Design of activated serine-containing catalytic triads with atomic-level accuracy.

A challenge in the computational design of enzymes is that multiple properties, including substrate binding, transition state stabilization and product release, must be simultaneously optimized, and this has limited the absolute activity of successful designs. Here, we focus on a single critical property of many enzymes: the nucleophilicity of an active site residue that initiates catalysis. We design proteins with idealized serine-containing catalytic triads and assess their nucleophilicity directly in native biological systems using activity-based organophosphate probes. Crystal structures of the most successful designs show unprecedented agreement with computational models, including extensive hydrogen bonding networks between the catalytic triad (or quartet) residues, and mutagenesis experiments demonstrate that these networks are critical for serine activation and organophosphate reactivity. Following optimization by yeast display, the designs react with organophosphate probes at rates comparable to natural serine hydrolases. Co-crystal structures with diisopropyl fluorophosphate bound to the serine nucleophile suggest that the designs could provide the basis for a new class of organophosphate capture agents.

Molecular deconvolution of the monoclonal antibodies that comprise the polyclonal serum response.

We have developed and validated a methodology for determining the antibody composition of the polyclonal serum response after immunization. Pepsin-digested serum IgGs were subjected to standard antigen-affinity chromatography, and resulting elution, wash, and flow-through fractions were analyzed by bottom-up, liquid chromatography-high-resolution tandem mass spectrometry. Identification of individual monoclonal antibodies required the generation of a database of IgG variable gene (V-gene) sequences constructed by NextGen sequencing of mature B cells. Antibody V-gene sequences are characterized by short complementarity determining regions (CDRs) of high diversity adjacent to framework regions shared across thousands of IgGs, greatly complicating the identification of antigen-specific IgGs from proteomically observed peptides. By mapping peptides marking unique V(H) CDRH3 sequences, we identified a set of V-genes heavily enriched in the affinity chromatography elution, constituting the serum polyclonal response. After booster immunization in a rabbit, we find that the antigen-specific serum immune response is oligoclonal, comprising antibodies encoding 34 different CDRH3s that group into 30 distinct antibody V(H) clonotypes. Of these 34 CDRH3s, 12 account for ∼60% of the antigen-specific CDRH3 peptide mass spectral counts. For comparison, antibodies with 18 different CDRH3s (12 clonotypes) were represented in the antigen-specific IgG fraction from an unimmunized rabbit that fortuitously displayed a moderate titer for BSA. Proteomically identified antibodies were synthesized and shown to display subnanomolar affinities. The ability to deconvolute the polyclonal serum response is likely to be of key importance for analyzing antibody responses after vaccination and for more completely understanding adaptive immune responses in health and disease.

Calibrating canines-a universal detector calibrant for detection dogs.

Since the advent of the Universal Detector Calibrant (UDC) by scientists at Florida International University in 2013, this tool has gone largely unrecognized and under-utilized by canine scent detection practitioners. The UDC is a chemical that enables reliability testing of biological and instrumental detectors. Training a biological detector, such as a scent detection canine, to respond to a safe, non-target, and uncommon compound has significant advantages. For example, if used prior to a search, the UDC provides the handler with the ability to confirm the detection dog is ready to work without placing target odor on site (i.e., a positive control), thereby increasing handler confidence in their canine and providing documentation of credibility that can withstand legal scrutiny. This review describes the UDC, summarizes its role in canine detection science, and addresses applications for UDC within scent detection canine development, training, and testing.

Detection of Bile Acids in Complex Matrices Using a Transcription Factor-Based Biosensor.

Bile acids play an important role in digestion and human health, are found throughout the gastrointestinal tract, and are excreted in feces. Therefore, bile acids are promising biomarkers for monitoring health and detecting fecal contamination in water sources. Here, we engineered a bile acid sensor by expressing the transcription factor BreR, a TetR-like repressor from Vibrio cholorae, in Escherichia coli. The sensor was further optimized by screening a promoter library. To further characterize the BreR sensor and increase its utility, we moved expression to a cell-free expression (CFE) system, resulting in an approximately 3 orders of magnitude increase in deoxycholic acid sensitivity. We next optimized this sensor to detect bile acids in fecal water, wastewater, and serum and transferred the CFE sensor to a paper-based assay to enhance fieldability.

