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At least 163 records · Page 9

High fidelity ground deposition measurement with robots after explosive radiological dispersion

A team of scientists from the Remote Sensing Laboratory at Joint Base Andrews, Maryland, has assembled a remote-controlled robot to field a few sodium iodide scintillators of different size and shape 18″ above ground for measurement of ground deposition of gamma-emitting particles after an explosion of a radiological dispersal device. This system uses a high-precision differential GPS device with submeter accuracy for radiation mapping. The system is most useful in characterizing large-area contamination and detecting gamma radioactivity in invisible, submicron particulate debris deposited on the ground at surface level or embedded in subsurface up to 3″ deep. The system was assembled as part of a larger effort to integrate advanced radiological detection devices into autonomous or remote-controlled robotic systems to eliminate or minimize the need for emergency responders to enter areas that pose significant health and safety risks to humans following a major radiological incident or accident. Research into autonomous algorithms is required to develop automated robotic systems for radiological survey and characterization activities in highly contaminated areas. The scope of this project also includes developing communications pathways and supporting infrastructure capabilities for different types of robotic technologies. The expected result is an advanced autonomous robotic system with integrated radiation detection electronics that allows emergency response personnel to view data remotely and in real time for radiological emergency response and consequence management purposes.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗

On the role of exchange of power and information signals in control and stability of the human-robot interaction

A human's ability to perform physical tasks is limited, not only by his intelligence, but by his physical strength. If, in an appropriate environment, a machine's mechanical power is closely integrated with a human arm's mechanical power under the control of the human intellect, the resulting system will be superior to a loosely integrated combination of a human and a fully automated robot. Therefore, we must develop a fundamental solution to the problem of 'extending' human mechanical power. The work presented here defines 'extenders' as a class of robot manipulators worn by humans to increase human mechanical strength, while the wearer's intellect remains the central control system for manipulating the extender. The human, in physical contact with the extender, exchanges power and information signals with the extender. The aim is to determine the fundamental building blocks of an intelligent controller, a controller which allows interaction between humans and a broad class of computer-controlled machines via simultaneous exchange of both power and information signals. The prevalent trend in automation has been to physically separate the human from the machine so the human must always send information signals via an intermediary device (e.g., joystick, pushbutton, light switch). Extenders, however are perfect examples of self-powered machines that are built and controlled for the optimal exchange of power and information signals with humans. The human wearing the extender is in physical contact with the machine, so power transfer is unavoidable and information signals from the human help to control the machine. Commands are transferred to the extender via the contact forces and the EMG signals between the wearer and the extender. The extender augments human motor ability without accepting any explicit commands: it accepts the EMG signals and the contact force between the person's arm and the extender, and the extender 'translates' them into a desired position. In this unique configuration, mechanical power transfer between the human and the extender occurs because the human is pushing against the extender. The extender transfers to the human's hand, in feedback fashion, a scaled-down version of the actual external load which the extender is manipulating. This natural feedback force on the human's hand allows him to 'feel' a modified version of the external forces on the extender. The information signals from the human (e.g., EMG signals) to the computer reflect human cognitive ability, and the power transfer between the human and the machine (e.g., physical interaction) reflects human physical ability. Thus the information transfer to the machine augments cognitive ability, and the power transfer augments motor ability. These two actions are coupled through the human cognitive/motor dynamic behavior. The goal is to derive the control rules for a class of computer-controlled machines that augment human physical and cognitive abilities in certain manipulative tasks.

