% more energy than the globally optimal dynamic programming solution; but a model of energy loss is required.

24 POWER TRANSMISSION AND DISTRIBUTION↗
DOE OSTI2024

Differential laboratory passaging of SARS-CoV-2 viral stocks impacts the in vitro assessment of neutralizing antibodies

Viral populations in natural infections can have a high degree of sequence diversity, which can directly impact immune escape. However, antibody potency is often tested in vitro with a relatively clonal viral populations, such as laboratory virus or pseudotyped virus stocks, which may not accurately represent the genetic diversity of circulating viral genotypes. This can affect the validity of viral phenotype assays, such as antibody neutralization assays. To address this issue, we tested whether recombinant virus carrying SARS-CoV-2 spike (VSV-SARS-CoV-2-S) stocks could be made more genetically diverse by passage, and if a stock passaged under selective pressure was more capable of escaping monoclonal antibody (mAb) neutralization than unpassaged stock or than viral stock passaged without selective pressures. We passaged VSV-SARS-CoV-2-S four times concurrently in three cell lines and then six times with or without polyclonal antiserum selection pressure. All three of the monoclonal antibodies tested neutralized the viral population present in the unpassaged stock. The viral inoculum derived from serial passage without antiserum selection pressure was neutralized by two of the three mAbs. However, the viral inoculum derived from serial passage under antiserum selection pressure escaped neutralization by all three mAbs. Deep sequencing revealed the rapid acquisition of multiple mutations associated with antibody escape in the VSV-SARS-CoV-2-S that had been passaged in the presence of antiserum, including key mutations present in currently circulating Omicron subvariants. These data indicate that viral stock that was generated under polyclonal antiserum selection pressure better reflects the natural environment of the circulating virus and may yield more biologically relevant outcomes in phenotypic assays. Thus, mAb assessment assays that utilize a more genetically diverse, biologically relevant, virus stock may yield data that are relevant for prediction of mAb efficacy and for enhancing biosurveillance.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Quantifying shifts in natural selection on codon usage between protein regions: a population genetics approach

Codon usage bias (CUB), the non-uniform usage of synonymous codons, occurs across all domains of life. Adaptive CUB is hypothesized to result from various selective pressures, including selection for efficient ribosome elongation, accurate translation, mRNA secondary structure, and/or protein folding. Given the critical link between protein folding and protein function, numerous studies have analyzed the relationship between codon usage and protein structure. The results from these studies have often been contradictory, likely reflecting the differing methods used for measuring codon usage and the failure to appropriately control for confounding factors, such as differences in amino acid usage between protein structures and changes in the frequency of different structures with gene expression. Here we take an explicit population genetics approach to quantify codon-specific shifts in natural selection related to protein structure in S. cerevisiae and E. coli. Unlike other metrics of codon usage, our approach explicitly separates the effects of natural selection, scaled by gene expression, and mutation bias while naturally accounting for a region’s amino acid usage. Bayesian model comparisons suggest selection on codon usage varies only slightly between helix, sheet, and coil secondary structures and, similarly, between structured and intrinsically-disordered regions. Similarly, in contrast to previous findings, we find selection on codon usage only varies slightly at the termini of helices in E. coli. Using simulated data, we show this previous work indicating “non-optimal” codons are enriched at the beginning of helices in S. cerevisiae was due to failure to control for various confounding factors (e.g. amino acid biases, gene expression, etc.), and rather than selection to modulate cotranslational folding. Our results reveal a weak relationship between codon usage and protein structure, indicating that differences in selection on codon usage between structures are slight. In addition to the magnitude of differences in selection between protein structures being slight, the observed shifts appear to be idiosyncratic and largely codon-specific rather than systematic reversals in the nature of selection. Overall, our work demonstrates the statistical power and benefits of studying selective shifts on codon usage or other genomic features from an explicitly evolutionary approach. Limitations of this approach and future potential research avenues are discussed.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Orthogonal replication with optogenetic selection evolves yeast JEN1 into a mevalonate transporter

Abstract The in vivo continuous evolution system OrthoRep (orthogonal replication) is a powerful strategy for rapid enzyme evolution inSaccharomyces cerevisiaethat diversifies genes at a rate exceeding the endogenous genome mutagenesis rate by several orders of magnitude. However, it is difficult to neofunctionalize genes using OrthoRep partly because of the way selection pressures are applied. Here we combine OrthoRep with optogenetics in a selection strategy we call OptoRep, which allows fine-tuning of selection pressure with light. With this capability, we evolved a truncated form of the endogenous monocarboxylate transporterJEN1 (JEN1t)into a de novo mevalonate importer. We demonstrate the functionality of the evolvedJEN1t(JEN1t Y180C/G ) in the production of farnesene, a renewable aviation biofuel, from mevalonate fed to fermentation media or produced by microbial consortia. This study shows that the light-induced complementation of OptoRep may improve the ability to evolve functions not currently accessible for selection, while its fine tunability of selection pressure may allow the continuous evolution of genes whose desired function has a restrictive range between providing effective selection and cellular viability.

