Tackling biological questions often requires a diverse set of capabilities to be brought together. One of Sandia’s key strengths is its ability to form and optimize multidisciplinary teams to carry out its mission. In integrating strengths in synthetic biology and gene-editing, infectious disease biology, computational biology and bioinformatics, microfluidics and microsystems and pre-treatment science and engineering, Sandia has created a differentiating environment for addressing challenging national security problems.
Synthetic Biology and Gene-Editing
Synthetic biology is used for a wide range of applications in bioenergy and biodefense. Applications include increasing the efficiency of algae cultivation and harvesting so that algae can become an economically viable biofuel source. Efforts are underway to develop synthetic pathways that that will allow biomass feedstocks to tolerate potential biorefinery conditions, such as extremes of temperature, and pH, as well as to design ideal enzymes through enzyme engineering — through methods such as directed evolution. Also, synthetic biology is used to introduce new synthetic pathways to ultimately allow the production of specialty chemicals and biopharmaceuticals from renewable biological “factories”.
We have been building extensive expertise in gene-editing through CRISPR-Cas systems. Gene editing is revolutionizing the bioscience and biomedical research landscape and holds great promise for “deleting” diseases from human bodies. In combination with nano-material delivery vehicles, this could be a powerful tool in the modern fight against a wide range of infectious diseases. Moreover, Sandia National Laboratories is also working to make this technology more secure and safer to ensure that one day it can be delivered into humans without triggering adverse immune system reactions or other undesirable side effects.
SNL’s core expertise in synthetic biology, process development and data science drive its strategy in biofuels and advanced bioproducts, in order to establish the viability of the BioEconomy. Our research focuses on enabling the entire value-chain by: (1) establishing platforms for accelerating process development via integrated biomanufacturing (2) aquatic and terrestrial biomass valorization to chemicals and fuels, and (3) advantaged bioproduct optimization. Our expertise in these areas is exemplified through the collaborative Agile BioFoundry (ABF) project.
As a key partner in the ABF, SNL leads advances in integrated biomanufacturing. ABF’s mission is to incorporate industrially–relevant production microbes, advanced tools for biological engineering and data analysis, and robust, scaled–up processes into a unified manufacturing approach.
CRISPR-Cas9 is a breakthrough technology used as a genome-editing tool to target and modify specific DNA sequences in different organisms. In addition to DNA, we discovered that certain Cas9 enzymes also target RNA, the ‘genetic middleman’ between DNA and proteins. This new capability for Cas9 enzymes opens the door for the development of novel RNA targeting therapies, viral countermeasures, cell and nucleic acid diagnostics, and gene regulation tools.
As part of the team’s capability in this area, new CRISPR tools and methods have been developed including CRISPR screens and an optical readout of gene-editing. Sandia hosted a CRISPR workshop in July 2018 to discuss the role of CRISPR against infectious disease, delivery vehicles, new CRISPR tools, and progress towards efficacy in animals.
Infectious Disease Biology
To respond to national security problems in infectious disease, we have developed extensive expertise understanding how a pathogen infects a host as well as how the host responds in turn to mount a defense to the infection. This is collectively known as pathogenesis and host-response.
Using a wide range of techniques and animal models in collaboration with Lawrence Livermore National Laboratory and other collaborative partners, Sandia has developed the knowledge base needed to mitigate the effects of infectious disease whether from naturally occurring outbreaks or bioterrorism.
The ultimate goal of our research is to assist in the formulation of new countermeasures and diagnostics for emerging infectious disease pathogens.
Host-pathogen interactions occur and can be studied at many different levels, from direct molecular interactions (e.g., binding of a pathogen’s toxin to a target protein in the host) to indirect effects of infection on the host (e.g., nutrient acquisition by the pathogen and its impact on host physiology).
The identification of cellular factors and pathways used by viruses and bacteria to replicate constitutes one of the core competencies of our group. This expertise is largely exploited for host-directed therapeutic target discovery and for the study of fundamental infection mechanisms linked to pathogenesis. Our research uses a broad spectrum of cellular, molecular biology, imaging and functional genomics techniques to identify and characterize novel host-pathogen interactions.
As an example, we have used genome-wide RNA interference screening to clarify the mechanism of infection for high-consequence pathogens such as Rift Valley fever virus (RVFV), an arbovirus of national biosecurity concern that can cause serious morbidity and mortality in humans and livestock.
