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At least 181 records · Page 10

Integrating continuous hypermutation with high–throughput screening for optimization of cis,cis –muconic acid production in yeast

Directed evolution is a powerful method to optimize proteins and metabolic reactions towards user-defined goals. It usually involves subjecting genes or pathways to iterative rounds of mutagenesis, selection and amplification. While powerful, systematic searches through large sequence-spaces is a labour-intensive task, and can be further limited by a priori knowledge about the optimal initial search space, and/or limits in terms of screening throughput. Here, we demonstrate an integrated directed evolution workflow for metabolic pathway enzymes that continuously generate enzyme variants using the recently developed orthogonal replication system, OrthoRep and screens for optimal performance in high-throughput using a transcription factor-based biosensor. We demonstrate the strengths of this workflow by evolving a rate-limiting enzymatic reaction of the biosynthetic pathway for cis,cis-muconic acid (CCM), a precursor used for bioplastic and coatings, in Saccharomyces cerevisiae. After two weeks of simply iterating between passaging of cells to generate variant enzymes via OrthoRep and high-throughput sorting of best-performing variants using a transcription factor-based biosensor for CCM, we ultimately identified variant enzymes improving CCM titers > 13-fold compared with reference enzymes. Taken together, the combination of synthetic biology tools as adopted in this study is an efficient approach to debottleneck repetitive workflows associated with directed evolution of metabolic enzymes.

59 BASIC BIOLOGICAL SCIENCES↗

Green Factor

Los Alamos is designing a screening platform coupled to our biosensor to customize production processes in cell-free environments that offer biomanufacturers higher titers and yields, better resource utilization and cost-effective bioprocess. Our technology aims at enabling cell-free biosynthetic systems by providing enzyme variants generated by coupling with our biosensor technology, that can utilize cheap raw material cofactor analogues. This will enable bioprocesses based on cell free synthetic biology for a sustainable bioeconomy.

42 ENGINEERING↗

Progress of Anaerobic capability at LANL

The anaerobic chamber at LANL is fully functional. The Wolf cell sorter is also functional and easy to use. Clostridium tyrobutyricum strains are growing at anaerobic conditions. Biosensors in Corynebacterium glutamicum have been developed with aerobic reporters. A biosensor for C.tyrobutyricum with Yfast will be developed soon.

59 BASIC BIOLOGICAL SCIENCES↗

Removal of Non-Specifically Bound Proteins Using Rayleigh Waves Generated on ST-Quartz Substrates

Label-free biosensors are plagued by the issue of non-specific protein binding which negatively affects sensing parameters such as sensitivity, selectivity, and limit-of-detection. In the current work, we explore the possibility of using the Rayleigh waves in ST-Quartz devices to efficiently remove non-specifically bound proteins via acoustic streaming. A coupled-field finite element (FE) fluid structure interaction (FSI) model of a surface acoustic wave (SAW) device based on ST-Quartz substrate in contact with a liquid loading was first used to predict trends in forces related to SAW-induced acoustic streaming. Based on model predictions, it is found that the computed SAW body force is sufficient to overcome adhesive forces between particles and a surface while lift and drag forces prevent reattachment for a range of SAW frequencies. We further performed experiments to validate the model predictions and observe that the excitation of Rayleigh SAWs removed non-specifically bound (NSB) antigens and antibodies from sensing and non-sensing regions, while rinsing and blocking agents were ineffective. An amplified RF signal applied to the device input disrupted the specific interactions between antigens and their capture antibody as well. ST-quartz allows propagation of Rayleigh and leaky SH-SAW waves in orthogonal directions. Thus, the results reported here could allow integration of three important biosensor functions on a single chip, i.e., removal of non-specific binding, mixing, and sensing in the liquid phase.

