121 results on '"Scott A, Walper"'
Search Results
2. Self assembling nanoparticle enzyme clusters provide access to substrate channeling in multienzymatic cascades
- Author
-
Joyce C. Breger, James N. Vranish, Eunkeu Oh, Michael H. Stewart, Kimihiro Susumu, Guillermo Lasarte-Aragonés, Gregory A. Ellis, Scott A. Walper, Sebastián A. Díaz, Shelby L. Hooe, William P. Klein, Meghna Thakur, Mario G. Ancona, and Igor L. Medintz
- Subjects
Science - Abstract
Channeling between enzymes is a uniquely nanoscale phenomenon that can improve multienzymatic reaction rates. Here, the authors demonstrate that multistep enzyme cascades can self-assemble with nanoparticles into nanoclusters that access channeling and improve the underlying catalytic flux by several fold.
- Published
- 2023
- Full Text
- View/download PDF
3. Bacterial Membrane Vesicles for In Vitro Catalysis
- Author
-
Meghna Thakur, Scott N. Dean, Julie C. Caruana, Scott A. Walper, and Gregory A. Ellis
- Subjects
outer membrane vesicles (OMVs) ,biocatalysis ,biomanufacturing ,Technology ,Biology (General) ,QH301-705.5 - Abstract
The use of biological systems in manufacturing and medical applications has seen a dramatic rise in recent years as scientists and engineers have gained a greater understanding of both the strengths and limitations of biological systems. Biomanufacturing, or the use of biology for the production of biomolecules, chemical precursors, and others, is one particular area on the rise as enzymatic systems have been shown to be highly advantageous in limiting the need for harsh chemical processes and the formation of toxic products. Unfortunately, biological production of some products can be limited due to their toxic nature or reduced reaction efficiency due to competing metabolic pathways. In nature, microbes often secrete enzymes directly into the environment or encapsulate them within membrane vesicles to allow catalysis to occur outside the cell for the purpose of environmental conditioning, nutrient acquisition, or community interactions. Of particular interest to biotechnology applications, researchers have shown that membrane vesicle encapsulation often confers improved stability, solvent tolerance, and other benefits that are highly conducive to industrial manufacturing practices. While still an emerging field, this review will provide an introduction to biocatalysis and bacterial membrane vesicles, highlight the use of vesicles in catalytic processes in nature, describe successes of engineering vesicle/enzyme systems for biocatalysis, and end with a perspective on future directions, using selected examples to illustrate these systems’ potential as an enabling tool for biotechnology and biomanufacturing.
- Published
- 2023
- Full Text
- View/download PDF
4. Enzymatic Laser‐Induced Graphene Biosensor for Electrochemical Sensing of the Herbicide Glyphosate
- Author
-
Zachary T. Johnson, Nathan Jared, John K. Peterson, Jingzhe Li, Emily A. Smith, Scott A. Walper, Shelby L. Hooe, Joyce C. Breger, Igor L. Medintz, Carmen Gomes, and Jonathan C. Claussen
- Subjects
biosensors ,glycine oxidase ,glyphosate ,herbicides ,laser‐induced graphene ,Technology ,Environmental sciences ,GE1-350 - Abstract
Abstract Glyphosate is a globally applied herbicide yet it has been relatively undetectable in‐field samples outside of gold‐standard techniques. Its presumed nontoxicity toward humans has been contested by the International Agency for Research on Cancer, while it has been detected in farmers’ urine, surface waters and crop residues. Rapid, on‐site detection of glyphosate is hindered by lack of field‐deployable and easy‐to‐use sensors that circumvent sample transportation to limited laboratories that possess the equipment needed for detection. Herein, the flavoenzyme, glycine oxidase, immobilized on platinum‐decorated laser‐induced graphene (LIG) is used for selective detection of glyphosate as it is a substrate for GlyOx. The LIG platform provides a scaffold for enzyme attachment while maintaining the electronic and surface properties of graphene. The sensor exhibits a linear range of 10–260 µm, detection limit of 3.03 µm, and sensitivity of 0.991 nA µm−1. The sensor shows minimal interference from the commonly used herbicides and insecticides: atrazine, 2,4‐dichlorophenoxyacetic acid, dicamba, parathion‐methyl, paraoxon‐methyl, malathion, chlorpyrifos, thiamethoxam, clothianidin, and imidacloprid. Sensor function is further tested in complex river water and crop residue fluids, which validate this platform as a scalable, direct‐write, and selective method of glyphosate detection for herbicide mapping and food analysis.
- Published
- 2022
- Full Text
- View/download PDF
5. Different Strategies Affect Enzyme Packaging into Bacterial Outer Membrane Vesicles
- Author
-
Scott N. Dean, Meghna Thakur, Joseph R. Spangler, Aaron D. Smith, Sean P. Garin, Scott A. Walper, and Gregory A. Ellis
- Subjects
outer membrane vesicles (OMVs) ,phosphotriesterase (PTE) ,diisopropyl fluorophosphatase (DFPase) ,Technology ,Biology (General) ,QH301-705.5 - Abstract
All Gram-negative bacteria are believed to produce outer membrane vesicles (OMVs), proteoliposomes shed from the outermost membrane. We previously separately engineered E. coli to produce and package two organophosphate (OP) hydrolyzing enzymes, phosphotriesterase (PTE) and diisopropylfluorophosphatase (DFPase), into secreted OMVs. From this work, we realized a need to thoroughly compare multiple packaging strategies to elicit design rules for this process, focused on (1) membrane anchors or periplasm-directing proteins (herein “anchors/directors”) and (2) the linkers connecting these to the cargo enzyme; both may affect enzyme cargo activity. Herein, we assessed six anchors/directors to load PTE and DFPase into OMVs: four membrane anchors, namely, lipopeptide Lpp’, SlyB, SLP, and OmpA, and two periplasm-directing proteins, namely, maltose-binding protein (MBP) and BtuF. To test the effect of linker length and rigidity, four different linkers were compared using the anchor Lpp’. Our results showed that PTE and DFPase were packaged with most anchors/directors to different degrees. For the Lpp’ anchor, increased packaging and activity corresponded to increased linker length. Our findings demonstrate that the selection of anchors/directors and linkers can greatly influence the packaging and bioactivity of enzymes loaded into OMVs, and these findings have the potential to be utilized for packaging other enzymes into OMVs.
- Published
- 2023
- Full Text
- View/download PDF
6. Variational Autoencoder for Generation of Antimicrobial Peptides
- Author
-
Scott N. Dean and Scott A. Walper
- Subjects
Chemistry ,QD1-999 - Published
- 2020
- Full Text
- View/download PDF
7. Self-assembled nanoparticle-enzyme aggregates enhance functional protein production in pure transcription-translation systems
- Author
-
Meghna Thakur, Joyce C. Breger, Kimihiro Susumu, Eunkeu Oh, Joseph R. Spangler, Igor L. Medintz, Scott A. Walper, and Gregory A. Ellis
- Subjects
Medicine ,Science - Abstract
Cell-free protein synthesis systems (CFPS) utilize cellular transcription and translation (TX-TL) machinery to synthesize proteins in vitro. These systems are useful for multiple applications including production of difficult proteins, as high-throughput tools for genetic circuit screening, and as systems for biosensor development. Though rapidly evolving, CFPS suffer from some disadvantages such as limited reaction rates due to longer diffusion times, significant cost per assay when using commercially sourced materials, and reduced reagent stability over prolonged periods. To address some of these challenges, we conducted a series of proof-of-concept experiments to demonstrate enhancement of CFPS productivity via nanoparticle assembly driven nanoaggregation of its constituent proteins. We combined a commercially available CFPS that utilizes purified polyhistidine-tagged (His-tag) TX-TL machinery with CdSe/CdS/ZnS core/shell/shell quantum dots (QDs) known to readily coordinate His-tagged proteins in an oriented fashion. We show that nanoparticle scaffolding of the CFPS cross-links the QDs into nanoaggregate structures while enhancing the production of functional recombinant super-folder green fluorescent protein and phosphotriesterase, an organophosphate hydrolase; the latter by up to 12-fold. This enhancement, which occurs by an undetermined mechanism, has the potential to improve CFPS in general and specifically CFPS-based biosensors (faster response time) while also enabling rapid detoxification/bioremediation through point-of-concern synthesis of similar catalytic enzymes. We further show that such nanoaggregates improve production in diluted CFPS reactions, which can help to save money and extend the amount of these costly reagents. The results are discussed in the context of what may contribute mechanistically to the enhancement and how this can be applied to other CFPS application scenarios.