Exploiting recent advances in genome editing technology, we now employ CRISPR-based functional genomics screening against intracellular bacteria (Burkholderia) and a wide range of viruses that include henipaviruses, bunyaviruses, arenaviruses, flaviviruses, alphaviruses, and filoviruses. Our CRISPR screening capabilities also involve the development of custom CRISPR guide RNA libraries for use with new CRISPR tools (e.g. RNA targeting Cas9 enzymes) or non-traditional cell lines. In collaboration with Lawrence Livermore National Laboratory (LLNL), we evaluate infections in animal models and BSL-3 select agent research facilities.
Sandia has developed extensive expertise in understanding the CRISPR based gene editing that is revolutionizing how we do many things and has incredible potential impact in treating or even curing infectious diseases, as well as cancer. We have special interest in emerging viruses and a focus on molecular viral-host interactions. Our research uses a broad spectrum of cellular, molecular biology, functional genomics, and nanoscience techniques to dissect host pathways involved in virus replication.
One of the major challenges associated with CRISPR based methods remains the ability to deliver viable constructs to where you want them without impacting other tissues in the host. One powerful way that Sandia is approaching this challenge is in the development of nanomaterial delivery vehicles. The integration of silica and other nanomaterials with biological molecules such as lipids and proteins holds immense promise for many applications, including ultra-small platforms for rapid delivery of drugs and vaccines. Using nature as a guide, Sandia researchers are developing a large variety of nanomaterial platforms for biological applications, including lipid-coated mesoporous silica nanoparticles (LC-MSNs) and virus-like particles (VLPs). LC-MSNs are porous nanoparticles protected by a coating similar to the membranes that surround live cells, with a protein that allows them to bind specifically with targeted cells. Virus-like particles (VLP) are non-infectious viral proteins that do not contain genetic material, yet mimic the surface coat of a natural virus. The protein expressions on the surface of VLPs generate immune responses similar to a virus playing a key role in many vaccine development and drug delivery strategies.
Developing nanoscale materials that can serve as targeted vehicles for advanced or even conventional therapeutics has great potential in the battle against infectious disease. Specific targeting moieties will enable the drugs to reach specific pathogens or tissues, potentially opening up a more extensive arsenal of antibiotics in the fight against multidrug resistant bacteria. Antibiotics that may be effective but are not used because of toxicity and side effects for the host might even become viable candidates since smaller—and therefore, less toxic—doses of drugs could be used if they can be sent exactly where they are needed.
Computational Biology and Bioinformatics
By applying our high performance computing capabilities to bioinformatics, molecular biophysics, biochemistry, modeling, and complex biological systems, Sandia is creating new computational biology tools and computing architectures. These novel tools drive experimental work by enabling researchers to describe, model, and predict the behavior of cells, networks, pathways, and molecules.
Sandia develops cutting-edge algorithms and simulation methods for deeper insight into many biological problems, such as CRISPR-Cas technology, next-generation sequencing, metabolic engineering, structural biology and enzyme engineering, and complex biological systems. These tools are being used in Sandia’s work to understand and combat emerging pathogens, recognize tool marks of genetic engineering, and enable biomass conversions.
We use state-of-the-art quantum chemistry calculations, ab initio molecular dynamics simulations, classical molecular dynamics simulations, and statistical mechanical free energy theory to identify and explain the mechanism, kinetics, and thermodynamics of chemical processes in biological systems.
Molecular simulations have high resolution in space and time, letting us observe dynamics over time in increments of one millionth to one billionth of a second and structure in hundredths of a nanometer. This allows us to see individual portions of a biological process that would be impossible to resolve in physical experiments and to determine which chemical structures contribute to the work involved. Experimental results provide data for validating and improving our theoretical approaches.
Information obtained from in silico studies may be used to learn how pathogens function, and that information can lead to new therapies to counter those pathogens. For example, at Sandia, we have used computation biology approaches to help understand biomolecular structure-dynamics-function relationships, membranes for selective transport, ion channels, enzyme-ligand binding and catalysis, solvation and transport theory.
