The plant polymer lignin is the most abundant renewable source of aromatics on the planet and conversion of it to valuable fuels and chemicals is critical to the economic viability of a lignocellulosic biofuels industry and to meeting the DOE’s 2022 goal of $\$2.50$/gallon mean biofuel selling price. Presently, there is no efficient way of converting lignin into valuable commodities. Current biological approaches require mixtures of expensive ligninolytic enzymes and engineered microbes. This project was aimed at circumventing these problems by discovering commensal relationships among fungi and bacteria involved in biological lignin utilization and using this knowledge to engineer microbial communities capable of converting lignin into renewable fuels and chemicals. Essentially, we aimed to learn from, mimic and improve on nature. We discovered fungi that synergistically work together to degrade lignin, engineered fungal systems to increase expression of the required enzymes and engineered organisms to produce products such as biodegradable plastics precursors.
The challenges of diagnosing infectious disease, especially in the developing world, and the shortcomings of available instrumentation have exposed the need for portable, easy-to-use diagnostic tools capable of detecting the wide range of causative microbes while operating in low resource settings. We present a centrifugal microfluidic platform that combines ultrasensitive immunoassay and isothermal amplification-based screening for the orthogonal detection of both protein and nucleic acid targets at the point-of-care. A disposable disc with automatic aliquoting inlets is paired with a non-contact heating system and precise rotary control system to yield an easy-to-use, field-deployable platform with versatile screening capabilities. The detection of three enterotoxins (cholera toxin, Staphylococcal enterotoxin B, and Shiga-like toxin 1) and three enteric bacteria (C. jejuni, E. coli, and S. typhimurium) were performed independently and shown to be highly sensitive (limit of detection = 1.35–5.50 ng/mL for immunoassays and 1–30 cells for isothermal amplification), highly exclusive in the presence of non-specific targets, and capable of handling a complex sample matrix like stool. The full panel of toxins and bacteria were reliably detected simultaneously on a single disc at clinically relevant sample concentrations in less than an hour. The ability of our technology to detect multiple analyte types in parallel at the point-of-care can serve a variety of needs, from routine patient care to outbreak triage, in a variety of settings to reduce disease impact and expedite effective treatment.
The RNA-guided DNA nuclease Cas9 is now widely used for the targeted modification of genomes of human cells and various organisms. Despite the extensive use of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) systems for genome engineering and the rapid discovery and engineering of new CRISPR-associated nucleases, there are no high-throughput assays for measuring enzymatic activity. The current laboratory and future therapeutic uses of CRISPR technology have a significant risk of accidental exposure or clinical off-target effects, underscoring the need for therapeutically effective inhibitors of Cas9. Here, we develop a fluorescence assay for monitoring Cas9 nuclease activity and demonstrate its utility with S. pyogenes (Spy), S. aureus (Sau), and C. jejuni (Cje) Cas9. The assay was validated by quantitatively profiling the species specificity of published anti-CRISPR (Acr) proteins, confirming the reported inhibition of Spy Cas9 by AcrIIA4 and Cje Cas9 by AcrIIC1 and no inhibition of Sau Cas9 by either anti-CRISPR. To identify drug-like inhibitors, we performed a screen of 189 606 small molecules for inhibition of Spy Cas9. Of 437 hits (0.2% hit rate), six were confirmed as Cas9 inhibitors in a direct gel electrophoresis secondary assay. The high-throughput nature of this assay makes it broadly applicable for the discovery of additional Cas9 inhibitors or the characterization of Cas9 enzyme variants.