59 BASIC BIOLOGICAL SCIENCES↗

Sensor Systems for Space Life Sciences

Sensors 2000! (S2K!) is a NASA Ames Research Center engineering initiative designed to provide biosensor and bio-instrumentation systems technology expertise to NASA's life sciences spaceflight programs. S2K! covers the full spectrum of sensor technology applications, ranging from spaceflight hardware design and fabrication to advanced technology development, transfer and commercialization. S2K! is currently developing sensor systems for space biomedical applications on BION (a Russian biosatellite focused on Rhesus Monkey physiology) and NEUROLAB (a Space Shuttle flight devoted to neuroscience). It's Advanced Technology Development-Biosensors (ATD-B) project focuses efforts in five principle areas: biotelemetry Systems, chemical and biological sensors, physiological sensors, advanced instrumentation architectures, and data and information management. Technologies already developed and tested included, application-specific sensors, preamplifier hybrids, modular programmable signal conditioners, power conditioning and distribution systems, and a fully implantable dual channel biotelemeter. Systems currently under development include a portable receiver system compatible with an off-the-shelf analog biotelemeter, a 4 channel digital biotelemetry system which monitors pH, a multichannel, g-processor based PCM biotelemetry system, and hand-held personal monitoring systems. S2K! technology easily lends itself to telescience and telemedicine applications as a front-end measurement and data acquisition device, suitable for obtaining and configuring physiological information, and processing that information under control from a remote location.

Somps, Chris J.↗

Advanced Technologies for Space Life Science Payloads on the International Space Station

SENSORS 2000! (S2K!) is a specialized, high-performance work group organized to provide advanced engineering and technology support for NASA's Life Sciences spaceflight and ground-based research and development programs. In support of these objectives, S2K! manages NASA's Advanced Technology Development Program for Biosensor and Biotelemetry Systems (ATD-B), with particular emphasis on technologies suitable for Gravitational Biology, Human Health and Performance, and Information Technology and Systems Management. A concurrent objective is to apply and transition ATD-B developed technologies to external, non-NASA humanitarian (medical, clinical, surgical, and emergency) situations and to stimulate partnering and leveraging with other government agencies, academia, and the commercial/industrial sectors. A phased long-term program has been implemented to support science disciplines and programs requiring specific biosensor (i.e., biopotential, biophysical, biochemical, and biological) measurements from humans, animals (mainly primates and rodents), and cells under controlled laboratory and simulated microgravity situations. In addition to the technology programs described above, NASA's Life and Microgravity Sciences and Applications Office has initiated a Technology Infusion process to identify and coordinate the utilization and integration of advanced technologies into its International Space Station Facilities. This project has recently identified a series of technologies, tasks, and products which, if implemented, would significantly increase the science return, decrease costs, and provide improved technological capability. This presentation will review the programs described above and discuss opportunities for collaboration, leveraging, and partnering with NASA.

Hines, John W.↗

Remote sensing of gene expression in Planta: transgenic plants as monitors of exogenous stress perception in extraterrestrial environments

Transgenic arabidopsis plants containing the alcohol dehydrogenase (Adh) gene promoter fused to the green fluorescent protein (GFP) reporter gene were developed as biological sensors for monitoring physiological responses to unique environments. Plants were monitored in vivo during exposure to hypoxia, high salt, cold, and abcissic acid in experiments designed to characterize the utility and responses of the Adh/GFP biosensors. Plants in the presence of environmental stimuli that induced the Adh promoter responded by expressing GFP, which in turn generated a detectable fluorescent signal. The GFP signal degraded when the inducing stimulus was removed. Digital imaging of the Adh/GFP plants exposed to each of the exogenous stresses demonstrated that the stress-induced gene expression could be followed in real time. The experimental results established the feasibility of using a digital monitoring system for collecting gene expression data in real time from Transgenic Arabidopsis Gene Expression System (TAGES) biosensor plants during space exploration experiments.