Kazerooni, H.↗

Advancing specialized biofoundries via automated adaptive laboratory evolution

Adaptive laboratory evolution (ALE) is a powerful strategy for improving microbial phenotypes by harnessing natural selection under defined environmental conditions. Through applying selection regimes, beneficial mutations accumulate, enabling the generation of strains with enhanced properties. However, conventional ALE is labor-intensive and difficult to scale, limiting reproducibility and broader discovery of evolutionary principles. Recent advances in robotics, automation, and computational infrastructure are transforming ALE into a scalable, data-rich experimental paradigm. Automated platforms enable standardized and complex protocols, real-time monitoring, and highly parallel evolution campaigns, improving consistency while generating longitudinal datasets that reveal convergent adaptive mechanisms. Here, we discuss the role of specialized biofoundries in advancing automated ALE and enabling large-scale evolutionary engineering. We review major automated ALE formats and outline key design principles for effective ALE biofoundries, highlighting how automated ALE can support autonomous experimentation and AI-guided strain engineering.

59 BASIC BIOLOGICAL SCIENCES↗

Robotic Waterjet System

NASA needed a way to safely strip old paint and thermal protection material from reusable components from the Space Shuttle; to meet this requirement, Marshall Space Flight Center teamed with United Technologies' USBI Company and developed a stripping system based on hydroblasting. United Technology spun off a new company, Waterjet Systems, to commercialize and market the technology. The resulting ARMS (Automated Robotic Maintenance Systems), employ waterblasts at 55,000 pounds per square inch controlled by target-sensitive robots. The systems are used on aircraft and engine parts, and the newest application is on ships, where it not only strips but catches the ensuing wastewater. This innovation results in faster, cheaper stripping with less clean-up and reduced environmental impact.

Source record↗

Testing of ROMPS robot mechanical interfaces and compliant device

The Robot Operated Materials Processing System (ROMPS) has been developed at Goddard Space Flight Center (GSFC) under a flight project to investigate commercially promising in-space material processes and to design reflyable robot automated systems to be used in the above processes for low-cost operations. The ROMPS is currently scheduled for flight in 1994 as a Hitchhiker payload in a Get Away Special (GAS) can. An important component of the ROMPS is a three degree-of-freedom (DOF) robot which will be responsible for carrying out the required tasks of in-space processing of selected materials. This report deals with testing of the mating capability of the ROMPS robot fingers with its various mechanical interfaces. In particular, the test plan will focus on studying the capability of a compliance mechanism mounted on the robot fingers in accommodating misalignments between the robot fingers and the interfaces during the mating. The report is organized as follows: Section 2 represents the main components of the ROMPS robot and briefly describes its operations. Section 3 presents the objectives of the test and outlines the test plan. The testbed comprising a Steward Platform-based high precision manipulator and associated data acquisition and control systems is described in Section 4. Section 5 presents results of numerous experiments conducted to study the mating capability of the robot fingers with its various interfaces under misalignments. The report is concluded with observations and recommendations based on the test results.

Nguyen, Charles C.↗

Virtual Presence: One Step Beyond Reality

Our primary objective was to team up a group consisting of scientists and engineers from two different NASA cultures, and simulate an interactive teleoperated robot conducting geologic field work on the Moon or Mars. The information derived from the experiment will benefit both the robotics team and the planetary exploration team in the areas of robot design and development, and mission planning and analysis. The Earth Sciences and Space and Life Sciences Division combines the past with the future contributing experience from Apollo crews exploring the lunar surface, knowledge of reduced gravity environments, the performance limits of EVA suits, and future goals for human exploration beyond low Earth orbit. The Automation, Robotics. and Simulation Division brings to the table the technical expertise of robotic systems, the future goals of highly interactive robotic capabilities, treading on the edge of technology by joining for the first time a unique combination of telepresence with virtual reality.