Biochemistry & Molecular Biology↗
DOE OSTI2023

Sizing and Selection of Pressure Relief Valves for High-Pressure Thermal–Hydraulic Systems

This study covers the critical concerns related to the sizing, selection, installation, maintenance, and testing of pressure safety valves (PSVs). The aim is to ensure the safety of pressurized systems, hydrostatic transmission systems, and hydraulic plants, including process plants, thermal power plants, and nuclear reactor systems. PSVs are devices that ensure the safety and reliability of pressurized vessels, lines, and systems during overpressure events. The task of selecting which PSV features are of greatest value for a specific purpose is complex—especially in the design of a high-pressure experimental thermal–hydraulic facility for hydrostatic and transient testing of the reactor system—when the systems are in the design and development phases and require qualification and demonstration to prove that they have reached a given level of technological readiness. The present study highlights the required steps for users to follow the associated rules, guidelines, and recommendations. As a part of this research, case studies are presented to help readers better understand the applicable strategy and standards. A discussion and a review of PSV performance degradation and failure are summarized to provide a better understanding of varied process applications and conditions, including fluid flow dynamics, boundary-layer formation and pressure drops, gas bubble formation and collapse, geometric configurations, inlet/outlet piping, abrupt pressure fluctuations, and acoustic resonance. Moreover, this study discusses the servicing and testing of PSVs in a multiphase pressurized system. Overall, it provides a basic overview of how PSVs ensure the safety of pressurized systems, supported by case studies and industrial practices.

02 PETROLEUM↗
DOE OSTI2023

Microdiverse bacterial clades prevail across Antarctic wetlands

Antarctica's extreme environmental conditions impose selection pressures on microbial communities. Indeed, a previous study revealed that bacterial assemblages at the Cierva Point Wetland Complex (CPWC) are shaped by strong homogeneous selection. Yet which bacterial phylogenetic clades are shaped by selection processes and their ecological strategies to thrive in such extreme conditions remain unknown. Here, we applied the phyloscore and feature-level βNTI indexes coupled with phylofactorization to successfully detect bacterial monophyletic clades subjected to homogeneous (HoS) and heterogenous (HeS) selection. Remarkably, only the HoS clades showed high relative abundance across all samples and signs of putative microdiversity. The majority of the amplicon sequence variants (ASVs) within each HoS clade clustered into a unique 97% sequence similarity operational taxonomic unit (OTU) and inhabited a specific environment (lotic, lentic or terrestrial). Our findings suggest the existence of microdiversification leading to sub-taxa niche differentiation, with putative distinct ecotypes (consisting of groups of ASVs) adapted to a specific environment. We hypothesize that HoS clades thriving in the CPWC have phylogenetically conserved traits that accelerate their rate of evolution, enabling them to adapt to strong spatio-temporally variable selection pressures. Variable selection appears to operate within clades to cause very rapid microdiversification without losing key traits that lead to high abundance. Variable and homogeneous selection, therefore, operate simultaneously but on different aspects of organismal ecology. The result is an overall signal of homogeneous selection due to rapid within-clade microdiversification caused by variable selection. It is unknown whether other systems experience this dynamic, and we encourage future work evaluating the transferability of our results.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Precise Motion Control of Hybrid Hydraulic Electric Architecture (HHEA)