Sandia has a robust bioinformatics research program. Our researchers develop new software pipelines for nucleic acid sequence analysis supporting genome editing detection and infectious disease research, including transcriptomics, genome and metagenome assembly and analysis, and a unique set of tools for characterizing the mobile segments of DNA that shape pathogen evolution. Mobile DNA analysis contributes to the renaissance in phage therapy in that these DNAs are often novel phage genomes. Data science approaches are applied to multi-‘omics datasets to understand microbial diversity or to predict phenotypes. Biomass conversion is supported with metabolic analysis and engineering, and structural and dynamic analysis of key enzymes.
Sandia develops bioinformatics tools and hosts specialized biological databases. For a broader picture of our bioinformatics research, explore our Bioinformatics website.
Microfluidics and Microsystems
The Sandia bioscience program leverages expertise in engineering and systems engineering built over more than 60 years to enable fully integrated devices serving both biodefense and biomass conversion needs. Our capabilities include Sandia’s state-of-the-art microfabrication facilities, Microsystems & Engineering Sciences Applications facility (MESA), on-site advanced manufacturing machine shops, and rapid prototyping laboratories.
Responding to the need to perform large numbers of biology experiments rapidly while using minimal amounts of biological sample, Sandia has developed microfluidic chips—tiny devices that take micro samples through the entire analysis process at high speed. Just as integrated microelectronic-chips revolutionized computing, microfluidic chips are transforming the field of bioanalysis—enabling rapid and economic research in new biological frontiers.
Sandia has an extensive background in harnessing the power of microfluidics to integrate and automate multiple bioprocesses, including mixing, dilution, concentration, transport, separation, and reaction, onto a single chip. Microfluidic assays can far outperform conventional methods in terms of speed, sample and regent consumption as well as efficiency. Sandia microfluidics research has enabled next-generation solutions in many areas: medical diagnostics, high throughput screening, single cell analysis, and synthetic biology, to name a few.
Sandia has an extensive repertoire of microfluidic chips and microcomponents. Devices with discrete functional elements such as valves, filter elements and frits, mixers and pumps as well as low-loss interconnects are just a few examples of the functional elements that have been designed and developed over the years.
We have developed designs that can move fluids and samples using voltage. This can be either as electrophoresis (separating molecules on the basis of their behavior in the electric field) or dielectrophoresis which has been used to demonstrate sorting of cell types in the presence of insulators that cause distortions of the electric field.
We are developing cutting-edge microfluidic chips and microcomponents for assembly of genes, screening of bacterial mutants for evolution of enzymes, high throughput enzyme and metabolite assays, and analysis of biomass constituents such as lignin. The research involves work with lab-on-a-chip techniques, microfabrication, cell culture, molecular biology, and adapting molecular biology assays to microfluidic format.
Droplet microfluidics is a class of methods that create and use discrete micron sized droplets as nano and sub-nanoliter reaction volumes. They can be created in large numbers very reproducibly and have utility in applications such as phage display or random library synthesis. In the image below droplets are generated in immiscible liquids (e.g. oil/water) to maintain separation from the others and to provide a means of moving them individually to where they will be used. Droplets of this design are typically created and moved using pressure driven flow.
Another example of droplet microfluidics – combined with another technique known as digital microfluidics– is shown in the video below with discrete droplets being generated and used not in an immiscible fluid but in air. In this case, droplets are moved by individually addressing interdigitated electrodes in sequence.
The video shows how individual drops can be manipulated, examined, moved and parked for later removal from the device using a capillary interface.
We have used our extensive systems engineering expertise to incorporate our microfluidic technologies into integrated, fieldable and often automated technologies for a variety of applications from environmental monitoring to point of care diagnostics.
Recent examples of technology include: hand portable rapid multiplexed immunoassay technology which typically does not require separate sample preparation (SpinDx); BaDx, a device that can confirm overnight the presence of B. anthracis in a device the size of a small stack of credit cards; and SMART Lamp which can rapidly detect a range of viral pathogens, including Zika, chikungunya, and dengue viruses, in a device controlled using a consumer smart phone.
Sandia has extensive expertise developing Bio and Chemical Sensors using alternative modalities and fabrication methods to the microfluidics work. Chemical detection based on our micro gas chromatography (micro GC) portfolio, developed over the past 20 years for high-performance, low false alarm field detection of national/homeland security threats has found additional utility in the biodetection and biosurveillance domains.
