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NASA NTRSDate not supplied

Studying Microbial Adaptation in the Laboratory: Sensor & Control Upgrades for an Experimental Evolution Biofluidics System

Experimental evolution (EE) involves iteratively exposing a microbial community to specific stressors to study its response to changes in environment over time. EE work is commonly done manually in the laboratory, but, when there are many environmental variables to measure and adjust, it is highly labor intensive, prone to human error, and challenging to scale. Single-purpose automated continuous culturing chambers exist, but implement only limited stressor types. A more general-purpose design is desirable. The BeING Lab at Ames Research Center created the prototype Automated Adaptive Directed Evolution Chamber (AADEC) to address these problems, beginning with Escherichia coli tolerance of short-wave ultraviolet (UV-C) radiation and of temperature. In newer versions, AADEC monitors microbial activity and can adjust the UV-C and temperature levels automatically. An optical density measurement is used to determine how many cells are present in the growth medium—over time, this corresponds to how many survive and reproduce. Oxidation-reduction potential provides information on consumed metabolic energy, and pH and electrical conductivity on metabolic products. Dissolved oxygen content is used to determine aerobic vs anaerobic growth. A Raspberry Pi computer processes all this data to set the UV-C stressor level. AADEC’s auxiliary systems include peristaltic pumps to change media and agitation to counteract cell settling. These actuators can also act as additional stressors. With the Raspberry Pi monitoring sensors and adjusting actuators in real time, AADEC takes measurements and controls the environment much more accurately than can be done with a manual EE implementation. The third and latest AADEC iteration is the first to simplify design and usage with circuits on PCBs and the ability to pre-program experimental protocols. Still planned is expansion to a multi-well design for the study of varying cell cultures in parallel, which will enable researchers to retain and re-inoculate cultures exhibiting the desired trait most strongly while flushing out others. AADEC’s special capabilities make it a valuable tool for studying life under multiple stressors, enabling scientists to replicate changes in climate on microbes for study in a lab setting.

Microbial Adaptation↗

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.↗

NASA NTRS2013

Elucidating Microbial Adaptation Dynamics via Autonomous Exposure and Sampling

The adaptation of micro-organisms to their environments is a complex process of interaction between the pressures of the environment and of competition. Reducing this multifactorial process to environmental exposure in the laboratory is a common tool for elucidating individual mechanisms of evolution, such as mutation rates. Although such studies inform fundamental questions about the way adaptation and even speciation occur, they are often limited by labor-intensive manual techniques. Current methods for controlled study of microbial adaptation limit the length of time, the depth of collected data, and the breadth of applied environmental conditions. Small idiosyncrasies in manual techniques can have large effects on outcomes; for example, there are significant variations in induced radiation resistances following similar repeated exposure protocols. We describe here a project under development to allow rapid cycling of multiple types of microbial environmental exposure. The system allows continuous autonomous monitoring and data collection of both single species and sampled communities, independently and concurrently providing multiple types of controlled environmental pressure (temperature, radiation, chemical presence or absence, and so on) to a microbial community in dynamic response to the ecosystem's current status. When combined with DNA sequencing and extraction, such a controlled environment can cast light on microbial functional development, population dynamics, inter- and intra-species competition, and microbe-environment interaction. The project's goal is to allow rapid, repeatable iteration of studies of both natural and artificial microbial adaptation. As an example, the same system can be used both to increase the pH of a wet soil aliquot over time while periodically sampling it for genetic activity analysis, or to repeatedly expose a culture of bacteria to the presence of a toxic metal, automatically adjusting the level of toxicity based on the number or growth rate of surviving cells. We are on our second prototype iteration, with demonstrated functions of microbial growth monitoring and dynamic exposure to UV-C radiation and temperature. We plan to add functionality for general chemical presence or absence by Nov. 2013. By making the project low-cost and open-source, we hope to encourage others to use it as a basis for future development of a common microbial environmental adaptation testbed.