- Published
- 2022
8. PepVAE: Variational Autoencoder Framework for Antimicrobial Peptide Generation and Activity Prediction
- Author
-
Scott N. Dean, Jerome Anthony E. Alvarez, Dan Zabetakis, Scott A. Walper, and Anthony P. Malanoski
- Subjects
antimicrobial peptides ,minimum inhibitory concentration ,generative deep learning ,activity prediction ,variational autoencoder ,Microbiology ,QR1-502 - Abstract
New methods for antimicrobial design are critical for combating pathogenic bacteria in the post-antibiotic era. Fortunately, competition within complex communities has led to the natural evolution of antimicrobial peptide (AMP) sequences that have promising bactericidal properties. Unfortunately, the identification, characterization, and production of AMPs can prove complex and time consuming. Here, we report a peptide generation framework, PepVAE, based around variational autoencoder (VAE) and antimicrobial activity prediction models for designing novel AMPs using only sequences and experimental minimum inhibitory concentration (MIC) data as input. Sampling from distinct regions of the learned latent space allows for controllable generation of new AMP sequences with minimal input parameters. Extensive analysis of the PepVAE-generated sequences paired with antimicrobial activity prediction models supports this modular design framework as a promising system for development of novel AMPs, demonstrating controlled production of AMPs with experimental validation of predicted antimicrobial activity.
- Published
- 2021
- Full Text
- View/download PDF
9. Harnessing the potential of Lactobacillus species for therapeutic delivery at the lumenal-mucosal interface
- Author
-
Joseph R Spangler, Julie C Caruana, Igor L Medintz, and Scott A Walper
- Subjects
cytokine stimulation ,delivery vehicle ,immunomodulation ,Lactobacillus ,microbiome ,probiotic ,Medicine ,Medicine (General) ,R5-920 - Abstract
Lactobacillus species have been studied for over 30 years in their role as commensal organisms in the human gut. Recently there has been a surge of interest in their abilities to natively and recombinantly stimulate immune activities, and studies have identified strains and novel molecules that convey particular advantages for applications as both immune adjuvants and immunomodulators. In this review, we discuss the recent advances in Lactobacillus-related activity at the gut/microbiota interface, the efforts to probe the boundaries of the direct and indirect therapeutic potential of these bacteria, and highlight the continued interest in harnessing the native capacity for the production of biogenic compounds shown to influence nervous system activity. Taken together, these aspects underscore Lactobacillus species as versatile therapeutic delivery vehicles capable of effector production at the lumenal-mucosal interface, and further establish a foundation of efficacy upon which future engineered strains can expand.
- Published
- 2021
- Full Text
- View/download PDF
10. Lactobacillus acidophilus Membrane Vesicles as a Vehicle of Bacteriocin Delivery
- Author
-
Scott N. Dean, Mary Ashley Rimmer, Kendrick B. Turner, Daniel A. Phillips, Julie C. Caruana, William Judson Hervey, Dagmar H. Leary, and Scott A. Walper
- Subjects
membrane vesicles ,bacteriocins ,lactic acid bacteria ,Lactobacillus acidophilus ,antimicrobial ,Microbiology ,QR1-502 - Abstract
Recent reports have shown that Gram-positive bacteria actively secrete spherical nanometer-sized proteoliposome membrane vesicles (MVs) into their surroundings. Though MVs are implicated in a broad range of biological functions, few studies have been conducted to examine their potential as delivery vehicles of antimicrobials. Here, we investigate the natural ability of Lactobacillus acidophilus MVs to carry and deliver bacteriocin peptides to the opportunistic pathogen, Lactobacillus delbrueckii. We demonstrate that upon treatment with lactacin B-inducing peptide, the proteome of the secreted MVs is enriched in putative bacteriocins encoded by the lab operon. Further, we show that purified MVs inhibit growth and compromise membrane integrity in L. delbrueckii, which is confirmed by confocal microscopy imaging and spectrophotometry. These results show that L. acidophilus MVs serve as conduits for antimicrobials to competing cells in the environment, suggesting a potential role for MVs in complex communities such as the gut microbiome. With the potential for controlling their payload through microbial engineering, MVs produced by L. acidophilus may be an interesting platform for effecting change in complex microbial communities or aiding in the development of new biomedical therapeutics.
- Published
- 2020
- Full Text
- View/download PDF
11. Bacterial Membrane Vesicles as Mediators of Microbe – Microbe and Microbe – Host Community Interactions
- Author
-
Julie C. Caruana and Scott A. Walper
- Subjects
outer membrane vesicles ,membrane vesicles ,community interactions ,host-microbe interactions ,host-pathogen interactions ,cell delivery ,Microbiology ,QR1-502 - Abstract
Bacterial membrane vesicles are proteoliposomal nanoparticles produced by both Gram-negative and Gram-positive bacteria. As they originate from the outer surface of the bacteria, their composition and content is generally similar to the parent bacterium’s membrane and cytoplasm. However, there is ample evidence that preferential packaging of proteins, metabolites, and toxins into vesicles does occur. Incorporation into vesicles imparts a number of benefits to the cargo, including protection from degradation by other bacteria, the host organism, or environmental factors, maintenance of a favorable microenvironment for enzymatic activity, and increased potential for long-distance movement. This enables vesicles to serve specialized functions tailored to changing or challenging environments, particularly in regard to microbial community interactions including quorum sensing, biofilm formation, antibiotic resistance, antimicrobial peptide expression and deployment, and nutrient acquisition. Additionally, based on their contents, vesicles play crucial roles in host-microbe interactions as carriers of virulence factors and other modulators of host cell function. Here, we discuss recent advances in our understanding of how vesicles function as signals both within microbial communities and between pathogenic or commensal microbes and their mammalian hosts. We also highlight a few areas that are currently ripe for additional research, including the mechanisms of selective cargo packaging into membrane vesicles and of cargo processing once it enters mammalian host cells, the function of vesicles in transfer of nucleic acids among bacteria, and the possibility of engineering commensal bacteria to deliver cargo of interest to mammalian hosts in a controlled manner.
- Published
- 2020
- Full Text
- View/download PDF
12. Enzymatic Bioremediation of Organophosphate Compounds—Progress and Remaining Challenges
- Author
-
Meghna Thakur, Igor L. Medintz, and Scott A. Walper
- Subjects
organophosphate ,enzyme ,chemical warfare agent ,bioremediation ,catalysis ,outer membrane vesicle ,Biotechnology ,TP248.13-248.65 - Abstract
Organophosphate compounds are ubiquitously employed as agricultural pesticides and maintained as chemical warfare agents by several nations. These compounds are highly toxic, show environmental persistence and accumulation, and contribute to numerous cases of poisoning and death each year. While their use as weapons of mass destruction is rare, these never fully disappear into obscurity as they continue to be tools of fear and control by governments and terrorist organizations. Beyond weaponization, their wide-scale dissemination as agricultural products has led to environmental accumulation and intoxication of soil and water across the globe. Therefore, there is a dire need for rapid and safe agents for environmental bioremediation, personal decontamination, and as therapeutic detoxicants. Organophosphate hydrolyzing enzymes are emerging as appealing targets to satisfy decontamination needs owing to their ability to hydrolyze both pesticides and nerve agents using biologically-derived materials safe for both the environment and the individual. As the release of genetically modified organisms is not widely accepted practice, researchers are exploring alternative strategies of organophosphate bioremediation that focus on cell-free enzyme systems. In this review, we first discuss several of the more prevalent organophosphorus hydrolyzing enzymes along with research and engineering efforts that have led to an enhancement in their activity, substrate tolerance, and stability. In the later half we focus on advances achieved through research focusing on enhancing the catalytic activity and stability of phosphotriesterase, a model organophosphate hydrolase, using various approaches such as nanoparticle display, DNA scaffolding, and outer membrane vesicle encapsulation.
- Published
- 2019
- Full Text
- View/download PDF
13. Response of Lactobacillus plantarum WCFS1 to the Gram-Negative Pathogen-Associated Quorum Sensing Molecule N-3-Oxododecanoyl Homoserine Lactone
- Author
-
Joseph R. Spangler, Scott N. Dean, Dagmar H. Leary, and Scott A. Walper
- Subjects
Lactobacillus plantarum ,quorum sense ,Pseudomonas aeruginosa ,transcriptomics ,proteomics ,homoserine lactone ,Microbiology ,QR1-502 - Abstract
The bacterial quorum sensing phenomenon has been well studied since its discovery and has traditionally been considered to include signaling pathways recognized exclusively within either Gram-positive or Gram-negative bacteria. These groups of bacteria synthesize structurally distinct signaling molecules to mediate quorum sensing, where Gram-positive bacteria traditionally utilize small autoinducing peptides (AIPs) and Gram-negatives use small molecules such as acyl-homoserine lactones (AHLs). The structural differences between the types of signaling molecules have historically implied a lack of cross-talk among Gram-positive and Gram-negative quorum sensing systems. Recent investigations, however, have demonstrated the ability for AIPs and AHLs to be produced by non-canonical organisms, implying quorum sensing systems may be more universally recognized than previously hypothesized. With that in mind, our interests were piqued by the organisms Lactobacillus plantarum, a Gram-positive commensal probiotic known to participate in AIP-mediated quorum sensing, and Pseudomonas aeruginosa, a characterized Gram-negative pathogen whose virulence is in part controlled by AHL-mediated quorum sensing. Both health-related organisms are known to inhabit the human gut in various instances, both are characterized to elicit distinct effects on host immunity, and some studies hint at the putative ability of L. plantarum to degrade AHLs produced by P. aeruginosa. We therefore wanted to determine if L. plantarum cultures would respond to the addition of N-(3-oxododecanoyl)-L-homoserine lactone (3OC12) from P. aeruginosa by analyzing changes on both the transcriptome and proteome over time. Based on the observed upregulation of various two-component systems, response regulators, and native quorum sensing related genes, the resulting data provide evidence of an AHL recognition and response by L. plantarum.