Cell-Free Protein Systems from Yersinia pestis are Functional and Growth-Temperature Dependent.

Cellular lysates capable of transcription and translation have become valuable tools for prototyping genetic circuits, screening engineered functional parts, and producing biological components. Here we report that lysates derived from Yersinia pestis CO92- are functional and can utilize both the E. coli σ70 and the bacteriophage T7 promoter systems to produce green fluorescent protein (GFP). Because of the natural lifestyle of Y. pestis, lysates were produced from cultures grown at 21 °C, 26 °C, and 37 °C to mimic the infection cycle. Regardless of the promoter system the GFP production from 37 °C was the most productive and the 26 °C lysate was the least. When reactions are initiated with 5 nM of DNA, the GFP output of the 37 °C lysate is comparable with the productivity of other non-E. coli systems. The data we present demonstrate that, without genetic modification to enhance productivity, cell-free extracts from Y. pestis are functional and dependent on the temperature at which the bacterium was grown.

Blind Spot: A Braille Patterned Novel Multiplex Lateral Flow Immunoassay Sensor Array for the Detection of Biothreat Agents.

Lateral flow immunoassays (LFIs) are simple, point-of-care diagnostic devices used for detecting biological agents or other analytes of interest in a sample. LFIs are predominantly singleplex assays, interrogating one target analyte at a time. There is a need for multiplex LFI devices, e.g., a syndromic panel to differentiate pathogens causing diseases exhibiting similar symptoms. Multiplex LFI devices would be especially valuable in instances where sample quantity is limiting and reducing assay time and costs is critical. There are limitations to the design parameters and performance characteristics of a multiplex LFI assay with many horizontal test lines due to constraints in capillary flow dynamics. To address some of the performance issues, we have developed a spot array multiplex LFI using Braille format (hence called Blind Spot) and a sensor, MACAW (Modular Automated Colorimetric Analyses Widget), that can analyze and interpret the results. As a proof of concept, we created a multiplex toxin panel, for detecting three toxins, using two letter codes for each. The results indicated that the six-plex, triple toxin assay performs as well as singleplex assays. The sensor-based calls are better compared to human interpretation in discriminating and interpreting ambiguous test results correctly especially at lower antigen concentrations and from strips with blemishes.

Impact of Porous Matrices and Concentration by Lyophilization on Cell-Free Expression.

Cell-free expression systems have drawn increasing attention as a tool to achieve complex biological functions outside of the cell. Several applications of the technology involve the delivery of functionality to challenging environments, such as field-forward diagnostics or point-of-need manufacturing of pharmaceuticals. To achieve these goals, cell-free reaction components are preserved using encapsulation or lyophilization methods, both of which often involve an embedding of components in porous matrices like paper or hydrogels. Previous work has shown a range of impacts of porous materials on cell-free expression reactions. Here, we explored a panel of 32 paperlike materials and 5 hydrogel materials for the impact on reaction performance. The screen included a tolerance to lyophilization for reaction systems based on both cell lysates and purified expression components. For paperlike materials, we found that (1) materials based on synthetic polymers were mostly incompatible with cell-free expression, (2) lysate-based reactions were largely insensitive to the matrix for cellulosic and microfiber materials, and (3) purified systems had an improved performance when lyophilized in cellulosic but not microfiber matrices. The impact of hydrogel materials ranged from completely inhibitory to a slight enhancement. The exploration of modulating the rehydration volume of lyophilized reactions yielded reaction speed increases using an enzymatic colorimetric reporter of up to twofold with an optimal ratio of 2:1 lyophilized reaction to rehydration volume for the lysate system and 1.5:1 for the purified system. The effect was independent of the matrices assessed. Testing with a fluorescent nonenzymatic reporter and no matrix showed similar improvements in both yields and reaction speeds for the lysate system and yields but not reaction speeds for the purified system. We finally used these observations to show an improved performance of two sensors that span reaction types, matrix, and reporters. In total, these results should enhance efforts to develop field-forward applications of cell-free expression systems.