Microbiology↗

NASA NTRS2000

Laboratory Astrochemistry: Interstellar PAH Analogs

Polycyclic aromatic hydrocarbons (PAHs) are now considered to be an important and ubiquitous component of the organic material in space. PAHs are found in a large variety of extraterrestrial materials such as interplanetary dust particles (IDPs) and meteoritic materials. PAHs are also good candidates to account for the infrared emission bands (UIRs) and the diffuse interstellar optical absorption bands (DIBs) detected in various regions of the interstellar medium. The recent observations made with the Infrared Space Observatory (ISO) have confirmed the ubiquitous nature of the UIR bands and their carriers. PAHs are though to form through chemical reactions in the outflow from carbon-rich stars in a process similar to soot formation. Once injected in the interstellar medium, PAHs are further processed by the interstellar radiation field, interstellar shocks and energetic particles. A major, dedicated, laboratory effort has been undertaken over the past years to measure the physical and chemical characteristics of these complex molecules and their ions under experimental conditions that mimic the interstellar conditions. These measurements require collision-free conditions where the molecules and ions are cold and chemically isolated. The spectroscopy of PAHs under controlled conditions represents an essential diagnostic tool to study the evolution of extraterrestrial PAHs. The Astrochemistry Laboratory program will be discussed through its multiple aspects: objectives, approach and techniques adopted, adaptability to the nature of the problem(s), results and implications for astronomy as well as for molecular spectroscopy. A review of the data generated through laboratory simulations of space environments and the role these data have played in our current understanding of the properties of interstellar PAHs will be presented. The discussion will also introduce the newest generation of laboratory experiments that are currently being developed in order to provide a closer simulation of space environments and a better support to space missions.

Salama, Farid↗

NASA NTRS2000

Laboratory Studies of Interstellar PAH Analogs

Salama, Farid↗

NASA NTRSDate not supplied

From the Interstellar Medium to Ocean Worlds: new challenges for Laboratory Astrophysics

From the Interstellar Medium to Ocean Worlds: new challenges for Laboratory Astrophysics For the past 35+ years, laboratory experiments in Astrophysics have been focusing on identifying and understanding the molecular species and chemistry occurring in interstellar environments, such as the diffuse interstellar medium (ISM) and dense molecular clouds. A significant portion of these laboratory efforts concerns investigations of carbon-based molecules ranging in size from a single carbon atom (e.g., CO, CO2, H2CO) to large polycyclic aromatic hydrocarbon (PAH) molecules potentially consisting of more than one hundred carbon atoms as well as their reactions with H2O and other interstellar species. The laboratory facilities built to conduct these investigations are capable of monitoring the chemistry and measuring the spectra under a wide range of interstellar conditions (e.g., low temperatures and pressures). The past 15 years of space exploration revealed the presence of at least nine other Ocean Worlds, besides Earth, in our Solar System. These worlds include moons and dwarf planets, ice-covered surfaces, subsurface oceans, as well as geysers venting H2O and other compounds, including organics in at least one environment, into space. Although these worlds represent environments that are significantly different from those traditionally considered the realm of Laboratory Astrophysics, their facilities are well situated to provide insight into the chemistry occurring in and around these worlds as well as experimental data for the interpretation of observations from missions visiting these Ocean World. This presentation will discuss the areas where Laboratory Astrophysics can provide experimental data to help understand the chemistry, and mission data of Ocean Worlds, and the challenges research into Ocean Worlds presents to our current facilities and how they can be overcome. Lastly, this presentation will introduce the new ICEE (In-situ Carbon Evolution Experiments) facility at NASA Ames Research Center as an example of how a lab dedicated to Laboratory Astrophysics can adapt itself to also meet the needs for Ocean Worlds research.

Andrew Lige Mattioda↗

NASA NTRS1989

The evolution of ecological tolerance in prokaryotes

The ecological ranges of Archaeobacteria and Eubacteria are constrained by a requirement for liquid water and the physico-chemical stability limits of biomolecules, but within this broad envelope, prokaryotes have evolved adaptations that permit them to tolerate a remarkable spectrum of habitats. Laboratory experiments indicate that prokaryotes can adapt rapidly to novel environmental conditions, yet geological studies suggest early diversification and long-term stasis within the prokaryotic kingdoms. These apparently contradictory perspectives can be reconciled by understanding that, in general, rates and patterns of prokaryotic evolution reflect the developmental history of the Earth's surface environments. Our understanding of modern microbial ecology provides a lens through which our accumulating knowledge of physiology, molecular phylogeny and the Earth's history can be integrated and focussed on the phenomenon of prokaryotic evolution.