- Published
- 2019
- Full Text
- View/download PDF
14. Microbial Nanocellulose Printed Circuit Boards for Medical Sensing.
- Author
-
Jonathan D. Yuen, Lisa C. Shriver-Lake, Scott A. Walper, Daniel Zabetakis, Joyce C. Breger, and David A. Stenger
- Published
- 2020
- Full Text
- View/download PDF
15. Conjugation of biotin-coated luminescent quantum dots with single domain antibody-rhizavidin fusions
- Author
-
Jinny L. Liu, Scott A. Walper, Kendrick B. Turner, Audrey Brozozog Lee, Igor L. Medintz, Kimihiro Susumu, Eunkeu Oh, Dan Zabetakis, Ellen R. Goldman, and George P. Anderson
- Subjects
Single domain antibodies ,Rhizavidin ,Quantum dots ,Surface plasmon resonance ,Biotechnology ,TP248.13-248.65 - Abstract
Straightforward and effective methods are required for the bioconjugation of proteins to surfaces and particles. Previously we demonstrated that the fusion of a single domain antibody with the biotin binding molecule rhizavidin provided a facile method to coat biotin-modified surfaces with a highly active and oriented antibody. Here, we constructed similar single domain antibody—rhizavidin fusions as well as unfused rhizavidin with a His-tag. The unfused rhizavidin produced efficiently and its utility for assay development was demonstrated in surface plasmon resonance experiments. The single domain antibody-rhizavidin fusions were utilized to coat quantum dots that had been prepared with surface biotins. Preparation of antibody coated quantum dots by this means was found to be both easy and effective. The prepared single domain antibody-quantum dot reagent was characterized by surface plasmon resonance and applied to toxin detection in a fluoroimmunoassay sensing format.
- Published
- 2016
- Full Text
- View/download PDF
16. Enzymatic bioconjugation to nanoparticles
- Author
-
Scott A. Walper, Igor L. Medintz, and Aaron D. Smith
- Subjects
chemistry.chemical_classification ,Enzyme ,Bioconjugation ,chemistry ,Nanoparticle ,Combinatorial chemistry - Published
- 2023
17. Exploring the performance of Escherichia coli outer membrane vesicles as a tool for vaccine development against Chagas disease
- Author
-
María Elisa Vázquez, Andrea Cecilia Mesías, Leonardo Acuña, Joseph Spangler, Brenda Zabala, Cecilia Parodi, Meghna Thakur, Eunkeu Oh, Scott Allan Walper, and Cecilia Pérez Brandán
- Subjects
Microbiology (medical) ,Trypanosoma cruzi ,vaccine ,outer membrane vesicle - Abstract
BACKGROUND Vaccine development is a laborious craftwork in which at least two main components must be defined: a highly immunogenic antigen and a suitable delivery method. Hence, the interplay of these elements could elicit the required immune response to cope with the targeted pathogen with a long-lasting protective capacity. OBJECTIVES Here we evaluate the properties of Escherichia coli spherical proteoliposomes - known as outer membrane vesicles (OMVs) - as particles with natural adjuvant capacities and as antigen-carrier structures to assemble an innovative prophylactic vaccine for Chagas disease. METHODS To achieve this, genetic manipulation was carried out on E. coli using an engineered plasmid containing the Tc24 Trypanosoma cruzi antigen. The goal was to induce the release of OMVs displaying the parasite protein on their surface. FINDINGS As a proof of principle, we observed that native OMVs - as well as those carrying the T. cruzi antigen - were able to trigger a slight, but functional humoral response at low immunization doses. Of note, compared to the non-immunized group, native OMVs-vaccinated animals survived the lethal challenge and showed minor parasitemia values, suggesting a possible involvement of innate trained immunity mechanism. MAIN CONCLUSION These results open the range for further research on the design of new carrier strategies focused on innate immunity activation as an additional immunization target and venture to seek for alternative forms in which OMVs could be used for optimizing vaccine development.
- Published
- 2023
18. Modulation of Intracellular Quantum Dot to Fluorescent Protein Förster Resonance Energy Transfer via Customized Ligands and Spatial Control of Donor–Acceptor Assembly
- Author
-
Lauren D. Field, Scott A. Walper, Kimihiro Susumu, Eunkeu Oh, Igor L. Medintz, and James B. Delehanty
- Subjects
fluorescence ,sensor ,FRET ,quantum dot ,protein ,membrane ,assembly ,noncovalent ,ligand ,Chemical technology ,TP1-1185 - Abstract
Understanding how to controllably modulate the efficiency of energy transfer in Förster resonance energy transfer (FRET)-based assemblies is critical to their implementation as sensing modalities. This is particularly true for sensing assemblies that are to be used as the basis for real time intracellular sensing of intracellular processes and events. We use a quantum dot (QD) donor -mCherry acceptor platform that is engineered to self-assemble in situ wherein the protein acceptor is expressed via transient transfection and the QD donor is microinjected into the cell. QD-protein assembly is driven by metal-affinity interactions where a terminal polyhistidine tag on the protein binds to the QD surface. Using this system, we show the ability to modulate the efficiency of the donor–acceptor energy transfer process by controllably altering either the ligand coating on the QD surface or the precise location where the QD-protein assembly process occurs. Intracellularly, a short, zwitterionic ligand mediates more efficient FRET relative to longer ligand species that are based on the solubilizing polymer, poly(ethylene glycol). We further show that a greater FRET efficiency is achieved when the QD-protein assembly occurs free in the cytosol compared to when the mCherry acceptor is expressed tethered to the inner leaflet of the plasma membrane. In the latter case, the lower FRET efficiency is likely attributable to a lower expression level of the mCherry acceptor at the membrane combined with steric hindrance. Our work points to some of the design considerations that one must be mindful of when developing FRET-based sensing schemes for use in intracellular sensing.
- Published
- 2015
- Full Text
- View/download PDF
19. Hybrid Nucleic Acid-Quantum Dot Assemblies as Multiplexed Reporter Platforms for Cell-Free Transcription Translation-Based Biosensors
- Author
-
Divita Mathur, Meghna Thakur, Sebastián A. Díaz, Kimihiro Susumu, Michael H. Stewart, Eunkeu Oh, Scott A. Walper, and Igor L. Medintz
- Subjects
Biomedical Engineering ,General Medicine ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Article - Abstract
Cell-free synthetic biology has emerged as a valuable tool for the development of rapid, portable biosensors that can be readily transported in the freeze-dried form to the point of need eliminating cold chain requirements. One of the challenges associated with cell-free sensors is the ability to simultaneously detect multiple analytes within a single reaction due to the availability of a limited set of fluorescent and colorimetric reporters. To potentially provide multiplexing capabilities to cell-free biosensors, we designed a modular semiconductor quantum dot (QD)-based reporter platform that is plugged in downstream of the transcription-translation functionality in the cell-free reaction and which converts enzymatic activity in the reaction into distinct optical signals. We demonstrate proof of concept by converting restriction enzyme activity, utilized as our prototypical sensing output, into optical changes across several distinct spectral output channels that all use a common excitation wavelength. These hybrid Förster resonance energy transfer (FRET)-based QD peptide PNA-DNA-Dye reporters (QD-PDDs) are completely self-assembled and consist of differentially emissive QD donors paired to a dye-acceptor displayed on a unique DNA encoding a given enzyme’s cleavage site. Three QD-based PDDs, independently activated by the enzymes BamHI, EcoRI, and NcoI, were prototyped in mixed enzyme assays where all three demonstrated the ability to convert enzymatic activity into fluorescent output. Simultaneous monitoring of each of the three paired QD-donor dye-acceptor spectral channels in cell-free biosensing reactions supplemented with added linear genes encoding each enzyme confirmed robust multiplexing capabilities for at least two enzymes when co-expressed. The modular QD-PDDs are easily adapted to respond to other restriction enzymes or even proteases if desired.