Rapid Visual Authentication Based on DNA Strand Displacement.

Novel ways to track and verify items of a high value or security is an ever-present need. Taggants made from deoxyribonucleic acid (DNA) have several advantageous properties, such as high information density and robust synthesis; however, existing methods require laboratory techniques to verify, limiting applications. Here, we leverage DNA nanotechnology to create DNA taggants that can be validated in the field in seconds to minutes with a simple equipment. The system is driven by toehold-mediated strand-displacement reactions where matching oligonucleotide sequences drive the generation of a fluorescent signal through the potential energy of base pairing. By pooling different "input" oligonucleotide sequences in a taggant and spatially separating "reporter" oligonucleotide sequences on a paper ticket, unique, sequence-driven patterns emerge for different taggant formulations. Algorithmically generated oligonucleotide sequences show no crosstalk and ink-embedded taggants maintain activity for at least 99 days at 60 °C (equivalent to nearly 2 years at room temperature). The resulting fluorescent signals can be analyzed by the eye or a smartphone when paired with a UV flashlight and filtered glasses.

A bacterial membrane sculpting protein with BAR domain-like activity.

Bin/Amphiphysin/RVS (BAR) domain proteins belong to a superfamily of coiled-coil proteins influencing membrane curvature in eukaryotes and are associated with vesicle biogenesis, vesicle-mediated protein trafficking, and intracellular signaling. Here, we report a bacterial protein with BAR domain-like activity, BdpA, from Shewanella oneidensis MR-1, known to produce redox-active membrane vesicles and micrometer-scale outer membrane extensions (OMEs). BdpA is required for uniform size distribution of membrane vesicles and influences scaffolding of OMEs into a consistent diameter and curvature. Cryo-TEM reveals that a strain lacking BdpA produces lobed, disordered OMEs rather than membrane tubules or narrow chains produced by the wild-type strain. Overexpression of BdpA promotes OME formation during planktonic growth of S. oneidensis where they are not typically observed. Heterologous expression results in OME production in Marinobacter atlanticus and Escherichia coli. Based on the ability of BdpA to alter membrane architecture in vivo, we propose that BdpA and its homologs comprise a newly identified class of bacterial BAR domain-like proteins.

Methodologies for preparation of prokaryotic extracts for cell-free expression systems.

Cell-free systems that mimic essential cell functions, such as gene expression, have dramatically expanded in recent years, both in terms of applications and widespread adoption. Here we provide a review of cell-extract methods, with a specific focus on prokaryotic systems. Firstly, we describe the diversity of Escherichia coli genetic strains available and their corresponding utility. We then trace the history of cell-extract methodology over the past 20 years, showing key improvements that lower the entry level for new researchers. Next, we survey the rise of new prokaryotic cell-free systems, with associated methods, and the opportunities provided. Finally, we use this historical perspective to comment on the role of methodology improvements and highlight where further improvements may be possible.

Detection of Chemical Warfare Agents by Colorimetric Sensor Arrays.

We report the successful use of colorimetric arrays to identify chemical warfare agents (CWAs). Methods were developed to interpret and analyze a 73-indicator array with an entirely automated workflow. Using a cross-validated first-nearest-neighbor algorithm for assessing detection and identification performances on 632 exposures, at 30 min postexposure we report, on average, 78% correct chemical identification, 86% correct class-level identification, and 96% correct red light/green light (agent versus non-agent) detection. Of 174 total independent agent test exposures, 164 were correctly identified from a 30 min exposure in the red light/green light context, yielding a 94% correct identification of CWAs. Of 149 independent non-agent exposures, 139 were correctly identified at 30 min in the red light/green light context, yielding a 7% false alarm rate. We find that this is a promising approach for the development of a miniaturized, field-portable analytical equipment suitable for soldiers and first responders.

Computer-based Engineering of Thermostabilized Antibody Fragments.

