Technological advancements towards paper-based biomolecular diagnostics

Clinically tractable diagnostics must be low-cost, rapid, sensitive, easy to use, and adaptable to new targets. With its rational design, synthetic biology holds promise for developing diagnostic technologies that can address these needs. In particular, progress in synthetic biology has led to improved circuit-building abilities and a large collection of biomolecular sensors. However, these technologies fundamentally require transcription and translation, limiting their applicability to cellular contexts In vitro cell-free expression systems that contain transcription and translation machinery provide the environment necessary for biologically-based technologies to function independently of living cells. Our lab recently developed a paper-based system for cell-free gene expression, which utilizes cell-free extracts that are freeze-dried on to paper and other porous substrates to allow for long-term preservation of synthetic circuits at room temperature. Our platform represents a scalable, cost-effective technology that is easy to use and is compatible with synthetic biology tools. In this dissertation, I present several advancements to this diagnostic platform that are geared towards improving the system’s clinical tractability. In the context of developing a diagnostic for Zika virus that could be deployed in low-resource settings, I demonstrate improvements to diagnostic sensitivity and rapid sample processing that allow for detection of low femtomolar quantities of active virus directly from blood plasma samples. I also describe preliminary results towards a streamlined one-pot amplification-sensing reaction, and propose the development of a paper-based diagnostic for antibiotic susceptibility testing

Lethality of MalE-LacZ hybrid protein shares mechanistic attributes with oxidative component of antibiotic lethality

Downstream metabolic events can contribute to the lethality of drugs or agents that interact with a primary cellular target. In bacteria, the production of reactive oxygen species (ROS) has been associated with the lethal effects of a variety of stresses including bactericidal antibiotics, but the relative contribution of this oxidative component to cell death depends on a variety of factors. Experimental evidence has suggested that unresolvable DNA problems caused by incorporation of oxidized nucleotides into nascent DNA followed by incomplete base excision repair contribute to the ROS-dependent component of antibiotic lethality. Expression of the chimeric periplasmic-cytoplasmic MalE-LacZ[subscript 72 – 47] protein is an historically important lethal stress originally identified during seminal genetic experiments that defined the SecY-dependent protein translocation system. Multiple, independent lines of evidence presented here indicate that the predominant mechanism for MalE-LacZ lethality shares attributes with the ROS-dependent component of antibiotic lethality. MalE-LacZ lethality requires molecular oxygen, and its expression induces ROS production. The increased susceptibility of mutants sensitive to oxidative stress to MalE-LacZ lethality indicates that ROS contribute causally to cell death rather than simply being produced by dying cells. Observations that support the proposed mechanism of cell death include MalE-LacZ expression being bacteriostatic rather than bactericidal in cells that over-express MutT, a nucleotide sanitizer that hydrolyzes 8-oxo-dGTP to the monophosphate, or that lack MutM and MutY, DNA glycosylases that process base pairs involving 8-oxo-dGTP. Our studies suggest stress-induced physiological changes that favor this mode of ROS-dependent death.National Institutes of Health (U.S.) (Grant R01CA021615)Defense Threat Reduction Agency (DTRA) (Grant HDTRA1-15-1-0051)National Science Foundation (U.S.) (Grant 1336493)National Institutes of Health (U.S.) (Grant K99GM118907

Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components

The recent Zika virus outbreak highlights the need for low-cost diagnostics that can be rapidly developed for distribution and use in pandemic regions. Here, we report a pipeline for the rapid design, assembly, and validation of cell-free, paper-based sensors for the detection of the Zika virus RNA genome. By linking isothermal RNA amplification to toehold switch RNA sensors, we detect clinically relevant concentrations of Zika virus sequences and demonstrate specificity against closely related Dengue virus sequences. When coupled with a novel CRISPR/Cas9-based module, our sensors can discriminate between viral strains with single-base resolution. We successfully demonstrate a simple, field-ready sample-processing workflow and detect Zika virus from the plasma of a viremic macaque. Our freeze-dried biomolecular platform resolves important practical limitations to the deployment of molecular diagnostics in the field and demonstrates how synthetic biology can be used to develop diagnostic tools for confronting global health crises.Defense Threat Reduction Agency (DTRA) (HDTRA1-14-1-0006)United States. National Institutes of Health (NIH AI100190