Budden, Nancy Ann↗

Gateway Autonomy for Enabling Deep Space Exploration

The Gateway spacecraft is an important stepping-stone to exploration of the solar system, integrating commercial and international partners into a tightly coupled system, enabling cislunar activities, and implementing key technologies for missions to Mars. Autonomy is a capability area necessary to handle long communication outages where intervention from Earth is impossible, to prepare to operate with long communication delays that will be common in interplanetary travel, and to make spaceflight more affordable and accessible by reducing sustaining operations costs. The Gateway Concept of Operations states that one of Gateway’s goals is to “focus on infrastructure and systems that will allow autonomous operations aboard the Gateway with robotics, automated systems, advanced communications, and distributed computing.” Gateway’s Vehicle Systems Manager (VSM) and associated Autonomous Spacecraft Management Architecture (ASMA) are key products towards delivering autonomous capability. The primary functions of the control architecture are Mission Management and Timeline Execution, Resource Management, Fault Management, and Vehicle Control and Operation (VCO). In each of these areas, there is an initial level of capability to be delivered at launch, with plans to continue development and grow to greater capability. The initial deployment of VSM will focus on maintaining vehicle safety by focusing on full fault management capabilities and deploying only enough resource and timeline planning functionality to support that. The final deployment of VSM will add significant planning and control optimization functionality to support nominal operations for up to 21 days without ground support, even accommodating fault and failure conditions. While the VSM is the vehicle-level representation of autonomous reasoning, distributed automation is essential to provide the right scope and abstraction of information to process. Module and system support of automation and simplicity of interfaces are two important design paradigms that Gateway is focusing on to garner a systems approach to autonomy. Distribution of reasoning can increase complexity, so Gateway is also taking a strict hierarchical approach to information flow and decision making. VSM is not the only capability necessary to achieve an autonomous spacecraft. Robotics support for maintenance of the spacecraft will be essential to provide continued vehicle functionality even when crew is not present. Technical and programmatic challenges exist when implementing autonomous robotics operations. These challenges include sufficient network flexibility to support data transfer to the rest of the vehicle to coordinate module-to-module robotic walk-offs and finding the proper interfaces to allow sufficient dexterity. Communication system upgrades planned for Gateway include Delay Tolerant Networking to best utilize the complex network of relays that will be part of mature cislunar operations. Distributed computing and management will provide failure tolerance, robustness, and growth of capabilities while still allowing significant reuse of heritage software on heritage systems as well as reuse of common applications across a spacecraft to minimize new development, but this requires adherence to key standards and interfaces. The Gateway program has demonstrated significant progress towards these capabilities and has identified challenges other spacecraft developers should be aware of from the start.

Molly Anderson↗

Gateway Autonomy for Enabling Deep Space Exploration

The Gateway spacecraft is an important stepping-stone to exploration of the solar system, integrating commercial and international partners into a tightly coupled system, enabling cislunar activities, and implementing key technologies for missions to Mars. Autonomy is a capability area necessary to handle long communication outages where intervention from Earth is impossible, to prepare to operate with long communication delays that will be common in interplanetary travel, and to make spaceflight more affordable and accessible by reducing sustaining operations costs. The Gateway Concept of Operations states that one of Gateway’s goals is to “focus on infrastructure and systems that will allow autonomous operations aboard the Gateway with robotics, automated systems, advanced communications, and distributed computing.” Gateway’s Vehicle Systems Manager (VSM) and associated Autonomous Spacecraft Management Architecture (ASMA) are key products towards delivering autonomous capability. The primary functions of the control architecture are Mission Management and Timeline Execution, Resource Management, Fault Management, and Vehicle Control and Operation (VCO). In each of these areas, there is an initial level of capability to be delivered at launch, with plans to continue development and grow to greater capability. The initial deployment of VSM will focus on maintaining vehicle safety by focusing on full fault management capabilities and deploying only enough resource and timeline planning functionality to support that. The final deployment of VSM will add significant planning and control optimization functionality to support nominal operations for up to 21 days without ground support, even accommodating fault and failure conditions. While the VSM is the vehicle-level representation of autonomous reasoning, distributed automation is essential to provide the right scope and abstraction of information to process. Module and system support of automation and simplicity of interfaces are two important design paradigms that Gateway is focusing on to garner a systems approach to autonomy. Distribution of reasoning can increase complexity, so Gateway is also taking a strict hierarchical approach to information flow and decision making. VSM is not the only capability necessary to achieve an autonomous spacecraft. Robotics support for maintenance of the spacecraft will be essential to provide continued vehicle functionality even when crew is not present. Technical and programmatic challenges exist when implementing autonomous robotics operations. These challenges include sufficient network flexibility to support data transfer to the rest of the vehicle to coordinate module-to-module robotic walk-offs and finding the proper interfaces to allow sufficient dexterity. Communication system upgrades planned for Gateway include Delay Tolerant Networking to best utilize the complex network of relays that will be part of mature cislunar operations. Distributed computing and management will provide failure tolerance, robustness, and growth of capabilities while still allowing significant reuse of heritage software on heritage systems as well as reuse of common applications across a spacecraft to minimize new development, but this requires adherence to key standards and interfaces. The Gateway program has demonstrated significant progress towards these capabilities and has identified challenges other spacecraft developers should be aware of from the start.