- Published
- 2022
20. Quantum Dots and Gold Nanoparticles as Scaffolds for Enzymatic Enhancement: Recent Advances and the Influence of Nanoparticle Size
- Author
-
Gregory A. Ellis, Scott N. Dean, Scott A. Walper, and Igor L. Medintz
- Subjects
enzyme ,nanoparticle ,biocatalysis ,quantum dots ,substrate channeling ,scaffold ,Chemical technology ,TP1-1185 ,Chemistry ,QD1-999 - Abstract
Nanoparticle scaffolds can impart multiple benefits onto immobilized enzymes including enhanced stability, activity, and recoverability. The magnitude of these benefits is modulated by features inherent to the scaffold−enzyme conjugate, amongst which the size of the nanoscaffold itself can be critically important. In this review, we highlight the benefits of enzyme immobilization on nanoparticles and the factors affecting these benefits using quantum dots and gold nanoparticles as representative materials due to their maturity. We then review recent literature on the use of these scaffolds for enzyme immobilization and as a means to dissect the underlying mechanisms. Detailed analysis of the literature suggests that there is a “sweet-spot” for scaffold size and the ratio of immobilized enzyme to scaffold, with smaller scaffolds and lower enzyme:scaffold ratios generally providing higher enzymatic activities. We anticipate that ongoing studies of enzyme immobilization onto nanoscale scaffolds will continue to sharpen our understanding of what gives rise to beneficial characteristics and allow for the next important step, namely, that of translation to large-scale processes that exploit these properties.
- Published
- 2020
- Full Text
- View/download PDF
21. Promoter Identification and Optimization for the Response of
- Author
-
Joseph R, Spangler, Denver N, Cooper, Anthony P, Malanoski, and Scott A, Walper
- Abstract
Quorum sensing (QS) in bacteria has been well studied as a cellular communication phenomenon for decades. In recent years, such systems have been repurposed for the use of biosensors in both cellular and cell-free contexts as well as for inducible protein expression in nontraditional chassis organisms. Such biosensors are particularly intriguing when considering the association between the pathogenesis of some bacteria and their signaling intermediates. Considering this relationship and considering the recent demonstration of the species
- Published
- 2022
22. Implementing Multi-Enzyme Biocatalytic Systems Using Nanoparticle Scaffolds
- Author
-
Joyce C, Breger, Gregory A, Ellis, Scott A, Walper, Kimihiro, Susumu, and Igor L, Medintz
- Subjects
Kinetics ,Quantum Dots ,Biocatalysis ,Nanoparticles - Abstract
Interest in multi-enzyme synthesis outside of cells (in vitro) is becoming far more prevalent as the field of cell-free synthetic biology grows exponentially. Such synthesis would allow for complex chemical transformations based on the exquisite specificity of enzymes in a "greener" manner as compared to organic chemical transformations. Here, we describe how nanoparticles, and in this specific case-semiconductor quantum dots, can be used to both stabilize enzymes and further allow them to self-assemble into nanocomplexes that facilitate high-efficiency channeling phenomena. Pertinent protocol information is provided on enzyme expression, choice of nanoparticulate material, confirmation of enzyme attachment to nanoparticles, assay format and tracking, data analysis, and optimization of assay formats to draw the best analytical information from the underlying processes.
- Published
- 2022
23. Cobalt(III)- and rhodium(III)-porphyrin complexes for the effective degradation of fentanyl: Biological inactivation and mechanistic insights
- Author
-
Hemant Pal, Anneli Nina, Okhil K. Nag, Christopher D. Chouinard, Amanda Pitt, Gregory A. Ellis, Scott A. Walper, Jeffrey Deschamps, Aurora Burkus-Matesevac, Kathy Maiello, James B. Delehanty, and D. Andrew Knight
- Subjects
Inorganic Chemistry ,Fentanyl ,Porphyrins ,Rhodium ,Cobalt ,Ligands ,Biochemistry - Abstract
Cobalt(III) and rhodium(III) complexes containing the water-soluble porphyrin ligand meso-tri(4-sulfonatophenyl)mono(4-carboxyphenyl)porphine (C
- Published
- 2022
24. Impact of Porous Matrices and Concentration by Lyophilization on Cell-Free Expression
- Author
-
Marilyn S. Lee, Jorge L. Chávez, Alvin T. Liem, Svetlana Harbaugh, Pierce A. Roth, Peter A. Emanuel, Matthew W. Lux, Aleksandr E. Miklos, Scott A. Walper, Glory E Mgboji, Kathryn Beabout, Vanessa L Funk, and Steven M Blum
- Subjects
Paper ,chemistry.chemical_classification ,Lysis ,business.product_category ,Cell-Free System ,Chemistry ,Biomedical Engineering ,Hydrogels ,Biosensing Techniques ,Quartz ,General Medicine ,Polymer ,Cell free ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Matrix (mathematics) ,Cross-Linking Reagents ,Freeze Drying ,Chemical engineering ,Self-healing hydrogels ,Microfiber ,Cellulose ,Porous medium ,business ,Porosity - Abstract
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.
- Published
- 2021
25. Gold Nanoparticle Templating Increases the Catalytic Rate of an Amylase, Maltase, and Glucokinase Multienzyme Cascade through Substrate Channeling Independent of Surface Curvature
- Author
-
Priscilla Choo, Scott A. Walper, Igor L. Medintz, Kimihiro Susumu, William P. Klein, David A. Hastman, Eunkeu Oh, Sebastián A. Díaz, and Teri W. Odom
- Subjects
biology ,Glucokinase ,Chemistry ,education ,Substrate channeling ,Nanoparticle ,General Chemistry ,Catalysis ,Enzyme catalysis ,Colloidal gold ,biology.protein ,Biophysics ,Amylase ,Maltase ,health care economics and organizations - Abstract
The templating of enzymes has shown myriad advantages, including increased stability and kinetic rates. Specifically, the use of nanoparticles (NPs) as templates has been shown to increase the kine...
- Published
- 2020
26. Selection and Characterization of Single Domain Antibodies Specific for Bacillus anthracis Spore Proteins
- Author
-
P. Audrey Brozozog Lee, George P. Anderson, Ellen R. Goldman, and Scott A. Walper
- Subjects
single domain antibodies ,Bacillus anthracis ,phage display ,biopanning ,MAGPIX ,Immunologic diseases. Allergy ,RC581-607 - Abstract
To obtain thermostable immunoreagents specific for the spore form of Bacillus anthracis two llamas were immunized with a combination of six different recombinant proteins. These proteins BclA, gerQ, SODA1, SOD15, BxpB and the protein p5303 have all been shown as components of the B. anthracis spore and could potentially serve as targets for the detection of spores in multiplexed biosensors. Peripheral blood lymphocytes were used to construct a phage display library from which single domain antibodies (sdAbs) targeting each of the proteins were isolated. Unique sdAbs exhibiting nanomolar or better affinities for the recombinant proteins were obtained and most of the isolated sdAbs retained their ability to bind antigen after cycles of heating as determined by enzyme linked immunosorbent assay (ELISA). SdAbs targeting the BclA and gerQ proteins were able to successfully detect bacterial spores, whether broken or intact, using a direct ELISA; the sdAbs were specific, showing binding only to B. anthracis spores and not to other Bacillus species. Additionally, SODA1 and p5303 binding sdAbs detected spores in sandwich assays serving as both captures and tracers. Used in combination, sdAbs targeting B. anthracis proteins could be integrated into emerging biosensors to improve specificity in multiplex assays.
- Published
- 2013
- Full Text
- View/download PDF
27. Packaging of Diisopropyl Fluorophosphatase (DFPase) in Bacterial Outer Membrane Vesicles Protects Its Activity at Extreme Temperature
- Author
-
Meghna Thakur, Scott N. Dean, Martin Moore, Joseph R. Spangler, Brandy J. Johnson, Igor L. Medintz, and Scott A. Walper
- Subjects
Biomaterials ,Bacterial Outer Membrane ,Phosphoric Triester Hydrolases ,Biomedical Engineering ,Loligo ,Temperature ,Animals - Abstract
Enzymatic decontamination of organophosphate compounds offers a biofriendly pathway to the neutralization of highly dangerous compounds. Environmental dissemination of enzymes, however, is an ongoing problem considering the costly process of production and chemical modification for stability that can diminish catalytic activity. As a result, there is interest in the potential for enzymatic encapsulation in situ or into nascent bacterial membrane vesicles to improve catalytic stability across various environmental challenges associated with storage and field deployment. In this study, we have engineered bacterial outer membrane vesicles (OMVs) to encapsulate the diisopropyl fluorophosphatase (DFPase), an enzyme originally isolated from squid
- Published
- 2022
28. Implementing Multi-Enzyme Biocatalytic Systems Using Nanoparticle Scaffolds
- Author
-
Joyce C. Breger, Gregory A. Ellis, Scott A. Walper, Kimihiro Susumu, and Igor L. Medintz
- Published
- 2022
29. Cobalt(Iii)- and Rhodium(Iii)-Porphyrin Complexes for the Effective Degradation of Fentanyl Biological Inactivation and Mechanistic Insights
- Author
-
D. Andrew Knight, Hemant Pal, Anneli Nina, Okhil K. Nag, Christopher D. Chouinard, Gregory A. Ellis, Scott A. Walper, Jeffrey Deschamps, Aurora Burkus-Matesevac, Kathy Maiello, and James B. Delehanty
- Subjects
History ,Polymers and Plastics ,Business and International Management ,Industrial and Manufacturing Engineering - Published
- 2022
30. Dendrimeric DNA-Based Nanoscaffolded BRET-FRET Optical Encryption Keys
- Author
-
Scott A. Walper, Matthew Chiriboga, Igor L. Medintz, William P. Klein, and Sebastián A. Díaz
- Subjects
business.industry ,Computer science ,Information security ,Encryption ,Optical encryption ,chemistry.chemical_compound ,Computer Science::Emerging Technologies ,Förster resonance energy transfer ,chemistry ,Coelenterazine ,General Materials Science ,Photonics ,business ,Computer Science::Cryptography and Security ,Computer network - Abstract
New types of unconventional encryption keys based on physically unclonable functions are critically needed for information security. Here we prototype such a key based on the (bio)photonic interact...