Einfluss der Verarbeitungstechnologie und Werkstoffzusammensetzung auf die Struktur-Eigenschafts-Beziehungen von thermoplastischen Nanoverbundwerkstoffen

Die Einarbeitung von nanoskaligen Füllstoffen zur Steigerung von polymeren Eigenschaftsprofilen ist sehr viel versprechend und stößt daher heutzutage sowohl in der Forschung als auch in der Industrie auf großes Interesse. Bedingt durch ausgeprägte Oberflächen und hohe Anziehungskräfte, liegen Nanopartikel allerdings nicht singulär sondern als Partikelanhäufungen, so genannten Agglomeraten oder Aggregaten, vor. Zur Erzielung der gewünschten Materialverbesserungen gilt es, diese aufzuspalten und homogen in der polymeren Matrix zu verteilen. Bei thermoplastischen Kunststoffen ist die gleichläufige Doppelschneckenextrusion eines der gängigsten Verfahren zur Einarbeitung von Additiven und Füllstoffen. Aus diesem Grund war es Ziel dieser Arbeit, mittels dieses Verfahrens verbesserte Verbundwerkstoffe mit Polyamid 66- und Polyetheretherketon-Matrix, durch Einarbeitung von nanoskaligem Titandioxid (15 und 300 nm), zu generieren. In einem ersten Schritt wurden die verfahrenstechnischen Parameter, wie Drehzahl und Durchsatz, sowie die Prozessführung und damit deren Einfluss auf die Materialeigenschaften beleuchtet. Der spezifische Energieeintrag ist ausschlaggebend zur Deagglomeration der Nanopartikel. Dieser zeigte leichte Abhängigkeiten von der Drehzahl und dem Durchsatz und verursachte bei der Einarbeitung der Partikel keine wesentlichen Unterschiede in der Aufspaltung der Partikel sowie gar keine in den resultierenden mechanischen Eigenschaften. Die Prozessführung wurde unterteilt in Mehrfach- und Einfachextrusion. Die Herstellung eines hochgefüllten Masterbatches, dessen mehrfaches Extrudieren und anschließendes Verdünnen, führte zu einer sehr guten Deagglomeration und stark verbesserten Materialeigenschaften. Mittels Simulation des Extrusionsprozesses konnte festgestellt werden, dass das Vorhandensein von ungeschmolzenem Granulat in der Verfahrenszone zu einer Schmelze/Nanopartikel/ Feststoffreibung führt, die die Ursache für eine sehr gute Aufspaltung der Partikel zu sein scheint. Durch Modifikation des Extrusionsprozesses erreichte die Einfachextrusion annähernd den Grad an Deagglomeration bei Mehrfachextrusion, wobei die Materialien bei letzterem Verfahren die besten Eigenschaftsprofile aufwiesen. In einem zweiten Schritt wurde ein Vergleich der Einflüsse von unterschiedlichen Partikelgrößen und –gehalten auf die polymeren Matrizes vollzogen. Die 15 nm Partikel zeigten signifikant bessere mechanische Ergebnisse auf als die 300 nm Partikel, und die Wirkungsweise des 15 nm Partikels auf Polyetheretherketon war stärker als auf Polyamid 66. Es konnten Steigerungen in Steifigkeit, Festigkeit und Zähigkeit erzielt werden. Rasterelektronenmikroskopische Aufnahmen bestätigten diese Ergebnisse. Eine Berechnung der Plan-Selbstkosten von einem Kilogramm PEEK-Nanoverbundwerkstoff im Vergleich zu einem Kilogramm unverstärktem PEEK verdeutlichte, dass ein Material kreiert wurde, welches deutlich verbesserte Eigenschaften bei gleichem Preis aufweist. Zusammenfassend konnte in dieser Arbeit ein tieferes Verständnis des Extrusionsvorganges zur Herstellung von kostengünstigen und verbesserten Thermoplasten durch das Einbringen von Nanopartikeln gewonnen werden