Off-highway heavy-duty vehicles have been long-standing users of hydraulic systems for power transmission and control. However, traditional hydraulic systems suffer from significant energy losses which lead to increased operating costs and a larger carbon footprint due to higher CO2 emissions. Improving the efficiency of these mobile machines is crucial not only for reducing their environmental impact but also for saving billions of dollars in operating costs. Currently, the state-of-the-art Load Sensing Architecture uses throttling valves for control, which significantly reduces its efficiency and does not recuperate energy from over-running loads. Researchers have developed several architectures such as Common Pressure Rail systems, Displacement Control, STEAM, and Electrohydraulic Architecture to improve the efficiency of off-road mobile machines. However, each of these architectures has its drawbacks. To increase system efficiency and take advantage of electrification benefits, our research group has developed a novel Hybrid Hydraulic-Electric Architecture (HHEA). The HHEA can significantly improve efficiency, decrease the size of electrical components, and maintain control performance. This new architecture has the potential to revolutionize the off-highway mobile machine industry and lead to a more sustainable future. The HHEA uses a set of common pressure rails to provide the majority of power to the actuators via power-dense hydraulics and uses electric motors for precise control and power modulation. In the context of off-road mobile machines, energy savings are undoubtedly important but it is equally important to consider the machines’ ability to perform tasks with precision and accuracy according to given commands. Therefore, precise motion control is of utmost importance to maintain the utility of Hybrid Hydraulic-Electric Architecture (HHEA). The HHEA presents a unique challenge to motion control due to the discrete pressure changes that occur when the system switches between selected pressure rails. These changes are made to minimize system inefficiencies or to keep the system within the torque capability of the electric motor. Hence, it is important to solve the motion control challenges for HHEA. This thesis aims at developing an effective motion control strategy for HHEA. The dissertation presents a two-tiered control strategy for HHEA, comprising a high-level and a low-level controller. The primary responsibility of the high- level controller is to optimize energy efficiency by making informed pressure rail selections. On the other hand, the low-level controller is focused on achieving precise motion control of the HHEA, which is crucial for realizing the desired reference trajectories. To achieve this, the low-level controller utilizes a passivity-based backstepping integral controller as the nominal control, which handles the motion control between two pressure rail switches. Additionally, a separate least norm controller is utilized as a transition controller to manage motion control during pressure rail transitions. The effectiveness of the combined control strategy is demonstrated through experiments conducted on two hardware-in-the-loop testbeds. Furthermore, the HHEA is installed on the boom and stick actuators of a backhoe arm to build a Human-in-the-Loop system that a human operator can control. A real-time rail switching algorithm is developed to determine pressure rail switching based on present duty cycle information from the operator. Modifications have been made to the human-machine interface to achieve more intuitive control. Modifications include performing control in the task-oriented coordinates, incorporating pressure feedback to enhance control with physical interaction, and using velocity field control to simplify multi-degree-of-freedom tasks and to enable novice operators to perform them with reduced risk, improved efficiency, and productivity. The research in this dissertation makes significant contributions to the field of off-road mobile machine control, providing a novel and effective control strategy for the HHEA, and demonstrating the potential for simplified machine operation.

24 POWER TRANSMISSION AND DISTRIBUTION↗
DOE OSTI2026

Postirradiation Examination of WIRE-21 Experiment Irradiated in the High Flux Isotope Reactor

Westinghouse Electric Company is developing wireless sensors to monitor the centerline temperature and internal pressure of commercial light-water reactor fuel rods during irradiation. Oak Ridge National Laboratory and Westinghouse Electric Company developed the Wireless Instrumented RB Experiment 2021 (WIRE-21) to test wireless temperature and pressure sensor technologies in a removable beryllium position in the High Flux Isotope Reactor. The experiment was irradiated for a total of 75 days, at temperatures ranging from approximately 150°C to 400°C, resulting in a peak fast (energy > 0.1 MeV) neutron fluence of about 3 × 10 21 n/cm 2 . The temperature was intentionally cycled multiple times to compare the response of the wireless temperature sensor to collocated thermocouples. Similarly, the pressure sensor was actuated in multiple steps to compare the response of the wireless measurement to excore pressure transducers (Petrie et al., 2023). After irradiation, the experiment was disassembled in the Irradiated Fuels Examination Laboratory (IFEL) hot cell at Oak Ridge National Laboratory with the intent to recover sensor and dosimetry components, document the as-irradiated condition of the hardware, and investigate possible causes of the sensor behavior observed during irradiation. The postirradiation examination successfully recovered and preserved key WIRE-21 components. After the housing was removed using a milling machine, the internal experiment sections were examined. All eight fiber-optic sensors were recovered, cut, and stored. The silicon carbide thermometry, temperature sensor, pressure sensor, lower spacer, and selected pressure and temperature cable sections were also removed and stored. The metal bellows of the pressure sensor was found to be plastically deformed, indicating that it had been properly pressurized during irradiation and generally behaved as expected. X-ray diffraction analysis of a section of one of the irradiated inductor cores within the pressure sensor was performed and confirmed the presence of phase-pure alpha ferrite (i.e., no unexpected phase transformations). Inductance testing was performed on the irradiated pressure sensor cores using an unirradiated test coil, and DC resistance measurements of the transceiver coils were also performed. The measurements with irradiated inductor cores assembled inside unirradiated coils showed slightly reduced inductance compared to measurements made with unirradiated cores, but the difference was not sufficient to explain the more significant reductions in inductance that were observed in-pile. Therefore, it is suspected that degradation of the inductor coils (specifically the wire wrapping) is responsible for the reduced inductance observed in-pile.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗
DOE OSTI2022