- Published
- 2019
31. Artificial Multienzyme Scaffolds: Pursuing in Vitro Substrate Channeling with an Overview of Current Progress
- Author
-
Gregory A. Ellis, Igor L. Medintz, Meghna Thakur, William P. Klein, Scott A. Walper, and Guillermo Lasarte-Aragonés
- Subjects
Synthetic biology ,Health services ,Materials science ,010405 organic chemistry ,Substrate channeling ,Nanotechnology ,General Chemistry ,010402 general chemistry ,01 natural sciences ,Catalysis ,0104 chemical sciences - Abstract
Artificial multienzyme scaffolds are being developed for in vitro cascaded biocatalytic activity and, in particular, accessing substrate channeling. This review covers progress in this field over t...
- Published
- 2019
32. Optimization of Heavy Metal Sensors Based on Transcription Factors and Cell-Free Expression Systems
- Author
-
Matthew W. Lux, Meghna Thakur, Stephanie D Cole, Casey B. Bernhards, Scott A. Walper, Jorge L. Chávez, Kathryn Beabout, and Kendrick B. Turner
- Subjects
Water contaminants ,Bacteria ,Cell-Free System ,Computer science ,Biomedical Engineering ,Heavy metals ,General Medicine ,Cell free ,DNA ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Combined approach ,Expression (mathematics) ,World health ,Plasmid dna ,Metals, Heavy ,Sensitivity (control systems) ,Biological system ,Plasmids ,Transcription Factors - Abstract
Many bacterial mechanisms for highly specific and sensitive detection of heavy metals and other hazards have been reengineered to serve as sensors. In some cases, these sensors have been implemented in cell-free expression systems, enabling easier design optimization and deployment in low-resource settings through lyophilization. Here, we apply the advantages of cell-free expression systems to optimize sensors based on three separate bacterial response mechanisms for arsenic, cadmium, and mercury. We achieved detection limits below the World Health Organization-recommended levels for arsenic and mercury and below the short-term US Military Exposure Guideline levels for all three. The optimization of each sensor was approached differently, leading to observations useful for the development of future sensors: (1) there can be a strong dependence of specificity on the particular cell-free expression system used, (2) tuning of relative concentrations of the sensing and reporter elements improves sensitivity, and (3) sensor performance can vary significantly with linear vs plasmid DNA. In addition, we show that simply combining DNA for the three sensors into a single reaction enables detection of each target heavy metal without any further optimization. This combined approach could lead to sensors that detect a range of hazards at once, such as a panel of water contaminants or all known variants of a target virus. For low-resource settings, such "all-hazard" sensors in a cheap, easy-to-use format could have high utility.
- Published
- 2021
33. Genetic Fusion of an Anti-BclA Single-Domain Antibody with Beta Galactosidase
- Author
-
George P. Anderson, Lisa C. Shriver-Lake, Scott A. Walper, Lauryn Ashford, Dan Zabetakis, Jinny L. Liu, Joyce C. Breger, P. Audrey Brozozog Lee, and Ellen R. Goldman
- Subjects
Bacillus anthracis ,immunoassay ,single-domain antibody ,genetic fusion ,Beta galactosidase ,Immunologic diseases. Allergy ,RC581-607 - Abstract
The Bacillus collagen-like protein of anthracis (BclA), found in Bacillus anthracis spores, is an attractive target for immunoassays. Previously, using phage display we had selected llama-derived single-domain antibodies that bound to B. anthracis spore proteins including BclA. Single-domain antibodies (sdAbs), the recombinantly expressed heavy domains from the unique heavy-chain-only antibodies found in camelids, provide stable and well-expressed binding elements with excellent affinity. In addition, sdAbs offer the important advantage that they can be tailored for specific applications through protein engineering. A fusion of a BclA targeting sdAb with the enzyme Beta galactosidase (β-gal) would enable highly sensitive immunoassays with no need for a secondary reagent. First, we evaluated five anti-BclA sdAbs, including four that had been previously identified but not characterized. Each was tested to determine its binding affinity, melting temperature, producibility, and ability to function as both capture and reporter in sandwich assays for BclA. The sdAb with the best combination of properties was constructed as a fusion with β-gal and shown to enable sensitive detection. This fusion has the potential to be incorporated into highly sensitive assays for the detection of anthrax spores.
- Published
- 2018
- Full Text
- View/download PDF
34. PepVAE: Variational Autoencoder Framework for Antimicrobial Peptide Generation and Activity Prediction
- Author
-
Dan Zabetakis, Scott A. Walper, Jerome Anthony E. Alvarez, Anthony P. Malanoski, and Scott N. Dean
- Subjects
Microbiology (medical) ,chemistry.chemical_classification ,Computer science ,Antimicrobial peptides ,Peptide ,Computational biology ,Experimental validation ,minimum inhibitory concentration ,Antimicrobial ,Autoencoder ,Microbiology ,QR1-502 ,antimicrobial peptides ,chemistry ,activity prediction ,generative deep learning ,variational autoencoder ,Original Research - Abstract
New methods for antimicrobial design are critical for combating pathogenic bacteria in the post-antibiotic era. Fortunately, competition within complex communities has led to the natural evolution of antimicrobial peptide (AMP) sequences that have promising bactericidal properties. Unfortunately, the identification, characterization, and production of AMPs can prove complex and time consuming. Here, we report a peptide generation framework, PepVAE, based around variational autoencoder (VAE) and antimicrobial activity prediction models for designing novel AMPs using only sequences and experimental minimum inhibitory concentration (MIC) data as input. Sampling from distinct regions of the learned latent space allows for controllable generation of new AMP sequences with minimal input parameters. Extensive analysis of the PepVAE-generated sequences paired with antimicrobial activity prediction models supports this modular design framework as a promising system for development of novel AMPs, demonstrating controlled production of AMPs with experimental validation of predicted antimicrobial activity.
- Published
- 2021
35. Gold nanoparticles capable of templating entire enzyme cascades and improving production yield through substrate channeling
- Author
-
Sebastián A. Díaz, David A. Hastman, Eunkeu Oh, Scott A. Walper, and Igor L. Medintz
- Subjects
chemistry.chemical_classification ,Enzyme ,Biocatalysis ,Chemistry ,Colloidal gold ,education ,Substrate channeling ,Kinetics ,technology, industry, and agriculture ,Biophysics ,Nanoparticle ,Maltase ,Conjugate - Abstract
We have demonstrated that multi-enzyme cascades can be templated on individual gold nanoparticles (NPs) with diameters below 100 nm. Utilizing a three enzyme cascade of amylase, maltase, and glucokinase we found a ~3-fold enhancement in product formation when all three enzymes were bound to the same NP as compared to controls. This strongly suggests that the increased kinetics was due to substrate channeling. Additional controls were realized to ensure that only when the enzymes were bound to the NPs was enhancement observed by modifying the ratio of enzyme to NP. Furthermore the experiments support a model where a single-layer of enzymes conjugate to the NPs independently of the enzyme to NP ratio. Being able to conjugate entire cascades on individual NPs should allow for optimized design of NPs and enzyme cascades for in vitro biocatalysis.