NASA Discipline Plant Biology↗

Ring-Interferometric Sol-Gel Bio-Sensor

A biosensor embodying the invention includes a sensing volume having an array of pores sized for immobilizing a first biological entity tending to bind to a second biological entity in such a manner as to change an index of refraction of the sensing volume. The biosensor further includes a ring interferometer, one volumetric section of the ring interferometer being the sensing volume, a laser for supplying light to the ring interferometer, and a photodetector for receiving light from the interferometer.

Bearman, Gregory↗

Thin membrane sensor with biochemical switch

A modular biosensor system for chemical or biological agent detection utilizes electrochemical measurement of an ion current across a gate membrane triggered by the reaction of the target agent with a recognition protein conjugated to a channel blocker. The sensor system includes a bioresponse simulator or biochemical switch module which contains the recognition protein-channel blocker conjugate, and in which the detection reactions occur, and a transducer module which contains a gate membrane and a measuring electrode, and in which the presence of agent is sensed electrically. In the poised state, ion channels in the gate membrane are blocked by the recognition protein-channel blocker conjugate. Detection reactions remove the recognition protein-channel blocker conjugate from the ion channels, thus eliciting an ion current surge in the gate membrane which subsequently triggers an output alarm. Sufficiently large currents are generated that simple direct current electronics are adequate for the measurements. The biosensor has applications for environmental, medical, and industrial use.

Case, George D.↗

Sensors Provide Early Warning of Biological Threats

Early Warning Inc. of Troy, New York, licensed powerful biosensor technology from Ames Research Center. Incorporating carbon nanotubes tipped with single strands of nucleic acid from waterborne pathogens, the sensor can detect even minute amounts of targeted, disease causing bacteria, viruses, and parasites. Early Warning features the NASA biosensor in its water analyzer, which can provide advance alert of potential biological hazards in water used for agriculture, food and beverages, showers, and at beaches and lakes -- within hours instead of the days required by conventional laboratory methods.

Source record↗

Replaceable Sensor System for Bioreactor Monitoring

A sensor system was proposed that would monitor spaceflight bioreactor parameters. Not only will this technology be invaluable in the space program for which it was developed, it will find applications in medical science and industrial laboratories as well. Using frequency-domain-based fluorescence lifetime technology, the sensor system will be able to detect changes in fluorescence lifetime quenching that results from displacement of fluorophorelabeled receptors bound to target ligands. This device will be used to monitor and regulate bioreactor parameters including glucose, pH, oxygen pressure (pO2), and carbon dioxide pressure (pCO2). Moreover, these biosensor fluorophore receptor-quenching complexes can be designed to further detect and monitor for potential biohazards, bioproducts, or bioimpurities. Biosensors used to detect biological fluid constituents have already been developed that employ a number of strategies, including invasive microelectrodes (e.g., dark electrodes), optical techniques including fluorescence, and membrane permeable systems based on osmotic pressure. Yet the longevity of any of these sensors does not meet the demands of extended use in spacecraft habitat or bioreactor monitoring. It was therefore necessary to develop a sensor platform that could determine not only fluid variables such as glucose concentration, pO2, pCO2, and pH but can also regulate these fluid variables with controlled feedback loop.

Mayo, Mike↗

Nanomaterial Based Sensors for NASA Missions

Nanomaterials such as carbon nanotubes (CNTs), carbon nanofibers (CNFs), graphene and metal nanowires have shown interesting electronic properties and therefore have been pursued for a variety of space applications requiring ultrasensitive and light-weight sensor and electronic devices. We have been pursuing development of chemical and biosensors using carbon nanotubes and carbon nanofibers for the last several years and this talk will present the benefits of nanomaterials these applications. More recently, printing approaches to manufacturing these devices have been explored as a strategy that is compatible to a microgravity environment. Nanomaterials are either grown in house or purchased and processed as electrical inks. Chemical modification or coatings are added to the nanomaterials to tailor the nanomaterial to the exact application. The development of printed chemical sensors and biosensors will be discussed for applications ranging from crew life support to exploration missions.

sensors↗

BioSentinel: An Adaptable Platform for Studying the Biological Effects of Deep Space Radiation