We used the molecular modeling program Rosetta to identify clusters of amino acid substitutions in antibody fragments (scFvs and scAbs) that improve global protein stability and resistance to thermal deactivation. Using this methodology, we increased the melting temperature (Tm) and resistance to heat treatment of an antibody fragment that binds to the Clostridium botulinum hemagglutinin protein (anti-HA33). Two designed antibody fragment variants with two amino acid replacement clusters, designed to stabilize local regions, were shown to have both higher Tm compared to the parental scFv and importantly, to retain full antigen binding activity after 2 hours of incubation at 70 °C. The crystal structure of one thermostabilized scFv variants was solved at 1.6 Å and shown to be in close agreement with the RosettaAntibody model prediction.

Alternative computational protocols for supercharging protein surfaces for reversible unfolding and retention of stability.

Reengineering protein surfaces to exhibit high net charge, referred to as "supercharging", can improve reversibility of unfolding by preventing aggregation of partially unfolded states. Incorporation of charged side chains should be optimized while considering structural and energetic consequences, as numerous mutations and accumulation of like-charges can also destabilize the native state. A previously demonstrated approach deterministically mutates flexible polar residues (amino acids DERKNQ) with the fewest average neighboring atoms per side chain atom (AvNAPSA). Our approach uses Rosetta-based energy calculations to choose the surface mutations. Both protocols are available for use through the ROSIE web server. The automated Rosetta and AvNAPSA approaches for supercharging choose dissimilar mutations, raising an interesting division in surface charging strategy. Rosetta-supercharged variants of GFP (RscG) ranging from -11 to -61 and +7 to +58 were experimentally tested, and for comparison, we re-tested the previously developed AvNAPSA-supercharged variants of GFP (AscG) with +36 and -30 net charge. Mid-charge variants demonstrated ∼3-fold improvement in refolding with retention of stability. However, as we pushed to higher net charges, expression and soluble yield decreased, indicating that net charge or mutational load may be limiting factors. Interestingly, the two different approaches resulted in GFP variants with similar refolding properties. Our results show that there are multiple sets of residues that can be mutated to successfully supercharge a protein, and combining alternative supercharge protocols with experimental testing can be an effective approach for charge-based improvement to refolding.

Design and assembly of large synthetic DNA constructs.

The availability of custom synthetic gene-length DNA products removes numerous bottlenecks in research efforts, making gene synthesis an increasingly common commercial service. However, the assembly of synthetic oligonucleotides into large, custom DNA constructs is not especially difficult, and performing "in-house" gene synthesis has time and cost advantages. This unit will treat both the concerns of design and physical assembly in gene synthesis, including how to design DNA sequences for synthesis and the design of overlapping oligonucleotide schemes to ensure facile assembly into the final product. Assembly is accomplished using a reliable series of PCR reactions, with a troubleshooting assembly protocol included, which not only assembles difficult sequences but allows identification of the source of a failure down to a pair of oligonucleotides.

Enrichment of error-free synthetic DNA sequences by CEL I nuclease.

As the availability of DNA sequence information has grown, so has the need to replicate DNA sequences synthetically. Synthetically produced DNA sequences allow the researcher to exert greater control over model systems and allow for the combinatorial design and construction of novel metabolic and regulatory pathways, as well as optimized protein-coding sequences for biotechnological applications. This utility has made synthetically produced DNA a hallmark of the molecular biosciences and a mainstay of synthetic biology. However, synthetically produced DNA has a significant shortcoming in that it typically has an error rate that is orders of magnitude higher when compared to DNA sequences derived directly from a biological source. This relatively high error rate adds to the cost and labor necessary to obtain sequence-verified clones from synthetically produced DNA sequences. This unit describes a protocol to enrich error-free sequences from a population of error-rich DNA via treatment with CEL I (Surveyor) endonuclease. This method is a straightforward and quick way of reducing the error content of synthetic DNA pools and reliably reduces the error rates by >6-fold per round of treatment.

SCHEMA-designed variants of human Arginase I and II reveal sequence elements important to stability and catalysis.