Molly Anderson↗

Self-driving laboratories to autonomously navigate the protein fitness landscape

Abstract Protein engineering has nearly limitless applications across chemistry, energy and medicine, but creating new proteins with improved or novel functions remains slow, labor-intensive and inefficient. Here we present the Self-driving Autonomous Machines for Protein Landscape Exploration (SAMPLE) platform for fully autonomous protein engineering. SAMPLE is driven by an intelligent agent that learns protein sequence–function relationships, designs new proteins and sends designs to a fully automated robotic system that experimentally tests the designed proteins and provides feedback to improve the agent’s understanding of the system. We deploy four SAMPLE agents with the goal of engineering glycoside hydrolase enzymes with enhanced thermal tolerance. Despite showing individual differences in their search behavior, all four agents quickly converge on thermostable enzymes. Self-driving laboratories automate and accelerate the scientific discovery process and hold great potential for the fields of protein engineering and synthetic biology.

Rapp, Jacob T. (ORCID:0000000226835204)↗

Anthropomorphic Robot Design and User Interaction Associated with Motion

Though in its original concept a robot was conceived to have some human-like shape, most robots now in use have specific industrial purposes and do not closely resemble humans. Nevertheless, robots that resemble human form in some way have continued to be introduced. They are called anthropomorphic robots. The fact that the user interface to all robots is now highly mediated means that the form of the user interface is not necessarily connected to the robots form, human or otherwise. Consequently, the unique way the design of anthropomorphic robots affects their user interaction is through their general appearance and the way they move. These robots human-like appearance acts as a kind of generalized predictor that gives its operators, and those with whom they may directly work, the expectation that they will behave to some extent like a human. This expectation is especially prominent for interactions with social robots, which are built to enhance it. Often interaction with them may be mainly cognitive because they are not necessarily kinematically intricate enough for complex physical interaction. Their body movement, for example, may be limited to simple wheeled locomotion. An anthropomorphic robot with human form, however, can be kinematically complex and designed, for example, to reproduce the details of human limb, torso, and head movement. Because of the mediated nature of robot control, there remains in general no necessary connection between the specific form of user interface and the anthropomorphic form of the robot. But their anthropomorphic kinematics and dynamics imply that the impact of their design shows up in the way the robot moves. The central finding of this report is that the control of this motion is a basic design element through which the anthropomorphic form can affect user interaction. In particular, designers of anthropomorphic robots can take advantage of the inherent human-like movement to 1) improve the users direct manual control over robot limbs and body positions, 2) improve users ability to detect anomalous robot behavior which could signal malfunction, and 3) enable users to be better able to infer the intent of robot movement. These three benefits of anthropomorphic design are inherent implications of the anthropomorphic form but they need to be recognized by designers as part of anthropomorphic design and explicitly enhanced to maximize their beneficial impact. Examples of such enhancements are provided in this report. If implemented, these benefits of anthropomorphic design can help reduce the risk of Inadequate Design of Human and Automation Robotic Integration (HARI) associated with the HARI-01 gap by providing efficient and dexterous operator control over robots and by improving operator ability to detect malfunctions and understand the intention of robot movement.