We have demonstrated one-dimensional microGC or two-dimensional high-speed microGC (over 75 compounds/sec), depending on the application. We use detectors that miniaturize well without compromises.Though the technologies were largely developed and demonstrated for detection of chemical warfare agents (such as G, V and blister agents) as well as toxic industrial chemicals for homeland security and military applications, these technologies have found utility in biological applications as well.
Wearable sensors for human performance have also been developed. Through a Cooperative Research and Development Agreement or CRADA, Sandia has developed in-mask sensors for monitoring the breath of pilots and divers for volatile signatures of health. Separate DTRA funded work was geared toward performance and health monitoring of warfighters. This work relies on our system approach of micro PC, microGCxGC and sensitive/miniature detectors (CIMS and PDID) to make high-performance breath analyzers.
Pain-free microneedles, with integrated sensors, will allow sampling of interstitial fluids in the skin as a facile clinical sample type and for the collection and analysis of human biomarkers. Electrochemical detection systems and arrays are integrated with microfluidics, electronics and microneedles. Electrochemical array sensors have also been integrated with microvalves, optical detection units, and electronics to create multiplexed detection systems.
Biological detection has been achieved by a variety of portable methods from acoustic-based assays, to volatile organic compounds, to electrochemical assays in non-invasive, or minimally-invasive formats.
Biological VOC (Volatile Organic Compounds) detection has been demonstrated for non-contact, non-invasive identification of bacterial pathogens, human biomarkers (e.g., from breath or skin), biomarkers from agricultural or food products, and more. This is based on the fact that biological systems produce unique VOCs. We use our miniature chemical vapor sensing platform with the same microGC core as mentioned above.
An extension of this work has been developed to identify biogenic VOC signatures of algae ponds to determine state-of-health measures and developing portable detection systems for early detection of pond crash. This ties in with our emphasis on using microGCxGC systems for field detection of biogenic volatiles.
Acoustic devices and sensors were developed for efficient cell lysis and low-resource biological sensing. Sandia’s credit-card-sized CleanBurst technology cracks open cells and spores at room temperature without reagents, drastically increasing available genetic and proteomic information relative to competing methods. Sandia’s SonoSense, also credit-card sized, can detect picogram to femtogram quantities of bacteria and viruses. Explore some of our microsystem technologies, such as those that are described above, that are available for licensing.
Pretreatment Science and Engineering
Sandia is developing the science and technology foundation to enable atom-economical lignocellulose conversion into fuels and bioproducts.
Current pretreatment and enzymatic approaches to biomass deconstruction remain challenging, as they are inefficient and expensive. What is needed is a sustainable, scalable, and affordable biomass deconstruction process.
SNL discovers, optimizes, and improves the process economics of ionic liquid biomass pretreatment systems; discovers, optimizes and improves the process economics of microbial and enzyme biomass pretreatment and lignocellulose–to–chemicals–and–fuels conversion systems; uses genetic engineering and bioinformatics tools to increase the efficacy of fungal biomass pretreatment biotechnology.
Sandia is developing an effective pretreatment that is affordable, scalable, and:
- Generates high yields of fermentable sugars
- Enables lignin valorization
- Minimizes downstream inhibition of enzymes and microbes needed for the fermentation processes
It has been discovered that certain ionic liquids (IL) —paired-ions salts that are liquid at room temperature —show great promise for liberating fermentable sugars from lignocellulose and improving advanced-biofuel economics.These have been shown to be able to substantially alter plant cell walls and have proved an effective way of delignifying biomass, even at relatively high biomass loading (e.g., 50wt%) and larger particle (>2cm) sizes (meaning extensive grinding or maceration of the biomass before pretreatment is not required).
- ILs generate the highest glucose yields
- ILs can handle an unparalleled range of biomass feedstocks
- ILs can effectively pretreat at high solids loading with large particle sizes
As promising as the results with ILs are, some challenges remain. Many ILs do still inhibit enzymes needed for downstream fermentation steps. A parallel effort is geared toward discovering more robust and efficient enzymes and microbes for pretreatment.
In addition, the current “standard,” imidazolium-based ionic liquids, effectively and efficiently deconstruct biomass into fuel sugars, but are expensive, and thus limited in industrial deployment. They are also made from nonrenewable sources such as petroleum or natural gas. “Bionic liquids” derived from lignin and hemicellulose show great promise for liberating fermentable sugars from lignocellulose.