NASA Discipline Number 52-30↗

NASA NTRS1992

Evolution of the Space Station Freedom module pattern

A reanalysis of the evolution configuration module pattern for Space Station Freedom is presented. The module pattern is to consist of one habitation module, one airlock, one cupola, two Space Shuttle Orbiter pressurized docking adapters, one assured crew return vehicle, and one pressurized logistics module. Three laboratory modules are also included. A phased plan has been developed on the basis of utilization analysis being performed and derived through actual and projected user mission requirements for the growth of pressurized elements. The evolution requirement impacts to the module pattern are outlined. Application of the growth philosophy and the physical limitations resulted in a number of core element ground rules for module pattern growth. Nodes and modules are to be grown symmetrically in order to optimize poor flight control characteristics and maximize the capability for crew dual egress between elements. Core module pattern elements are not to be grown along Y-Y axis from nodes 1 or 2 due to physical clearance problems with the thermal control system radiators operational envelope.

Gould, Marston↗

NASA NTRS2000

Laboratory Astrochemistry: Interstellar PAHs

Polycyclic aromatic hydrocarbons (PAHs) are now considered to be an important and ubiquitous component of the organic material in space. PAHs are found in a large variety of extraterrestrial materials such as interplanetary dust particles (IDPs) and meteoritic materials. PAHs are also good candidates to account for the infrared emission bands (UIRs) and the diffuse interstellar optical absorption bands (DIBs) detected in various regions of the interstellar medium. The recent observations made with the Infrared Space Observatory (ISO) have confirmed the ubiquitous nature of the UIR bands and their carriers. PAHs are thought to form through chemical reactions in the outflow from carbon-rich stars in a process similar to soot formation. Once injected in the interstellar medium, PAHs are further processed by the interstellar radiation field, interstellar shocks and energetic particles. A major, dedicated, laboratory effort has been undertaken to measure the physical and chemical characteristics of these complex molecules and their ions under experimental conditions that mimic the interstellar conditions. These measurements require collision-free conditions where the molecules and ions are cold and chemically isolated. The spectroscopy of PAHs under controlled conditions represents an essential diagnostic tool to study the evolution of extraterrestrial PAHs. The Astrochemistry Laboratory program will be discussed through its multiple aspects: (1) objectives, (2) approach and techniques adopted, (3) adaptability to the nature of the problem(s), and (4) results and implications for astronomy as well as for molecular spectroscopy. A review of the data generated through laboratory simulations of space environments and the role these data have played in our current understanding of the properties of interstellar PAHs will be presented. The discussion will also introduce the newest generation of laboratory experiments that are currently being developed in order to provide a closer simulation of space environments and a better support to space missions.

Salama, Farid↗

NASA NTRSDate not supplied

Supervised Autonomous Assembly to Create and Evolve Persistent Assets

Supervised autonomous assembly (SAA) will create a paradigm shift in the planning and design of future persistent assets (PAs), both in near zero-g environments and on planetary surfaces. SAA refers to an autonomy approach that has the benefits of autonomous assembly as well as the benefits provided by a supervisor (operator) who is available to resolve unexpected situations. SAA provides both increased design freedom as well as reduced programmatic risk. SAA enables evolution of future PAs over decades as in-space operations transition from single purpose missions to creation of PAs, such as laboratories and experimental stations which more closely resembling terrestrial laboratories that can easily adapt and evolve to new missions leveraging repeated visits to the PA. The ability to evolve enables PAs to rapidly respond to changing objectives resulting from new questions as our understanding improves. A recently initiated National Aeronautics and Space Administration (NASA) project in the Space Technology Mission Directorate (STMD) Game Changing Development (GCD) Program called the Precision Assembled Space Structure (PASS), leverages the advantages of SAA to develop technologies that enable efficient creation and evolution of hexagonal topologies; both planar (example: fuel depots) and curved (examples: telescopes and shelters). PASS will be used to provide context for the philosophy and concepts discussed as well as the decision and selections made. PASS objectives are: a) Develop confidence in SAA and on-orbit servicing, assembly and manufacturing (OSAM) technologies by executing a test campaign that uses a path-to-flight autonomous precision assembly process directly applicable to future space telescopes. b) Test autonomous technologies including automated path planning and error recovery, to emphasize a robust approach that relies on generic robots and special purpose tools. c) Validate critical component models using a digital twin that includes the assembled primary mirror support structure and assembly process. A digital twin is a high-fidelity simulation of the asset capable of predicting the on-orbit performance. The paper concludes after identifying the critical need for a modest assembly flight experiment to validate and develop confidence in the SAA paradigm, thus accelerating adoption of the benefits described. SAA is a game changing paradigm that enhances the ability of an organization to infuse new technology through rapid evolution of PAs while leveraging OSAM technologies.

