Modeling Hybridization Kinetics of Gene Probes in a DNA Biochip Using FEMLAB.

Microfluidic DNA biochips capable of detecting specific DNA sequences are useful in medical diagnostics, drug discovery, food safety monitoring and agriculture. They are used as miniaturized platforms for analysis of nucleic acids-based biomarkers. Binding kinetics between immobilized single stranded DNA on the surface and its complementary strand present in the sample are of interest. To achieve optimal sensitivity with minimum sample size and rapid hybridization, ability to predict the kinetics of hybridization based on the thermodynamic characteristics of the probe is crucial. In this study, a computer aided numerical model for the design and optimization of a flow-through biochip was developed using a finite element technique packaged software tool (FEMLAB; package included in COMSOL Multiphysics) to simulate the transport of DNA through a microfluidic chamber to the reaction surface. The model accounts for fluid flow, convection and diffusion in the channel and on the reaction surface. Concentration, association rate constant, dissociation rate constant, recirculation flow rate, and temperature were key parameters affecting the rate of hybridization. The model predicted the kinetic profile and signal intensities of eighteen 20-mer probes targeting vancomycin resistance genes (VRGs). Predicted signal intensities and hybridization kinetics strongly correlated with experimental data in the biochip (R² = 0.8131).

Escherichia coli expressing endoglucanase gene from Thai higher termite bacteria for enzymatic and microbial hydrolysis of cellulosic materials

Background: Endoglucanase plays a major role in initiating cellulose hydrolysis. Various wild-type strains were searched to produce this enzyme, but mostly low extracellular enzyme activities were obtained. To improve extracellular enzyme production for potential industrial applications, the endoglucanase gene of Bacillus subtilis M015, isolated from Thai higher termite, was expressed in a periplasmic-leaky Escherichia coli. Then, the crude recombinant endoglucanase (EglS) along with a commercial cellulase (Cel) was used for hydrolyzing celluloses and microbial hydrolysis using whole bacterial cells. Results: E. coli Glu5 expressing endoglucanase at high levels was successfully constructed. It produced EglS (55 kDa) with extracellular activity of 18.56 U/mg total protein at optimal hydrolytic conditions (pH 4.8 and 50°C). EglS was highly stable (over 80% activity retained) at 40­50°C after 100 h. The addition of EglS significantly improved the initial sugar production rates of Cel on the hydrolysis of carboxymethyl cellulose (CMC), microcrystalline cellulose, and corncob about 5.2-, 1.7-, and 4.0-folds, respectively, compared to those with Cel alone. E. coli Glu5 could secrete EglS with high activity in the presence of glucose (1% w/v) and Tween 80 (5% w/v) with low glucose consumption. Microbial hydrolysis of CMC using E. coli Glu5 yielded 26 mg reducing sugar/g CMC at pH 7.0 and 37°C after 48 h. Conclusions: The recombinant endoglucanase activity improved by 17 times compared with that of the native strain and could greatly enhance the enzymatic hydrolysis of all studied celluloses when combined with a commercial cellulase.

Diagnostic microarray for 14 water and foodborne pathogens using a flatbed scanner.

Parallel detection approaches are of interest to many researchers interested in identifying multiple water and foodborne pathogens simultaneously. Availability and cost-effectiveness are two key factors determining the usefulness of such approaches for laboratories with limited resources. In this study, we developed and validated a high-density microarray for simultaneous screening of 14 bacterial pathogens using an approach that employs gold labeling with silver enhancement (GLS) protocol. In total, 8887 probes (50-mer) were designed using an in-house database of virulence and marker genes (VMGs), and synthesized in quadruplicate on glass slides using an in-situ synthesis technology. Target VMG amplicons were obtained using multiplex polymerase chain reaction (PCR), labeled with biotin, and hybridized to the microarray. The signals generated after gold deposition and silver enhancement, were quantified using a flatbed scanner having 2-µm resolution. Data analysis indicated that reliable presence/absence calls could be made, if: i) over four probes were used per gene, ii) the signal-to-noise ratio (SNR) cutoff was greater than or equal to two, and iii) the positive fraction (PF), i.e., number of probes with SNR≥2 for a given VMG was greater than 0.75. Hybridization of the array with blind samples resulted in 100% correct calls, and no false positive. Because amplicons were obtained by multiplex PCR, sensitivity of this method is similar to PCR. This assay is an inexpensive and reliable technique for high throughput screening of multiple pathogens.

Static self-directed sample dispensing into a series of reaction wells on a microfluidic card for parallel genetic detection of microbial pathogens.

A microfluidic card is described for simultaneous and rapid genetic detection of multiple microbial pathogens. The hydrophobic surface of native acrylic and a novel microfluidic mechanism termed "airlock" were used to dispense sample into a series of 64 reaction wells without the use of valves, external pumping peripherals, multiple layers, or vacuum assistance. This airlock mechanism was tested with dilutions of whole human blood, saliva, and urine, along with mock samples of varying viscosities and surface tensions. Samples spiked with genomic DNA (gDNA) or crude lysates from clinical bacterial isolates were tested with loop mediated isothermal amplification assays (LAMP) designed to target virulence and antibiotic resistance genes. Reactions were monitored in real time using the Gene-Z, which is a portable smartphone-driven system. Samples loaded correctly into the microfluidic card in 99.3% of instances. Amplification results confirmed no carryover of pre-dispensed primer between wells during sample loading, and no observable diffusion between adjacent wells during the 60 to 90 min isothermal reaction. Sensitivity was comparable between LAMP reactions tested within the microfluidic card and in conventional vials. Tests demonstrate that the airlock card works with various sample types, manufacturing techniques, and can potentially be used in many point-of-care diagnostics applications.