Current multiplexed diagnostics for Zika, dengue, and chikungunya viruses are situated outside the intersection of affordability, high performance, and suitability for use at the point-of-care in resource-limited settings. Consequently, insufficient diagnostic capabilities are a key limitation facing current Zika outbreak management strategies. Here we demonstrate highly sensitive and specific detection of Zika, chikungunya, and dengue viruses by coupling reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with our recently developed quenching of unincorporated amplification signal reporters (QUASR) technique. We conduct reactions in a simple, inexpensive and portable "LAMP box" supplemented with a consumer class smartphone. The entire assembly can be powered by a 5 V USB source such as a USB power bank or solar panel. Our smartphone employs a novel algorithm utilizing chromaticity to analyze fluorescence signals, which improves the discrimination of positive/negative signals by 5-fold when compared to detection with traditional RGB intensity sensors or the naked eye. The ability to detect ZIKV directly from crude human sample matrices (blood, urine, and saliva) demonstrates our device's utility for widespread clinical deployment. Together, these advances enable our system to host the key components necessary to expand the use of nucleic acid amplification-based detection assays towards point-of-care settings where they are needed most.
Multiple displacement amplification (MDA) is a widely used technique for amplification of DNA from samples containing limited amounts of DNA (e.g., uncultivable microbes or clinical samples) before whole genome sequencing. Despite its advantages of high yield and fidelity, it suffers from high amplification bias and non-specific amplification when amplifying sub-nanogram of template DNA. Here, we present a microfluidic digital droplet MDA (ddMDA) technique where partitioning of the template DNA into thousands of sub-nanoliter droplets, each containing a small number of DNA fragments, greatly reduces the competition among DNA fragments for primers and polymerase thereby greatly reducing amplification bias. Consequently, the ddMDA approach enabled a more uniform coverage of amplification over the entire length of the genome, with significantly lower bias and non-specific amplification than conventional MDA. For a sample containing 0.1 pg/μL of E. coli DNA (equivalent of ~3/1000 of an E. coli genome per droplet), ddMDA achieves a 65-fold increase in coverage in de novo assembly, and more than 20-fold increase in specificity (percentage of reads mapping to E. coli) compared to the conventional tube MDA. ddMDA offers a powerful method useful for many applications including medical diagnostics, forensics, and environmental microbiology.
Reverse-transcription-loop-mediated isothermal amplification (RT-LAMP) has frequently been proposed as an enabling technology for simplified diagnostic tests for RNA viruses. However, common detection techniques used for LAMP and RT-LAMP have drawbacks, including poor discrimination capability, inability to multiplex targets, high rates of false positives, and (in some cases) the requirement of opening reaction tubes postamplification. Here, we present a simple technique that allows closed-tube, target-specific detection, based on inclusion of a dye-labeled primer that is incorporated into a target-specific amplicon if the target is present. A short, complementary quencher hybridizes to unincorporated primer upon cooling down at the end of the reaction, thereby quenching fluorescence of any unincorporated primer. Our technique, which we term QUASR (for quenching of unincorporated amplification signal reporters, read "quasar"), does not significantly reduce the amplification efficiency or sensitivity of RT-LAMP. Equipped with a simple LED excitation source and a colored plastic gel filter, the naked eye or a camera can easily discriminate between positive and negative QUASR reactions, which produce a difference in signal of approximately 10:1 without background subtraction. We demonstrate that QUASR detection is compatible with complex sample matrices such as human blood, using a novel LAMP primer set for bacteriophage MS2 (a model RNA virus particle). Furthermore, we demonstrate single-tube duplex detection of West Nile virus (WNV) and chikungunya virus (CHIKV) RNA.
Portable, sensitive, easy-to-use diagnostics are urgently needed to meet the growing need for advanced healthcare in the developing world. As the recent outbreaks of infectious diseases have demonstrated, early detection and treatment are vital tools to containing outbreaks and minimizing loss of life. Toward addressing these concerns, we have developed a centrifugal microfluidic platform capable of detecting both proteins and nucleic acids signatures from biological threats. This platform utilizes a novel sedimentation assay format to integrate sample preparation into a single step. Platform performance is competitive with traditional benchtop techniques.
Arboviruses (viruses spread by arthropod vectors like mosquitoes and ticks) represent a significant burden to public health and agriculture. In most cases, vector control is the only effective measure to stop the spread of these pathogens. We present a novel approach to field-based detection of mosquito-borne viruses, using a device called the "Smart Trap" which automates all steps of a sugarbased surveillance assay, providing daily reports from a network of bait stations placed in the field.