Structural Modeling↗

NASA NTRS2012

Designing Mission Operations for the Gravity Recovery and Interior Laboratory Mission

NASA's Gravity Recovery and Interior Laboratory (GRAIL) mission, to understand the internal structure and thermal evolution of the Moon, offered unique challenges to mission operations. From launch through end of mission, the twin GRAIL orbiters had to be operated in parallel. The journey to the Moon and into the low science orbit involved numerous maneuvers, planned on tight timelines, to ultimately place the orbiters into the required formation-flying configuration necessary. The baseline GRAIL mission is short, only 9 months in duration, but progressed quickly through seven very unique mission phases. Compressed into this short mission timeline, operations activities and maneuvers for both orbiters had to be planned and coordinated carefully. To prepare for these challenges, development of the GRAIL Mission Operations System began in 2008. Based on high heritage multi-mission operations developed by NASA's Jet Propulsion Laboratory and Lockheed Martin, the GRAIL mission operations system was adapted to meet the unique challenges posed by the GRAIL mission design. This paper describes GRAIL's system engineering development process for defining GRAIL's operations scenarios and generating requirements, tracing the evolution from operations concept through final design, implementation, and validation.

MOS↗

NASA NTRS2000

A Flexible Model of a CMOS Field Programmable Transistor Array Targeted for Hardware Evolution

This article focuses on the properties of a fine grained reconfigurable transistor array currently under test at the Jet Propulsion Laboratory (JPL).

evolvable hardware reconfigurable hardware genetic↗

NASA NTRS2000

Evolution of thermotolerance in hot spring cyanobacteria of the genus Synechococcus

The extension of ecological tolerance limits may be an important mechanism by which microorganisms adapt to novel environments, but it may come at the evolutionary cost of reduced performance under ancestral conditions. We combined a comparative physiological approach with phylogenetic analyses to study the evolution of thermotolerance in hot spring cyanobacteria of the genus Synechococcus. Among the 20 laboratory clones of Synechococcus isolated from collections made along an Oregon hot spring thermal gradient, four different 16S rRNA gene sequences were identified. Phylogenies constructed by using the sequence data indicated that the clones were polyphyletic but that three of the four sequence groups formed a clade. Differences in thermotolerance were observed for clones with different 16S rRNA gene sequences, and comparison of these physiological differences within a phylogenetic framework provided evidence that more thermotolerant lineages of Synechococcus evolved from less thermotolerant ancestors. The extension of the thermal limit in these bacteria was correlated with a reduction in the breadth of the temperature range for growth, which provides evidence that enhanced thermotolerance has come at the evolutionary cost of increased thermal specialization. This study illustrates the utility of using phylogenetic comparative methods to investigate how evolutionary processes have shaped historical patterns of ecological diversification in microorganisms.