Synthetic biology platform technologies for antimicrobial applications

The growing prevalence of antibiotic resistance calls for new approaches in the development of antimicrobial therapeutics. Likewise, improved diagnostic measures are essential in guiding the application of targeted therapies and preventing the evolution of therapeutic resistance. Discovery platforms are also needed to form new treatment strategies and identify novel antimicrobial agents. By applying engineering principles to molecular biology, synthetic biologists have developed platforms that improve upon, supplement, and will perhaps supplant traditional broad-spectrum antibiotics. Efforts in engineering bacteriophages and synthetic probiotics demonstrate targeted antimicrobial approaches that can be fine-tuned using synthetic biology-derived principles. Further, the development of paper-based, cell-free expression systems holds promise in promoting the clinical translation of molecular biology tools for diagnostic purposes. In this review, we highlight emerging synthetic biology platform technologies that are geared toward the generation of new antimicrobial therapies, diagnostics, and discovery channels.Defense Threat Reduction Agency (DTRA) (Grant HDTRA1-14-1-0006)Defense Threat Reduction Agency (DTRA) (Grant HDTRA1-15-1-0051

Trapping of bacteria near the constriction.

<p>Fluorescence intensity (arb) (a) and time–lapse images (b) for aggregation of <i>S. mitis</i> SF100, PA14 WT, and PA14 <i>mucA</i> when exposed to a constant voltage difference of 45 V for t = 10–110 seconds. Away from the constriction, electrophoretic force acting on the bacteria dominates over electroosmotic fluid flow and moves the bacteria from right to left towards the negative electrode. Close to the constriction, positive dielectrophoresis pulls the bacteria back into the constriction. Trapping behavior was observed in PA14 <i>mucA</i> and SF100, but not in PA14 WT, while aggregation was only observed in PA14 <i>mucA</i>. The region of integration is shown in (b).</p

Summary of the investigated bacterial strains.

<p>Summary of the investigated bacterial strains.</p

Linear sweep trapping intensity varies across strains of bacteria.

<p>Fluorescence intensity as a function of time as voltage was swept at 1/s for <i>P. aeruginosa</i> PA01 WT (a), <i>cupA1</i> (b), <i>flgK</i> (c), <i>mucA</i> (d), <i>pelA</i> (e), <i>pilC</i> (f), <i>pvrR</i> (g), <i>S. mitis</i> SF100 (h), and PS344 (i) compared to <i>P. aeruginosa</i> PA14 WT.</p

3DiDEP chip design.

<p>Photographs of microfluidic chip (a) during fabrication, and (b) micrograph of the contraction region of the channel. (c) CAD rendering of the micro channel highlighting the constriction region. (d) Sample inverted fluorescent image of <i>S. mitis</i> SF100 trapping during 3DiDEP. Scale bars are 1 cm (a), and 100 <i>μ</i>m (b and d).</p

A low-cost paper-based synthetic biology platform for analyzing gut microbiota and host biomarkers

There is a need for large-scale, longitudinal studies to determine the mechanisms by which the gut microbiome and its interactions with the host affect human health and disease. Current methods for profiling the microbiome typically utilize next-generation sequencing applications that are expensive, slow, and complex. Here, we present a synthetic biology platform for affordable, on-demand, and simple analysis of microbiome samples using RNA toehold switch sensors in paper-based, cell-free reactions. We demonstrate species-specific detection of mRNAs from 10 different bacteria that affect human health and four clinically relevant host biomarkers. We develop a method to quantify mRNA using our toehold sensors and validate our platform on clinical stool samples by comparison to RT-qPCR. We further highlight the potential clinical utility of the platform by showing that it can be used to rapidly and inexpensively detect toxin mRNA in the diagnosis of Clostridium difficile infections.National Institutes of Health (U.S.) (Grant T32-DK007191