automation/robotic integration↗

In-space construction and dynamics of large space structures

The types of equipment and structures that will be required to construct very large spacecraft in space are discussed. One of the basic issues that must be resolved is the appropriate mix of humans and machines in the construction process. While the use of robots offers the potential for reducing the number of extra-vehicular activity (EVA) hours required for particular construction operations, the availability of humans greatly increases the reliability of complex construction tasks. A hybrid system is described which makes the best use of man and machine to provide a highly reliable and versatile construction approach. Such a system will provide an efficient method for constructing large spacecraft until fully automated, robotic devices can be perfected. Details are given on an extensive ground test program which was designed to evaluate and demonstrate large spacecraft construction. A discussion is presented on the use of the Space Station Freedom, or an appropriate derivative, as a construction facility. Finally, a construction scenario and assembly timelines are presented for constructing a 20-meter-diameter high precision reflector.

Mikulas, Martin M., Jr.↗

Self-Driving Laboratory for Polymer Electronics

Owing to the chemical pluripotency and viscoelastic nature of electronic polymers, polymer electronics have shown unique advances in many emerging applications such as skin-like electronics, large-area printed energy devices, and neuromorphic computing devices, but their development period is years-long. Recent advancements in automation, robotics, and learning algorithms have led to a growing number of self-driving (autonomous) laboratories that have begun to revolutionize the development and accelerated discovery of materials. In this perspective, we first introduce the current state of autonomous laboratories. Then we analyze why it is challenging to conduct polymer electronics research by an autonomous laboratory and highlight the needs. Here, we further discuss our efforts in building an autonomous laboratory, namely Polybot, for the automated synthesis and characterization of electronic polymers and their processing and fabrication into electronic devices. Finally, we share our vision in using a self-driving laboratory for different types of polymer electronics research.

36 MATERIALS SCIENCE↗

A Viable Scheme for Elemental Extraction and Purification Using In-Situ Planetary Resources

NASA's new strategic direction includes establishing a self-sufficient, affordable and safe human and robotic presence outside the low earth orbit. Some of the items required for a self-sufficient extra-terrestrial habitat will include materials for power generation (e.g. Si for solar cells) and habitat construction (e.g. Al, Fe, and Ti). In this paper we will present a viable elemental extraction and refining process from in-situ regolith which would be optimally continuous, robotically automated, and require a minimum amount of astronaut supervision and containment facilities, The approach is based on using a concentrated heat source and translating sample geometry to enable simultaneous oxide reduction and elemental refining. Preliminary results will be presented to demonstrate that the proposed zone refining process is capable of segregating or refining important elements such as Si (for solar cell fabrication) and Fe (for habitat construction). A conceptual scheme will be presented whereby such a process could be supported by use of solar energy and a precursor robotic mission on the surface of the moon.

Sen, S.↗

JSC flight experiment recommendation in support of Space Station robotic operations

The man-tended configuration (MTC) of Space Station Freedom (SSF) provides a unique opportunity to move robotic systems from the laboratory into the mainstream space program. Restricted crew access due to the Shuttle's flight rate, as well as constrained on-orbit stay time, reduces the productivity of a facility dependent on astronauts to perform useful work. A natural tendency toward robotics to perform maintenance and routine tasks will be seen in efforts to increase SSF usefulness. This tendency will provide the foothold for deploying space robots. This paper outlines a flight experiment that will capitalize on the investment in robotic technology made by NASA over the past ten years. The flight experiment described herein provides the technology demonstration necessary for taking advantage of the expected opportunity at MTC. As a context to this flight experiment, a broader view of the strategy developed at the JSC is required. The JSC is building toward MTC by developing a ground-based SSF emulation funded jointly by internal funds, NASA/Code R, and NASA/Code M. The purpose of this ground-based Station is to provide a platform whereby technology originally developed at JPL, LaRC, and GSFC can be integrated into a near flight-like condition. For instance, the Automated Robotic Maintenance of Space Station (ARMSS) project integrates flat targets, surface inspection, and other JPL technologies into a Station analogy for evaluation. Also, ARMSS provides the experimental platform for the Capaciflector from GSPC to be evaluated for its usefulness in performing ORU change out or other tasks where proximity detection is required. The use and enhancement of these ground-based SSF models are planned for use through FY-93. The experimental data gathered from tests in these facilities will provide the basis for the technology content of the proposed flight experiment.