- Published
- 2021
36. Quantification of Interlaboratory Cell-Free Protein Synthesis Variability
- Author
-
Vanessa L Funk, Jorge L. Chávez, Peter A. Emanuel, Kathryn Beabout, Brian A. Geier, Stephanie D Cole, Pierce A. Roth, Scott A. Walper, Zachary K Smith, Alvin T. Liem, Matthew W. Lux, Kendrick B. Turner, and Svetlana Harbaugh
- Subjects
Protocol (science) ,Cell-free protein synthesis ,Cell-Free System ,Standardization ,Computer science ,Biomedical Engineering ,Proteins ,Reproducibility of Results ,DNA ,General Medicine ,Cell free ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Protein Biosynthesis ,Quantitative assessment ,Pairwise comparison ,Biomanufacturing ,Biochemical engineering ,Laboratories - Abstract
Cell-free protein synthesis (CFPS) platforms, once primarily a research tool to produce difficult to express proteins, are increasingly being pursued by the synthetic biology community for applications including biomanufacturing, rapid screening systems, and field-ready sensors. While consistency within individual studies is apparent in the literature, challenges with reproducing results between laboratories, or even between individuals within a laboratory, are discussed openly by practitioners. As the field continues to grow and move toward applications, a quantitative understanding of expected variability for CFPS and the relative contribution of underlying sources will become increasingly important. Here we offer the first quantitative assessment of interlaboratory variability in CFPS. Three laboratories implemented a single CFPS protocol and performed a series of exchanges, both of material and personnel, designed to quantify relative contributions to variability associated with the site, operator, cell extract preparation, and supplemental reagent preparation. We found that materials prepared at each laboratory, exchanged pairwise, and tested at each site resulted in 40.3% coefficient of variation compared to 7.64% for a single operator across days using a single set of materials. Reagent preparations contributed significantly to observed variability; extract preparations, however, surprisingly did not explain any of the observed variability, even when prepared in different laboratories by different operators. Subsequent exchanges showed that both the site and the operator each contributed to observed interlaboratory variability. In addition to providing the first quantitative assessment of interlaboratory variability in CFPS, these results establish a baseline for individual operator variability across days that can be used as an initial benchmark for community-driven standardization efforts. We anticipate that our results will narrow future avenues of investigation to develop best practices that will ultimately drive down interlaboratory variability, accelerating research progress and informing the suitability of CFPS for real-world applications.
- Published
- 2019
37. Isolation and characterization of Lactobacillus-derived membrane vesicles
- Author
-
Scott A. Walper, Eunkeu Oh, Scott N. Dean, Dagmar H. Leary, and Claretta J. Sullivan
- Subjects
0301 basic medicine ,Limosilactobacillus reuteri ,lcsh:Medicine ,Proteomics ,Article ,Biological pathway ,Cell membrane ,03 medical and health sciences ,0302 clinical medicine ,Bacterial Proteins ,Lactobacillus ,medicine ,lcsh:Science ,Multidisciplinary ,biology ,Bacteria ,Chemistry ,Vesicle ,Probiotics ,lcsh:R ,Membrane Proteins ,food and beverages ,biology.organism_classification ,Subcellular localization ,Lactobacillus acidophilus ,Lacticaseibacillus casei ,030104 developmental biology ,medicine.anatomical_structure ,Biochemistry ,lcsh:Q ,030217 neurology & neurosurgery ,Lactobacillus plantarum - Abstract
Bacterial membrane vesicles have been implicated in a broad range of functions in microbial communities from pathogenesis to gene transfer. Though first thought to be a phenomenon associated with Gram-negative bacteria, vesicle production in Staphylococcus aureus, Lactobacillus plantarum, and other Gram-positives has recently been described. Given that many Lactobacillus species are Generally Regarded as Safe and often employed as probiotics, the engineering of Lactobacillus membrane vesicles presents a new avenue for the development of therapeutics and vaccines. Here we characterize and compare the membrane vesicles (MVs) from three different Lactobacillus species (L. acidophilus ATCC 53544, L. casei ATCC 393, and L. reuteri ATCC 23272), with the aim of developing future strategies for vesicle engineering. We characterize the vesicles from each Lactobacillus species comparing the physiochemical properties and protein composition of each. More than 80 protein components from Lactobacillus-derived MVs were identified, including some that were enriched in the vesicles themselves suggesting vesicles as a vehicle for antimicrobial delivery. Additionally, for each species vesicular proteins were categorized based on biological pathway and examined for subcellular localization signals in an effort to identify possible sorting mechanisms for MV proteins.
- Published
- 2019
38. Development and evaluation of single domain antibodies for vaccinia and the L1 antigen.
- Author
-
Scott A Walper, Jinny L Liu, Daniel Zabetakis, George P Anderson, and Ellen R Goldman
- Subjects
Medicine ,Science - Abstract
There is ongoing interest to develop high affinity, thermal stable recognition elements to replace conventional antibodies in biothreat detection assays. As part of this effort, single domain antibodies that target vaccinia virus were developed. Two llamas were immunized with killed viral particles followed by boosts with the recombinant membrane protein, L1, to stimulate the immune response for envelope and membrane proteins of the virus. The variable domains of the induced heavy chain antibodies were selected from M13 phage display libraries developed from isolated RNA. Selection via biopanning on the L1 antigen produced single domain antibodies that were specific and had affinities ranging from 4×10(-9) M to 7.0×10(-10) M, as determined by surface plasmon resonance. Several showed good ability to refold after heat denaturation. These L1-binding single domain antibodies, however, failed to recognize the killed vaccinia antigen. Useful vaccinia binding single domain antibodies were isolated by a second selection using the killed virus as the target. The virus binding single domain antibodies were incorporated in sandwich assays as both capture and tracer using the MAGPIX system yielding limits of detection down to 4×10(5) pfu/ml, a four-fold improvement over the limit obtained using conventional antibodies. This work demonstrates the development of anti-vaccinia single domain antibodies and their incorporation into sandwich assays for viral detection. It also highlights the properties of high affinity and thermal stability that are hallmarks of single domain antibodies.
- Published
- 2014
- Full Text
- View/download PDF
39. PepVAE3: variational autoencoder framework for antimicrobial peptide generation and activity prediction
- Author
-
Anthony P. Malanoski, Scott A. Walper, Scott N. Dean, Dan Zabetakis, and Jerome Anthony E. Alvarez
- Subjects
chemistry.chemical_classification ,chemistry ,Computer science ,Peptide ,Computational biology ,Experimental validation ,Antimicrobial ,Autoencoder - Abstract
New methods for antimicrobial design are critical for combating pathogenic bacteria in the post-antibiotic era. Fortunately, competition within complex communities has led to the natural evolution of antimicrobial peptide (AMP) sequences that have promising bactericidal properties. Unfortunately, the identification, characterization, and production of AMPs can prove complex and time consuming. Here we report a peptide generation framework, PepVAE3, based around variational autoencoder (VAE) and antimicrobial activity prediction models for designing novel AMPs using only sequences and experimental minimum inhibitory concentration (MIC) data as input. Sampling from distinct regions of the learned latent space allows for controllable generation of new AMP sequences with minimal input parameters. Extensive analysis of the PepVAE3-generated sequences paired with antimicrobial activity prediction models supports this modular design framework as a promising system for development of novel AMPs, demonstrating controlled production of AMPs with experimental validation of predicted antimicrobial activity.
- Published
- 2021
40. Isolation and Characterization of Membrane Vesicles from
- Author
-
Julie C, Caruana, Scott N, Dean, and Scott A, Walper
- Subjects
Methods Article - Abstract
Throughout their life cycle, bacteria shed portions of their outermost membrane comprised of proteins, lipids, and a diversity of other biomolecules. These biological nanoparticles have been shown to have a range of highly diverse biological activities, including pathogenesis, community regulation, and cellular defense (among others). In recent publications, we have isolated and characterized membrane vesicles (MVs) from several species of Lactobacilli, microbes classified as commensals within the human gut microbiome ( Dean et al., 2019 and 2020). With increasing scientific understanding of host-microbe interactions, the gut-brain axis, and tailored probiotics for therapeutic or performance increasing applications, the protocols described herein will be useful to researchers developing new strategies for gut community engineering or the targeted delivery of bio-active molecules. Graphic abstract: [Image: see text]
- Published
- 2021
41. Harnessing the potential of Lactobacillus species for therapeutic delivery at the lumenal-mucosal interface
- Author
-
Julie C. Caruana, Scott A. Walper, Igor L. Medintz, and Joseph R. Spangler
- Subjects
0301 basic medicine ,030106 microbiology ,microbiome ,Review ,Biology ,immunomodulation ,Microbiology ,law.invention ,03 medical and health sciences ,Probiotic ,Human gut ,law ,Lactobacillus ,vaccine ,Microbiome ,Lactobacillus species ,therapy ,cytokine stimulation ,Delivery vehicle ,delivery vehicle ,biology.organism_classification ,030104 developmental biology ,Cytokine stimulation ,probiotic ,Biotechnology - Abstract
Lactobacillus species have been studied for over 30 years in their role as commensal organisms in the human gut. Recently there has been a surge of interest in their abilities to natively and recombinantly stimulate immune activities, and studies have identified strains and novel molecules that convey particular advantages for applications as both immune adjuvants and immunomodulators. In this review, we discuss the recent advances in Lactobacillus-related activity at the gut/microbiota interface, the efforts to probe the boundaries of the direct and indirect therapeutic potential of these bacteria, and highlight the continued interest in harnessing the native capacity for the production of biogenic compounds shown to influence nervous system activity. Taken together, these aspects underscore Lactobacillus species as versatile therapeutic delivery vehicles capable of effector production at the lumenal-mucosal interface, and further establish a foundation of efficacy upon which future engineered strains can expand., Lay abstract Bacteria such as Lactobacillus species have traditionally maintained positive association with gastrointestinal health. This review summarizes recent research relating to human health associated Lactobacillus administration on cellular and systematic levels. As techniques in molecular and synthetic biology continue to advance, the potential to increase the therapeutic scope and efficacy of Lactobacillus cultures increases regarding target molecule production, immune system influence, vaccination and nervous system activities.