Arginases catalyze the divalent cation-dependent hydrolysis of L-arginine to urea and L-ornithine. There is significant interest in using arginase as a therapeutic antineogenic agent against L-arginine auxotrophic tumors and in enzyme replacement therapy for treating hyperargininemia. Both therapeutic applications require enzymes with sufficient stability under physiological conditions. To explore sequence elements that contribute to arginase stability we used SCHEMA-guided recombination to design a library of chimeric enzymes composed of sequence fragments from the two human isozymes Arginase I and II. We then developed a novel active learning algorithm that selects sequences from this library that are both highly informative and functional. Using high-throughput gene synthesis and our two-step active learning algorithm, we were able to rapidly create a small but highly informative set of seven enzymatically active chimeras that had an average variant distance of 40 mutations from the closest parent arginase. Within this set of sequences, linear regression was used to identify the sequence elements that contribute to the long-term stability of human arginase under physiological conditions. This approach revealed a striking correlation between the isoelectric point and the long-term stability of the enzyme to deactivation under physiological conditions.

Structure-based design of supercharged, highly thermoresistant antibodies.

Mutation of surface residues to charged amino acids increases resistance to aggregation and can enable reversible unfolding. We have developed a protocol using the Rosetta computational design package that "supercharges" proteins while considering the energetic implications of each mutation. Using a homology model, a single-chain variable fragment antibody was designed that has a markedly enhanced resistance to thermal inactivation and displays an unanticipated ≈30-fold improvement in affinity. Such supercharged antibodies should prove useful for assays in resource-limited settings and for developing reagents with improved shelf lives.

Gene synthesis: methods and applications.

DNA synthesis techniques and technologies are quickly becoming a cornerstone of modern molecular biology and play a pivotal role in the field of synthetic biology. The ability to synthesize whole genes, novel genetic pathways, and even entire genomes is no longer the dream it was 30 years ago. Using little more than a thermocycler, commercially synthesized oligonucleotides, and DNA polymerases, a standard molecular biology laboratory can synthesize several kilobase pairs of synthetic DNA in a week using existing techniques. Herein, we review the techniques used in the generation of synthetic DNA, from the chemical synthesis of oligonucleotides to their assembly into long, custom sequences. Software and websites to facilitate the execution of these approaches are explored, and applications of DNA synthesis techniques to gene expression and synthetic biology are discussed. Finally, an example of automated gene synthesis from our own laboratory is provided.

Structure of the Escherichia coli phosphonate binding protein PhnD and rationally optimized phosphonate biosensors.

The phnD gene of Escherichia coli encodes the periplasmic binding protein of the phosphonate (Pn) uptake and utilization pathway. We have crystallized and determined structures of E. coli PhnD (EcPhnD) in the absence of ligand and in complex with the environmentally abundant 2-aminoethylphosphonate (2AEP). Similar to other bacterial periplasmic binding proteins, 2AEP binds near the center of mass of EcPhnD in a cleft formed between two lobes. Comparison of the open, unliganded structure with the closed 2AEP-bound structure shows that the two lobes pivot around a hinge by ~70° between the two states. Extensive hydrogen bonding and electrostatic interactions stabilize 2AEP, which binds to EcPhnD with low nanomolar affinity. These structures provide insight into Pn uptake by bacteria and facilitated the rational design of high signal-to-noise Pn biosensors based on both coupled small-molecule dyes and autocatalytic fluorescent proteins.

Monoclonal antibodies isolated without screening by analyzing the variable-gene repertoire of plasma cells.

Isolation of antigen-specific monoclonal antibodies (mAbs) and antibody fragments relies on high-throughput screening of immortalized B cells or recombinant antibody libraries. We bypassed the screening step by using high-throughput DNA sequencing and bioinformatic analysis to mine antibody variable region (V)-gene repertoires from bone marrow plasma cells (BMPC) of immunized mice. BMPCs, which cannot be immortalized, produce the vast majority of circulating antibodies. We found that the V-gene repertoire of BMPCs becomes highly polarized after immunization, with the most abundant sequences represented at frequencies between approximately 1% and >10% of the total repertoire. We paired the most abundant variable heavy (V(H)) and variable light (V(L)) genes based on their relative frequencies, reconstructed them using automated gene synthesis, and expressed recombinant antibodies in bacteria or mammalian cells. Antibodies generated in this manner from six mice, each immunized with one of three antigens were overwhelmingly antigen specific (21/27 or 78%). Those generated from a mouse with high serum titers had nanomolar binding affinities.