NASA's BioSentinel mission is a 6U nanosatellite with autonomous life support that will utilize the budding yeast Saccharomyces cerevisiae to study the DNA damage response to the deep space radiation environment. BioSentinel is planned to launch in 2019 as a secondary payload on the Space Launch System's first Exploration Mission (EM-1), and will undergo a lunar fly-by and enter heliocentric orbit after deployment. As the first biological mission beyond Low Earth Orbit (LEO) in nearly half a century, this mission will help fill critical gaps in knowledge about the effects of uniquely composed, chronic, low-flux deep space radiation on biological systems. Yeast is well-suited for this mission due to its desiccation tolerance and space-flight heritage. As a eukaryotic model organism, it also serves as a robust analog for human cells. Data gathered on this mission will thus inform us of the hazards involved in long-duration human exploration in deep space, and the protections necessary to mitigate them. Due to its low-cost, flexible and advanced technology, the 4U BioSensor payload contained within the nanosatellite is adaptable to other model microorganisms, exploration platforms and environments relevant to human exploration, such as the ISS, the Lunar Orbital Platform - Gateway and future lunar landers. In order to query the DNA damage response to deep space radiation, BioSentinel contains a wild type yeast strain as a positive control, and a radiation sensitive rad51 mutant strain that is defective for DNA repair. Yeast cells are desiccated in microfluidic cards, and rehydrated with growth medium and metabolic indicator dye at the desired time points during the mission. A thermal control system supports these stasis and growth states, and an optical system continuously measures cell growth and metabolism. An onboard radiation spectrometer and dosimeter allows us to correlate the dose, energy and particle-type of deep space radiation to the biological response. Data received from the deep space biosensor will be compared to control payloads on Earth and the ISS. Ongoing science testing for the BioSentinel project includes optimization for cell viability, desiccation tolerance, and long-term biocompatibility, as well as radiation experiments to understand the sensitivity and responsiveness of cells to varying radiation doses and particle types.

BioSentinel↗

BioSentinel

The BioSentinel mission was selected in 2013 as one of three secondary payloads to fly on the Space Launch Systems first Exploration Mission (EM-1) planned for launch in December 2017. The primary objective of BioSentinel is to develop a biosensor using a simple model organism to detect, measure, and correlate the impact of space radiation to living organisms overlong durations beyond Low Earth Orbit(LEO). While progress identifying and characterizing biological radiation effects using Earth-based facilities has been significant, no terrestrial source duplicates the unique space radiation environment.The BioSentinel biosensor uses the budding

Hanel, Robert↗

BioSentinel ISS Control Experiment Investigation Summary Form

The main science objective of BioSentinel is to develop biosensors to study the effects of the space radiation environment on biological organisms. Given its similarities to human cells, we decided to use the budding yeast Saccharomyces cerevisiae. Yeast cells share homology to human cells, particularly in the response to DNA damage caused by ionizing radiation. Most importantly, yeast cells can be desiccated and survive in that form for long periods of time. Thus, budding yeast is the ideal model organism for long-term experiments in preparation for future manned missions to deep space. In addition to the ISS mission, our biosensors will fly in a nanosatellite designed for deep space, which is manifested to fly as a secondary payload onboard NASA’s Artemis-1 rocket.