Synechococcus Group/classification/growth & develo↗

NASA NTRS1989

Ecological considerations for possible Martian biota

Current climatic and geological evidence suggests that, like early Earth, conditions on ancient Mars may also have been favorable for the origin and evolution of life. The primordial atmospheres of the two planets were quite similar, composed primarily of CO2, N2, and water vapor at a total atmospheric pressure of approximately 1 bar. Each of these gases are important for the evolution of biological systems. With the exception of nitrogen, there seems to have been a sufficient supply of the biogenic elements C, H, O, P, and S (CHOPS) on early Mars for life to have evolved. It was postulated that primordial Mars contained only 18 mb of nitrogen in the form of N2 given that only fixed nitrogen is utilized by living systems. Laboratory tests performed at a total pressure of 1 bar and various partial pressures of dinitrogen (pN2 1-780 mb) show that nitrogen fixing organisms grow at pN2's of 18 mb or less, although the biomass and growth rates are decreased. The calcualted in vivo Km's ranged from 46 mb to 130 mb. If organisms adapted on Earth to a pH2 of 780 mb are capable of growing at these low partial pressures, it is conceivable that nitrogen was not the limiting factor in the evolution of life on early Mars.

Klingler, June M.↗

NASA NTRS2018

Enabling Experimental Evolution: Multi-Parameter Sensor System Integration into a Culture/Stressor Biofluidics System

Experimental evolution (EE) exposes microbial communities to ecological stressors, simulating dynamics up to near-extinction events. Combined with comparative sequencing and other molecular tools, such data can inform the genetic and other biological mechanisms underlying extremophile adaptation, and other observed effects. Automating this type of experiment using biofluidics can mitigate many traditional obstacles, including delays in assay results and environment adjustment and the need for many replicates. A first-generation device for automating EE procedures, the Automated Adaptive Directed Evolution Chamber (AADEC), was developed at NASA Ames. UV-C radiation was the stressor, an LED-photodiode array measured optical density, magnetic agitation and peristaltic pump systems ensured nutrient availability, and Arduino microcontrollers provided control. Escherichia coli in LB kanamycin media was used for testing and performance verification. A manual laboratory procedure with timed exposure to UV-C was performed to typify tolerance acquisition. Approximately a 106 factor increase in survival ratio was recorded over multiple iterations. Currently, a second-generation device is being developed integrating more real-time sensors: redox potential (ORP), indicating available/consumed metabolic energy; dissolved oxygen (DO), indicating aerobic/anaerobic growth; pH, indicating metabolic products; and electrical conductivity (EC), another indicator of metabolic products. The EC sensor system was constructed and calibrated in-house and matched commercial sensors in the required range. A Raspberry Pi computer automated the electrical system, allowing real-time data acquisition. The fluidics card was made of CNC-milled polycarbonate for biocompatibility. Each sensor parameter can also be used as a selection pressure alone or in combination with others to create extreme microbial environments. As a proof of concept, this work demonstrated sensor operation in one pair of growth-sensor chambers. It can be expanded to a multi-chamber system to enable inter-culture comparisons and multi-population studies. The prior Arduino system will be ported to the RPi system. Future stressors to be added include thermal, reactive oxygen species, and varying nutrient availability.

Govinda Raj, Chinmayee↗

NASA NTRS2017

The Evolution of Planetary Protection Implementation on Mars Landed Missions

NASA has developed requirements dedicated to the prevention of forward and backward contamination during space exploration. Historically, international agreements provided guidelines to prevent contamination of the Moon and other celestial bodies, as well as the Earth (e.g., sample return missions). The UN Outer Space Treaty was established in 1967 and the Committee on Space Research (COSPAR) maintains a planetary protection policy complying with Article IX of this treaty. By avoiding forward contamination, the integrity of scientific exploration is preserved. Planetary Protection mission requirements are levied on missions to control contamination. These requirements are dependent on the science of the mission and on the celestial bodies encountered or targeted along the way. Consequently, categories are assigned to missions, and specific implementation plans are developed to meet the planetary protection requirements. NASA missions have evolved over time with increasingly more demanding scientific objectives and more complex flight systems to achieve those objectives and, thus, planetary protection methods and processes used for implementation have become much more intricate, complicated, and challenging. Here, we will portray the evolution of planetary protection implementation at JPL in several important areas throughout the course of NASA sponsored robotic Mars lander or rover missions, starting from Mars Pathfinder through the beginning of Mars 2020. Highlighted in the discussion will be process changes in planetary protection requirements development and flow down. Development and implementation of new and improved methods used in the reduction of spacecraft bioburden will be discussed as well as approaches and challenges that come along with setting up remote laboratories to perform bioassays. The consequences and forward planning of delays on missions will be highlighted as well as lessons learned on the impact of communication and training in achieving planetary protection requirements. The evolution of methods used for the detection of microbial bioburden on spacecraft hardware will be considered. These methods use standard microbiology as well as the adaptation of advances in biotechnology, molecular biology, and bioinformatics. Technical approaches developed for the prevention of contamination and recontamination of hardware during Assembly, Test, and Launch Operations will be discussed.