High-Throughput Microbial Community Analyses to Establish a Natural Fungal and Bacterial Consortium from Sewage Sludge Enriched with Three Pharmaceutical Compounds

Emerging and unregulated contaminants end up in soils via stabilized/composted sewage sludges, paired with possible risks associated with the development of microbial resistance to antimicrobial agents or an imbalance in the microbial communities. An enrichment experiment was performed, fortifying the sewage sludge with carbamazepine, ketoprofen and diclofenac as model compounds, with the aim to obtain strains with the capability to transform these pollutants. Culturable microorganisms were obtained at the end of the experiment. Among fungi, Cladosporium cladosporioides, Alternaria alternata and Penicillium raistrickii showed remarkable degradation rates. Population shifts in bacterial and fungal communities were also studied during the selective pressure using Illumina MiSeq. These analyses showed a predominance of Ascomycota (Dothideomycetes and Aspergillaceae) and Actinobacteria and Proteobacteria, suggesting the possibility of selecting native microorganisms to carry out bioremediation processes using tailored techniques.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Trait drift in microalgae and applications for strain improvement

Microalgae are increasingly used to generate a wide range of commercial products, and there is growing evidence that microalgae-based products can be produced sustainably. However, industrial production of microalgal biomass is not as developed as other biomanufacturing platform technologies. In addition, results of bench-scale research often fail to translate to large-scale or mass production systems. This disconnect may result from trait drift and evolution occurring, through time, in response to unique drivers in each environment, such as cultivation regimes, weather, and pests. Moreover, outdoor and indoor cultivation of microalgae has the potential to impose negative selection pressures, which makes the maintenance of desired traits a challenge. In this context, this review sheds the light on our current understanding of trait drift and evolution in microalgae. Here, we delineate the basics of phenotype plasticity and evolution, with a focus on how microalgae respond under various conditions. In addition, we review techniques that exploit phenotypic plasticity and evolution for strain improvement in view of industrial commercial applications, highlighting associated advantages and shortcomings. Finally, we suggest future research directions and recommendations to overcome unwanted trait drift and evolution in microalgae cultivation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Volatile traits expand the microbial playbook

Microbial metabolic functions are increasingly conceptualized as fitness-regulating traits. However, volatile compounds (the volatilome), despite their key roles in metabolism and ecology, are often overlooked in trait-based frameworks. We propose that volatile traits not only reflect ecological strategies but also shape them by mediating responses to selection pressures. Their volatility affects diffusion, substrate access, and interactions across space, conferring selective advantages as resources or waste products. We outline approaches to incorporate volatile traits into predictive models to improve understanding of microbial selection and community dynamics. Furthermore, this integration enables a more holistic view of microbial life by accounting for the ecological and evolutionary consequences of volatile-mediated processes.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2026

Genomic signatures in Variovorax enabling colonization of the Populus endosphere

Microbial colonization of plant roots involves strong selective pressures that shape the structure and function of root-associated communities. In particular, the endosphere represents a highly selective environment requiring host entry and in planta persistence. However, strain-specific microbial traits that enable endosphere colonization remain poorly understood. Here, we use a defined, genome-resolved community of 28 Variovorax strains isolated from the roots of Populus deltoides and Populus trichocarpa (poplar trees) to determine which strains partition between rhizosphere and endosphere compartments and to identify the genomic traits associated with endosphere specialization. By combining strain-resolved metagenomic profiling, comparative genomics, and functional assays, we demonstrate that dominant endosphere colonizers are enriched in genes related to nutrient metabolism, redox balance, transcriptional regulation, and a conserved L-fucose utilization pathway experimentally shown to enhance root colonization. Not all strains succeed through the same strategy. Community-wide functional profiling revealed a distinct and reduced set of traits in the endosphere, including orthogroups associated with low-abundance strains that were overlooked in strain-level analyses. These findings reveal that multiple ecological strategies, such as metabolic competition, regulatory adaptation, and niche specialization, can support endosphere colonization. Our results advance the understanding of how bacterial colonization traits are distributed and deployed within a plant microbiome and suggest that host filtering selects for distinct, and sometimes complementary, microbial strategies. This work supports a shift toward mechanistic, genome-resolved models of microbiome assembly and offers a framework for linking microbial function to host colonization success.