BioSentinel↗

Developing Technologies for Biological Experiments in Deep Space

NASA Ames Research Center is the leader in developing autonomous nanosatellites or CubeSats to address strategic knowledge gaps about the effects of space travel on biological organisms, including GeneSat, PharmaSat, and EcAMSat. Now that NASA has set its sights on human exploration in deep space, such missions require significant technological and biomedical countermeasures to protect astronauts from chronic radiation exposure. CubeSats can inform these countermeasures by querying relevant space environments with model organisms and/or biosensors. BioSentinel will be the first interplanetary CubeSat to study the biological response to space radiation outside low Earth orbit in almost 50 years. BioSentinel is an autonomous platform able to support biology and to investigate the effects of space radiation on a model organism in interplanetary deep space. It will fly onboard Artemis-1, from which it will be deployed on a lunar fly-by trajectory and into a heliocentric orbit. BioSentinel, a 6U CubeSat (1U = 10-cm cube), will measure the DNA damage response to ambient space radiation in a model organism, which will be compared to information provided by an onboard radiation spectrometer and to data obtained on the ISS and on Earth. Even though the primary objective of the mission is to develop an autonomous spacecraft capable of conducting biological experiments in deep space, the 4U BioSensor science payload contained within the free-flyer is an adaptable instrument that can perform bio measurements with different microorganisms and in multiple space environments, including the ISS, lunar gateway, and on the surface of the Moon. Thus, nanosatellites like BioSentinel can be used to study the effects of both reduced gravity and space radiation and can house different organisms to answer specific science questions. In addition to their flexibility, nanosatellites also provide a low-cost alternative to more complex and larger missions, and require minimal crew support, if any

biosenses↗

Developing Technologies for Biological Experiments in Deep Space

NASA Ames Research Center is the leader in developing autonomous nanosatellites or CubeSats to address strategic knowledge gaps about the effects of space travel on biological organisms, including GeneSat, PharmaSat, and EcAMSat. Now that NASA has set its sights on human exploration in deep space, such missions require significant technological and biomedical countermeasures to protect astronauts from chronic radiation exposure. CubeSats can inform these countermeasures by querying relevant space environments with model organisms and/or biosensors. BioSentinel will be the first interplanetary CubeSat to study the biological response to space radiation outside low Earth orbit in almost 50 years. BioSentinel is an autonomous platform able to support biology and to investigate the effects of space radiation on a model organism in interplanetary deep space. It will fly onboard Artemis-1, from which it will be deployed on a lunar fly-by trajectory and into a heliocentric orbit. BioSentinel, a 6U CubeSat (1U = 10-cm cube), will measure the DNA damage response to ambient space radiation in a model organism, which will be compared to information provided by an onboard radiation spectrometer and to data obtained on the ISS and on Earth. Even though the primary objective of the mission is to develop an autonomous spacecraft capable of conducting biological experiments in deep space, the 4U BioSensor science payload contained within the free-flyer is an adaptable instrument that can perform bio measurements with different microorganisms and in multiple space environments, including the ISS, lunar gateway, and on the surface of the Moon. Thus, nanosatellites like BioSentinel can be used to study the effects of both reduced gravity and space radiation and can house different organisms to answer specific science questions. In addition to their flexibility, nanosatellites also provide a low-cost alternative to more complex and larger missions, and require minimal crew support, if any.

biosenses↗

Beyond BioSentinel: Iterative Development of Automated Microfluidics

NASA Ames has flown a series of Bio-CubeSats that performed biology experiments supported by automated fluidic systems. Since Genesat-1 in 2006, these payloads have increased in complexity and functionality, building upon previous successes, and applying lessons learned. BioSentinel was the most recent of this series, launched into heliocentric orbit onboard Artemis-1 in 2022. This presentation will discuss how the fluidic technology developed for these Bio-CubeSat missions, including the multi-layer polycarbonate manifolds at the heart of the BioSentinel BioSensor, have spurred the development of several additional projects. Most directly is the modified BioSensor that will be a part of LEIA, which will perform its Lunar biology experiment onboard a Commercial Lunar Payload Services lander. Several search-for-life manifolds have been designed to prepare samples from icy moons for downstream analyses. Two early career Polaris projects are developing fluidics to perform genetic sequencing on samples from multigenerational cell culture and to extract and quantify target miRNAs to support astronaut radiation health assessment. Improving the readiness of these systems has been accelerated by adopting the established flight heritage and microgravity-compatibility of the Bio-CubeSat fluidic hardware and designs, while focusing development efforts on the integration of novel functionalities and components.

microfluidics↗