Kazarians, Gayane A.↗

NASA NTRSDate not supplied

Automating the Study of Microbial Adaptation Dynamics on and off the ISS

The International Space Station (ISS) not only serves as a unique environment for humans, but also the microorganisms that join alongside. Many microbes present on the spacecraft arrive via humans, and as they interact with different surfaces they begin to inhabit those locations. Much like how human health has shown to be impacted by these extreme environments, microbial viability and response to stress also changes. Experimental evolution (EE) can aid in studying how microbes’ growth and activity changes within the ISS environments by applying controlled stressors to microbial cultures and monitoring their response over generations. EE studies are commonly done manually in laboratories, but, with multiple environmental variables to measure and adjust, it becomes highly labor-intensive, prone to human error, and challenging to scale. A multipurpose automated EE system named the AADEC has been developed to address these problems. This system integrates multiple sensors into a single fluidic chamber using UV-C flux, temperature, and media composition as stressors. AADEC contains five sensors: oxidation-reduction potential, electrical conductivity, pH, dissolved oxygen, and optical density. On their own, each is able to provide certain information on growth rate or metabolism; together, they show in detail how stressors affect life. AADEC studies can be conducted on Earth and repeated aboard the ISS to see how behavior changes when exposed to space mission stressors such as microgravity and radiation. AADEC’s auxiliary systems include peristaltic pumps for media exchange, magnetic rods for agitation, and a Raspberry Pi microprocessor to monitor, store, and adjust stressor levels real-time. This allows researchers to gather information within rapid generations, data and accuracy which is challenging to achieve through manual studies. With further miniaturization and automation, such as a more robust single-piece fluidics card, AADEC has the potential to be developed as a spacecraft payload. Support: NASA Ames CIF Award

Automating↗

NASA NTRS2016

Instrumentation for Examining Microbial Response to Changes In Environmental Pressures

The Automated Adaptive Directed Evolution Chamber (AADEC) is a device that allows operators to generate a micro-scale analog of real world systems that can be used to model the local-scale effects of climate change on microbial ecosystems. The AADEC uses an artificial environment to expose cultures of micro-organisms to environmental pressures, such as UV-C radiation, chemical toxins, and temperature. The AADEC autonomously exposes micro-organisms to selection pressures. This improves upon standard manual laboratory techniques: the process can take place over a longer period of time, involve more stressors, implement real-time adjustments based on the state of the population, and minimize the risk of contamination. We currently use UV-C radiation as the main selection pressure, UV-C is well studied both for its cell and DNA damaging effects as a type of selection pressure and for its related effectiveness as a mutagen; having these functions united makes it a good choice for a proof of concept. The AADEC roadmap includes expansion to different selection pressures, including heavy metal toxicity, temperature, and other forms of radiation.The AADEC uses closed-loop control to feedback the current state of the culture to the AADEC controller that modifies selection pressure intensity during experimentation, in this case culture density and growth rate. Culture density and growth rate are determined by measuring the optical density of the culture using 600 nm light. An array of 600 nm LEDs illuminate the culture and photodiodes are used to measure the shadow on the opposite side of the chamber.Previous experiments showed that we can produce a million fold increase to UV-C radiation over seven iterations. The most recent implements a microfluidic system that can expose cultures to multiple different selection pressures, perform non-survival based selection, and autonomously perform hundreds of exposure cycles. A scalable pump system gives the ability to pump in various different growth media to individual cultures and introduce chemical toxins during experimentation; AADEC can perform freeze and thaw cycles. We improved our baseline characterization by building a custom UV-C exposure hood, a shutter operates on a preset timer allowing the user to set exposure intensity consistently for multiple iterations.

Environment↗