- Published
- 2021
42. Microbial Nanocellulose Printed Circuit Boards for Medical Sensing
- Author
-
Scott A. Walper, David A. Stenger, Joyce C. Breger, Daniel Zabetakis, Jonathan D. Yuen, and Lisa C. Shriver-Lake
- Subjects
Computer science ,Internet of Things ,Nanotechnology ,Biocompatible Materials ,02 engineering and technology ,Biosensing Techniques ,lcsh:Chemical technology ,010402 general chemistry ,01 natural sciences ,Biochemistry ,flexible electronics ,Article ,Analytical Chemistry ,Nanocellulose ,Body Temperature ,Printed circuit board ,Heart Rate ,Heart rate monitoring ,Humans ,lcsh:TP1-1185 ,Electronics ,Electrical and Electronic Engineering ,Cellulose ,Instrumentation ,nanocellulose ,Monitoring, Physiologic ,business.industry ,021001 nanoscience & nanotechnology ,Biocompatible material ,Atomic and Molecular Physics, and Optics ,Flexible electronics ,0104 chemical sciences ,Nanostructures ,biosensing ,0210 nano-technology ,business - Abstract
We demonstrate the viability of using ultra-thin sheets of microbially grown nanocellulose to build functional medical sensors. Microbially grown nanocellulose is an interesting alternative to plastics, as it is hydrophilic, biocompatible, porous, and hydrogen bonding, thereby allowing the potential development of new application routes. Exploiting the distinguishing properties of this material enables us to develop solution-based processes to create nanocellulose printed circuit boards, allowing a variety of electronics to be mounted onto our nanocellulose. As proofs of concept, we have demonstrated applications in medical sensing such as heart rate monitoring and temperature sensing&mdash, potential applications fitting the wide-ranging paradigm of a future where the Internet of Things is dominant.
- Published
- 2020
- Full Text
- View/download PDF
43. Lactobacillus acidophilus Membrane Vesicles as a Vehicle of Bacteriocin Delivery
- Author
-
Dagmar H. Leary, Kendrick B. Turner, William Judson Hervey, Mary Ashley Rimmer, Daniel A. Phillips, Scott A. Walper, Scott N. Dean, and Julie C. Caruana
- Subjects
Microbiology (medical) ,Operon ,lcsh:QR1-502 ,Peptide ,membrane vesicles ,Microbiology ,lcsh:Microbiology ,bacteriocins ,03 medical and health sciences ,Lactobacillus acidophilus ,Bacteriocin ,Lactobacillus ,Secretion ,030304 developmental biology ,Original Research ,chemistry.chemical_classification ,0303 health sciences ,biology ,030306 microbiology ,food and beverages ,biology.organism_classification ,lactic acid bacteria ,Biochemistry ,chemistry ,Proteome ,bacteria ,antimicrobial ,Bacteria - Abstract
Recent reports have shown that Gram-positive bacteria actively secrete spherical nanometer-sized proteoliposome membrane vesicles (MVs) into their surroundings. Though MVs are implicated in a broad range of biological functions, few studies have been conducted to examine their potential as delivery vehicles of antimicrobials. Here, we investigate the natural ability of Lactobacillus acidophilus MVs to carry and deliver bacteriocin peptides to the opportunistic pathogen, Lactobacillus delbrueckii. We demonstrate that upon treatment with lactacin B-inducing peptide, the proteome of the secreted MVs is enriched in putative bacteriocins encoded by the lab operon. Further, we show that purified MVs inhibit growth and compromise membrane integrity in L. delbrueckii, which is confirmed by confocal microscopy imaging and spectrophotometry. These results show that L. acidophilus MVs serve as conduits for antimicrobials to competing cells in the environment, suggesting a potential role for MVs in complex communities such as the gut microbiome. With the potential for controlling their payload through microbial engineering, MVs produced by L. acidophilus may be an interesting platform for effecting change in complex microbial communities or aiding in the development of new biomedical therapeutics.
- Published
- 2020
44. Bacterial Membrane Vesicles and Their Applications as Vaccines and in Biotechnology
- Author
-
Julie C. Caruana and Scott A. Walper
- Subjects
Vaccine research ,business.industry ,Physical health ,Membrane vesicle ,Business ,Human society ,Biotechnology - Abstract
Human society has coevolved with our invisible, microbial neighbors. From their use in the processing of food and drink to the manufacture of commercial products and therapeutics, bacteria, yeast, and fungi are invaluable tools to many aspects of our existence. Despite our long history together, researchers are only recently beginning to understand the complexities of these relationships and how the microbial world can fully be exploited. In the last 50 years alone, researchers have shown that bacteria can be used to manufacture drugs to aid in the treatment of medical disorders such as diabetes, that environmental microbes can be used to clean up chemical spills and disasters, and that the microbial communities that reside within our bodies are capable of influencing our mental and physical health. With this greater understanding comes new avenues of research to utilize these microbes to advance human society. Here we discuss efforts to utilize both native and engineered membrane vesicles shed by bacteria and their potential applications to several areas of biotechnology. We highlight the use of bacterial membrane vesicles in vaccine research and as emerging therapeutics as well as exploring their potential commercial applications and benefits.
- Published
- 2020
45. Rugged single domain antibody detection elements for Bacillus anthracis spores and vegetative cells.
- Author
-
Scott A Walper, George P Anderson, P Audrey Brozozog Lee, Richard H Glaven, Jinny L Liu, Rachel D Bernstein, Dan Zabetakis, Linwood Johnson, Jill M Czarnecki, and Ellen R Goldman
- Subjects
Medicine ,Science - Abstract
Significant efforts to develop both laboratory and field-based detection assays for an array of potential biological threats started well before the anthrax attacks of 2001 and have continued with renewed urgency following. While numerous assays and methods have been explored that are suitable for laboratory utilization, detection in the field is often complicated by requirements for functionality in austere environments, where limited cold-chain facilities exist. In an effort to overcome these assay limitations for Bacillus anthracis, one of the most recognizable threats, a series of single domain antibodies (sdAbs) were isolated from a phage display library prepared from immunized llamas. Characterization of target specificity, affinity, and thermal stability was conducted for six sdAb families isolated from rounds of selection against the bacterial spore. The protein target for all six sdAb families was determined to be the S-layer protein EA1, which is present in both vegetative cells and bacterial spores. All of the sdAbs examined exhibited a high degree of specificity for the target bacterium and its spore, with affinities in the nanomolar range, and the ability to refold into functional antigen-binding molecules following several rounds of thermal denaturation and refolding. This research demonstrates the capabilities of these sdAbs and their potential for integration into current and developing assays and biosensors.
- Published
- 2012
- Full Text
- View/download PDF
46. Bacterial Membrane Vesicles as Mediators of Microbe - Microbe and Microbe - Host Community Interactions
- Author
-
Scott A. Walper and Julie C. Caruana
- Subjects
Microbiology (medical) ,lcsh:QR1-502 ,Virulence ,Review ,membrane vesicles ,Microbiology ,lcsh:Microbiology ,host-microbe interactions ,03 medical and health sciences ,cell delivery ,host-pathogen interactions ,030304 developmental biology ,community interactions ,0303 health sciences ,biology ,030306 microbiology ,Host (biology) ,Chemistry ,Vesicle ,Biofilm ,biology.organism_classification ,Cell biology ,Quorum sensing ,Cytoplasm ,antimicrobial ,Bacteria ,Function (biology) ,outer membrane vesicles - Abstract
Bacterial membrane vesicles are proteoliposomal nanoparticles produced by both Gram-negative and Gram-positive bacteria. As they originate from the outer surface of the bacteria, their composition and content is generally similar to the parent bacterium’s membrane and cytoplasm. However, there is ample evidence that preferential packaging of proteins, metabolites, and toxins into vesicles does occur. Incorporation into vesicles imparts a number of benefits to the cargo, including protection from degradation by other bacteria, the host organism, or environmental factors, maintenance of a favorable microenvironment for enzymatic activity, and increased potential for long-distance movement. This enables vesicles to serve specialized functions tailored to changing or challenging environments, particularly in regard to microbial community interactions including quorum sensing, biofilm formation, antibiotic resistance, antimicrobial peptide expression and deployment, and nutrient acquisition. Additionally, based on their contents, vesicles play crucial roles in host-microbe interactions as carriers of virulence factors and other modulators of host cell function. Here, we discuss recent advances in our understanding of how vesicles to function as signals both within microbial communities and between pathogenic or commensal microbes and their mammalian hosts. We also highlight a few areas that are currently ripe for additional research, including the mechanisms of selective cargo packaging into membrane vesicles and of cargo processing once it enters mammalian host cells, the function of vesicles in transfer of nucleic acids among bacteria, and the possibility of engineering commensal bacteria to deliver cargo of interest to mammalian hosts in a controlled manner.