Berka, Reginald B.↗

Human machine interaction via the transfer of power and information signals

Robot manipulators are designed to perform tasks which would otherwise be executed by a human operator. No manipulator can even approach the speed and accuracy with which humans execute these tasks. But manipulators have the capability to exceed human ability in one particular area: strength. Through any reasonable observation and experience, the human's ability to perform a variety of physical tasks is limited not by his intelligence, but by his physical strength. If, in the appropriate environment, we can more closely integrate the mechanical power of a machine with intellectually driven human hand under the supervisory control of the human's intellect, we will then have a system which is superior to a loosely-integrated combination of a human and his fully automated robot as in the present day robotic systems. We must therefore develop a fundamental approach to the problem of this extending human mechanical power in certain environments. Extenders will be a class of robots worn by humans to increase human mechanical ability, while the wearer's intellect remains the central intelligent control system for manipulating the extender. The human body, in physical contact with the extender, exchanges information signals and power with the extender. Commands are transferred to the extender via the contact forces between the wearer and the extender as opposed to use of joystick (master arm), push-button or key-board to execute such commands that were used in previous man amplifiers. Instead, the operator becomes an integral part of the extender while executing the task. In this unique configuration the mechanical power transfer between the human and extender occurs in addition to information signal transfer. When the wearer uses the extender to touch and manipulate an object, the extender transfers to the wearer's hand, in feedback fashion, a scaled-down value of the actual external load which the extender is manipulating. This natural feedback force on the wearer's hand allows him to feel the scaled-down value of the external forces in the manipulations. Extenders can be utilized to maneuver very heavy loads in factories, shipyards, airports, and construction sites. In some instances, for example, extenders can replace forklifts. The experimental results for a prototype extender are discussed.

Kazerooni, H.↗

Human-Automation Allocations for Current Robotic Space Operations

Within the Human Research Program, one risk delineates the uncertainty surrounding crew working with automation and robotics in spaceflight. The Risk of Inadequate Design of Human and Automation/Robotic Integration (HARI) is concerned with the detrimental effects on crew performance due to ineffective user interfaces, system designs and/or functional task allocation, potentially compromising mission success and safety. Risk arises because we have limited experience with complex automation and robotics. One key gap within HARI, is the gap related to functional allocation. The gap states: We need to evaluate, develop, and validate methods and guidelines for identifying human-automation/robot task information needs, function allocation, and team composition for future long duration, long distance space missions. Allocations determine the human-system performance as it identifies the functions and performance levels required by the automation/robotic system, and in turn, what work the crew is expected to perform and the necessary human performance requirements. Allocations must take into account each of the human, automation, and robotic systems capabilities and limitations. Some functions may be intuitively assigned to the human versus the robot, but to optimize efficiency and effectiveness, purposeful role assignments will be required. The role of automation and robotics will significantly change in future exploration missions, particularly as crew becomes more autonomous from ground controllers. Thus, we must understand the suitability of existing function allocation methods within NASA as well as the existing allocations established by the few robotic systems that are operational in spaceflight. In order to evaluate future methods of robotic allocations, we must first benchmark the allocations and allocation methods that have been used. We will present 1) documentation of human-automation-robotic allocations in existing, operational spaceflight systems; and 2) To gather existing lessons learned and best practices in these role assignments, from spaceflight operational experience of crew and ground teams that may be used to guide development for future systems. NASA and other space agencies have operational spaceflight experience with two key Human-Automation-Robotic (HAR) systems: heavy lift robotic arms and planetary robotic explorers. Additionally, NASA has invested in high-fidelity rover systems that can carry crew, building beyond Apollo's lunar rover. The heavy lift robotic arms reviewed are: Space Station Remote Manipulator System (SSRMS), Japanese Remote Manipulator System (JEMRMS), and the European Robotic Arm (ERA, designed but not deployed in space). The robotic rover systems reviewed are: Mars Exploration Rovers, Mars Science Laboratory rover, and the high-fidelity K10 rovers. Much of the design and operational feedback for these systems have been communicated to flight controllers and robotic design teams. As part of the mitigating the HARI risk for future human spaceflight operations, we must document function allocations between robots and humans that have worked well in practice.