comparative genomics↗
DOE OSTI2026

Single nucleotide variants drive evolutionary phage-host arms race in anaerobic carbon dioxide-converting microbiome

Microbial bioconversions are shaped by environmental perturbations and the adaptation of resident microbiomes. Prokaryotes coexist with bacteriophages, yet their coevolutionary trajectories remain underexplored. Here, we investigate the effects of a cultivation vessel leak on an anaerobic consortium performing carbon dioxide reduction. Using time-series shotgun metagenomic sequencing, we reconstruct microbial and viral genomes to track community shifts. We further apply single-nucleotide variant profiling and CRISPR array analysis to monitor viral microdiversity and host defense mechanisms. After bioaugmentation restores bioconversion efficiency, the consortium undergoes pronounced restructuring, with new dominant taxa emerging from the rare biosphere. We identify patterns consistent with phage predation selectively removing certain species, while others exhibit resilience to infection. This shift aligns with a widespread viral outbreak and a transient increased frequency of single nucleotide variants in bacterial CRISPR–Cas defense genes. Expansion of CRISPR spacers further supports that CRISPR-mediated processes influence microbial resilience. Concurrently, phages infecting resilient hosts exhibited adaptive evolution, marked by high genetic heterogeneity. Selective pressure varies across their genomes, targeting infectivity genes and protospacer-adjacent motifs. These findings highlight a dynamic evolutionary arms race driven by the selection of beneficial genetic variants, providing a mechanistic framework for multi-omics investigations, and informing biotechnological applications, including phage-based microbiome manipulation.

Ghiotto, G↗
DOE OSTI2023

Pressured cytotoxic T cell epitope strength among SARS-CoV-2 variants correlates with COVID-19 severity

Heterogeneity in susceptibility among individuals to COVID-19 has been evident through the pandemic worldwide. Cytotoxic T lymphocyte (CTL) responses generated against pathogens in certain individuals are known to impose selection pressure on the pathogen, thus driving emergence of new variants. In this study, we probe the role played by host genetic heterogeneity in terms of HLA-genotypes in determining differential COVID-19 severity in patients. We use bioinformatic tools for CTL epitope prediction to identify epitopes under immune pressure. Using HLA-genotype data of COVID-19 patients from a local cohort, we observe that the recognition of pressured epitopes from the parent strain Wuhan-Hu-1 correlates with COVID-19 severity. We also identify and rank list HLA-alleles and epitopes that offer protectivity against severe disease in infected individuals. Finally, we shortlist a set of 6 pressured and protective epitopes that represent regions in the viral proteome that are under high immune pressure across SARS-CoV-2 variants. In conclusion, identification of such epitopes, defined by the distribution of HLA-genotypes among members of a population, could potentially aid in prediction of indigenous variants of SARS-CoV-2 and other pathogens.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Natural transformation as a tool in Acinetobacter baylyi : Evolution by amplification of gene copy number

For many years, the natural competency of Acinetobacter baylyi ADP1 facilitated studies of bacterial metabolism, biochemistry, and physiology. With the advent of synthetic biology, new opportunities arise to exploit the remarkable transformability and chromosomal plasticity of this model organism. In this chapter, we describe a recently developed method, “Evolution by Amplification and Synthetic Biology” (EASy). EASy allows the targeted amplification of chromosomal segments that give rise to new phenotypes. Increased gene dosage regulates protein expression in a rudimentary fashion by establishing a chromosomal array in which copy number adjusts via recombination between repeated DNA sequences. Selective conditions enrich for cells within the population that confer a growth advantage. Under continuous selective pressure, beneficial mutations may accumulate in any genomic region. Such mutations favor decreases in the average copy number of the target region. Thus, the genetic flexibility afforded by transient copy number variation helps accelerate the selection of engineered strains with desired traits during laboratory evolution. As a result, thanks to the extremely simple genetic manipulation of Acinetobacter baylyi ADP1, the EASy method can be readily implemented by researchers without the need for advanced instrumentation or complex cloning techniques.

59 BASIC BIOLOGICAL SCIENCES↗