- Published
- 2019
47. Detecting Biothreat Agents: From Current Diagnostics to Developing Sensor Technologies
- Author
-
Scott A. Walper, Clare E. Rowland, Igor L. Medintz, Guillermo Lasarte Aragonés, Carl W. Brown, Kim E. Sapsford, and Joyce C. Breger
- Subjects
Point-of-Care Systems ,Early detection ,Biological Warfare Agents ,Bioengineering ,Context (language use) ,Biosensing Techniques ,02 engineering and technology ,01 natural sciences ,Limit of Detection ,Humans ,Instrumentation ,Toxins, Biological ,Immunoassay ,Fluid Flow and Transfer Processes ,Bacteria ,Process Chemistry and Technology ,010401 analytical chemistry ,021001 nanoscience & nanotechnology ,Pathogenicity ,0104 chemical sciences ,Risk analysis (engineering) ,Virus Diseases ,Viruses ,Terrorism ,Business ,0210 nano-technology - Abstract
Although a fundamental understanding of the pathogenicity of most biothreat agents has been elucidated and available treatments have increased substantially over the past decades, they still represent a significant public health threat in this age of (bio)terrorism, indiscriminate warfare, pollution, climate change, unchecked population growth, and globalization. The key step to almost all prevention, protection, prophylaxis, post-exposure treatment, and mitigation of any bioagent is early detection. Here, we review available methods for detecting bioagents including pathogenic bacteria and viruses along with their toxins. An introduction placing this subject in the historical context of previous naturally occurring outbreaks and efforts to weaponize selected agents is first provided along with definitions and relevant considerations. An overview of the detection technologies that find use in this endeavor along with how they provide data or transduce signal within a sensing configuration follows. Current "gold" standards for biothreat detection/diagnostics along with a listing of relevant FDA approved in vitro diagnostic devices is then discussed to provide an overview of the current state of the art. Given the 2014 outbreak of Ebola virus in Western Africa and the recent 2016 spread of Zika virus in the Americas, discussion of what constitutes a public health emergency and how new in vitro diagnostic devices are authorized for emergency use in the U.S. are also included. The majority of the Review is then subdivided around the sensing of bacterial, viral, and toxin biothreats with each including an overview of the major agents in that class, a detailed cross-section of different sensing methods in development based on assay format or analytical technique, and some discussion of related microfluidic lab-on-a-chip/point-of-care devices. Finally, an outlook is given on how this field will develop from the perspective of the biosensing technology itself and the new emerging threats they may face.
- Published
- 2018
48. Exploiting the Feedstock Flexibility of the Emergent Synthetic Biology Chassis Vibrio natriegens for Engineered Natural Product Production
- Author
-
Gary J. Vora, Scott A. Walper, Joseph R. Spangler, Igor L. Medintz, Tanya Tschirhart, and Gregory A. Ellis
- Subjects
Chassis ,Indoles ,Pharmaceutical Science ,Vibrio natriegens ,Article ,03 medical and health sciences ,Synthetic biology ,chemistry.chemical_compound ,violacein ,Marine bacteriophage ,Drug Discovery ,Vibrio campbellii ,Pharmacology, Toxicology and Pharmaceutics (miscellaneous) ,030304 developmental biology ,Vibrio ,0303 health sciences ,Biological Products ,Natural product ,biology ,030306 microbiology ,Chromobacterium ,biology.organism_classification ,beta Carotene ,Biosynthetic Pathways ,beta-carotene ,chemistry ,marine bacteria ,Multigene Family ,Synthetic Biology ,Biochemical engineering ,Chromobacterium violaceum - Abstract
A recent goal of synthetic biology has been to identify new chassis that provide benefits lacking in model organisms. Vibrio natriegens is a marine Gram-negative bacterium which is an emergent synthetic biology chassis with inherent benefits: An extremely fast growth rate, genetic tractability, and the ability to grow on a variety of carbon sources (&ldquo, feedstock flexibility&rdquo, ). Given these inherent benefits, we sought to determine its potential to heterologously produce natural products, and chose beta-carotene and violacein as test cases. For beta-carotene production, we expressed the beta-carotene biosynthetic pathway from the sister marine bacterium Vibrio campbellii, as well as the mevalonate biosynthetic pathway from the Gram-positive bacterium Lactobacillus acidophilus to improve precursor abundance. Violacein was produced by expressing a biosynthetic gene cluster derived from Chromobacterium violaceum. Not only was V. natriegens able to heterologously produce these compounds in rich media, illustrating its promise as a new chassis for small molecule drug production, but it also did so in minimal media using a variety of feedstocks. The ability for V. natriegens to produce natural products with multiple industrially-relevant feedstocks argues for continued investigations into the production of more complex natural products in this chassis.
- Published
- 2019
- Full Text
- View/download PDF
49. Enhanced Catalysis from Multienzyme Cascades Assembled on a DNA Origami Triangle
- Author
-
William P. Klein, Rasmus P. Thomsen, Scott A. Walper, Igor L Medintz, Mario G. Ancona, Jørgen Kjems, James N. Vranish, and Kendrick B. Turner
- Subjects
EXPRESSION ,Models, Molecular ,ENZYME ,Kinetics ,General Physics and Astronomy ,02 engineering and technology ,010402 general chemistry ,01 natural sciences ,NANOSTRUCTURES ,Catalysis ,Substrate Specificity ,Synthetic biology ,chemistry.chemical_compound ,Multienzyme Complexes ,origami ,BETA-AMYLASE ,Molecule ,DNA origami ,General Materials Science ,A-DNA ,cell-free ,Computer Simulation ,PROTEOLYSIS ,Probability ,chemistry.chemical_classification ,PURIFICATION ,Chemistry ,General Engineering ,DNA ,021001 nanoscience & nanotechnology ,channeling ,TRANSPORT ,0104 chemical sciences ,cascade ,Enzyme ,kinetics ,ESCHERICHIA-COLI ,Biophysics ,Nucleic Acid Conformation ,COMPLEXES ,0210 nano-technology ,GLUCOKINASE - Abstract
Developing reliable methods of constructing cell-free multienzyme biocatalytic systems is a milestone goal of synthetic biology. It would enable overcoming the limitations of current cell-based systems, which suffer from the presence of competing pathways, toxicity, and inefficient access to extracellular reactants and removal of products. DNA nanostructures have been suggested as ideal scaffolds for assembling sequential enzymatic cascades in close enough proximity to potentially allow for exploiting of channeling effects; however, initial demonstrations have provided somewhat contradictory results toward confirming this phenomenon. In this work, a three-enzyme sequential cascade was realized by site-specifically immobilizing DNA-conjugated amylase, maltase, and glucokinase on a self-assembled DNA origami triangle. The kinetics of seven different enzyme configurations were evaluated experimentally and compared to simulations of optimized activity. A 30-fold increase in the pathway's kinetic activity was observed for enzymes assembled to the DNA. Detailed kinetic analysis suggests that this catalytic enhancement originated from increased enzyme stability and a localized DNA surface affinity or hydration layer effect and not from a directed enzyme-to-enzyme channeling mechanism. Nevertheless, the approach used to construct this pathway still shows promise toward improving other more elaborate multienzymatic cascades and could potentially allow for the custom synthesis of complex (bio)molecules that cannot be realized with conventional organic chemistry approaches.
- Published
- 2019
50. DNA–Nanoparticle Composites Synergistically Enhance Organophosphate Hydrolase Enzymatic Activity
- Author
-
Scott A. Walper, Eunkeu Oh, Joyce C. Breger, Nabil Bassim, Anirban Samanta, Igor L. Medintz, and Kimihiro Susumu
- Subjects
0301 basic medicine ,chemistry.chemical_classification ,biology ,Nanoparticle ,Bioinorganic chemistry ,02 engineering and technology ,021001 nanoscience & nanotechnology ,Enzyme assay ,Turn (biochemistry) ,03 medical and health sciences ,chemistry.chemical_compound ,Synthetic biology ,030104 developmental biology ,Enzyme ,chemistry ,biology.protein ,General Materials Science ,A-DNA ,Composite material ,0210 nano-technology ,DNA - Abstract
Cell-free synthetic biology relies on optimally exploiting enzymatic activity, and recent demonstrations that nanoparticle (NP) and DNA scaffolding can enhance enzyme activity suggest new avenues toward this. A modular architecture consisting of a DNA cage displaying semiconductor quantum dots (QDs) that, in turn, ratiometrically display the organophosphate hydrolase phosphotriesterase (PTE) was utilized as a model system. Increasing DNA cage concentration relative to QD-PTE and creating a dense composite enhanced PTE rates up to 12.5-fold, suggesting strong synergy between the NP and DNA components; this putatively arises from increased enzymatic stability and alleviation of its rate-limiting step. Such bioinorganic composites may offer new scaffolding approaches for synthetic biology.
- Published
- 2018
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.