robotic allocation↗

Robotic vision techniques for space operations

Automation and robotics for space applications are being pursued for increased productivity, enhanced reliability, increased flexibility, higher safety, and for the automation of time-consuming tasks and those activities which are beyond the capacity of the crew. One of the key functional elements of an automated robotic system is sensing and perception. As the robotics era dawns in space, vision systems will be required to provide the key sensory data needed for multifaceted intelligent operations. In general, the three-dimensional scene/object description, along with location, orientation, and motion parameters will be needed. In space, the absence of diffused lighting due to a lack of atmosphere gives rise to: (a) high dynamic range (10(exp 8)) of scattered sunlight intensities, resulting in very high contrast between shadowed and specular portions of the scene; (b) intense specular reflections causing target/scene bloom; and (c) loss of portions of the image due to shadowing and presence of stars, Earth, Moon, and other space objects in the scene. In this work, developments for combating the adverse effects described earlier and for enhancing scene definition are discussed. Both active and passive sensors are used. The algorithm for selecting appropriate wavelength, polarization, look angle of vision sensors is based on environmental factors as well as the properties of the target/scene which are to be perceived. The environment is characterized on the basis of sunlight and other illumination incident on the target/scene and the temperature profiles estimated on the basis of the incident illumination. The unknown geometrical and physical parameters are then derived from the fusion of the active and passive microwave, infrared, laser, and optical data.

Krishen, Kumar↗

A colorimetric method to measure in vitro nitrogenase functionality for engineering nitrogen fixation

Biological nitrogen fixation (BNF) is the reduction of N 2 into NH 3 in a group of prokaryotes by an extremely O 2 -sensitive protein complex called nitrogenase. Transfer of the BNF pathway directly into plants, rather than by association with microorganisms, could generate crops that are less dependent on synthetic nitrogen fertilizers and increase agricultural productivity and sustainability. In the laboratory, nitrogenase activity is commonly determined by measuring ethylene produced from the nitrogenase-dependent reduction of acetylene (ARA) using a gas chromatograph. The ARA is not well suited for analysis of large sample sets nor easily adapted to automated robotic determination of nitrogenase activities. Here, we show that a reduced sulfonated viologen derivative (S 2 V red ) assay can replace the ARA for simultaneous analysis of isolated nitrogenase proteins using a microplate reader. We used the S 2 V red to screen a library of NifH nitrogenase components targeted to mitochondria in yeast. Two NifH proteins presented properties of great interest for engineering of nitrogen fixation in plants, namely NifM independency, to reduce the number of genes to be transferred to the eukaryotic host; and O 2 resistance, to expand the half-life of NifH iron-sulfur cluster in a eukaryotic cell. This study established that NifH from Dehalococcoides ethenogenes did not require NifM for solubility, [Fe-S] cluster occupancy or functionality, and that NifH from Geobacter sulfurreducens was more resistant to O 2 exposure than the other NifH proteins tested. It demonstrates that nitrogenase components with specific biochemical properties such as a wider range of O 2 tolerance exist in Nature, and that their identification should be an area of focus for the engineering of nitrogen-fixing crops.

59 BASIC BIOLOGICAL SCIENCES↗