Combined in vitro transcription and reverse transcription to amplify and label complex synthetic oligonucleotide probe libraries.

Oligonucleotide microarrays allow the production of complex custom oligonucleotide libraries for nucleic acid detection-based applications such as fluorescence in situ hybridization (FISH). We have developed a PCR-free method to make single-stranded DNA (ssDNA) fluorescent probes through an intermediate RNA library. A double-stranded oligonucleotide library is amplified by transcription to create an RNA library. Next, dye- or hapten-conjugate primers are used to reverse transcribe the RNA to produce a dye-labeled cDNA library. Finally the RNA is hydrolyzed under alkaline conditions to obtain the single-stranded fluorescent probes library. Starting from unique oligonucleotide library constructs, we present two methods to produce single-stranded probe libraries. The two methods differ in the type of reverse transcription (RT) primer, the incorporation of fluorescent dye, and the purification of fluorescent probes. The first method employs dye-labeled reverse transcription primers to produce multiple differentially single-labeled probe subsets from one microarray library. The fluorescent probes are purified from excess primers by oligonucleotide-bead capture. The second method uses an RNA:DNA chimeric primer and amino-modified nucleotides to produce amino-allyl probes. The excess primers and RNA are hydrolyzed under alkaline conditions, followed by probe purification and labeling with amino-reactive dyes. The fluorescent probes created by the combination of transcription and reverse transcription can be used for FISH and to detect any RNA and DNA targets via hybridization.

Secretory expression and characterization of two hemicellulases, xylanase, and ß-xylosidase, isolated from Bacillus subtilis M015.

Microbial hydrolysis of lignocellulosic biomass is becoming increasingly important for the production of renewable biofuels to address global energy concerns. Hemicellulose is the second most abundant lignocellulosic biopolymer consisting of mostly xylan and other polysaccharides. A variety of enzymes is involved in complete hydrolysis of xylan into its constituent sugars for subsequent biofuel fermentation. Two enzymes, endo-ß-xylanase and ß-xylosidase, are particularly important in hydrolyzing the xylan backbone into xylooligosaccharides and individual xylose units. In this study, we describe the cloning, expression, and characterization of xylanase and ß-xylosidase isolated from Bacillus subtilis M015 in Escherichia coli. The genes were identified to encode a 213 amino acid protein for xylanase (glycoside hydrolase (GH) family 11) and a 533 amino acid protein for ß-xylosidase (GH family 43). Recombinant enzymes were produced by periplasmic-leaky E. coli JE5505 and therefore secreted into the supernatant during growth. Temperature and pH optima were determined to be 50 °C and 5.5-6 for xylanase and 35 °C and 7.0-7.5 for ß-xylosidase using beech wood xylan and p-nitrophenyl-ß-D-xylopyranoside as the substrates, respectively. We have also investigated the synergy of two enzymes on xylan hydrolysis and observed 90 % increase in total sugar release (composed of xylose, xylobiose, xylotriose, and xylotetraose) for xylanase/ß-xylosidase combination as opposed to xylanase alone.

Methods for the preparation of large quantities of complex single-stranded oligonucleotide libraries.

Custom-defined oligonucleotide collections have a broad range of applications in fields of synthetic biology, targeted sequencing, and cytogenetics. Also, they are used to encode information for technologies like RNA interference, protein engineering and DNA-encoded libraries. High-throughput parallel DNA synthesis technologies developed for the manufacture of DNA microarrays can produce libraries of large numbers of different oligonucleotides, but in very limited amounts. Here, we compare three approaches to prepare large quantities of single-stranded oligonucleotide libraries derived from microarray synthesized collections. The first approach, alkaline melting of double-stranded PCR amplified libraries with a biotinylated strand captured on streptavidin coated magnetic beads results in little or no non-biotinylated ssDNA. The second method wherein the phosphorylated strand of PCR amplified libraries is nucleolyticaly hydrolyzed is recommended when small amounts of libraries are needed. The third method combining in vitro transcription of PCR amplified libraries to reverse transcription of the RNA product into single-stranded cDNA is our recommended method to produce large amounts of oligonucleotide libraries. Finally, we propose a method to remove any primer binding sequences introduced during library amplification.

Detection and characterization of human pathogenic viruses circulating in community wastewater using multi target microarrays and polymerase chain reaction.

Sewage pollution remains the most significant source of human waterborne pathogens. This study describes the detection and characterization of human enteric viruses in community wastewaters using cell culture coupled with multiple target microarrays (with a total of 780 unique probes targeting 27 different groups of both DNA and RNA viruses) and polymerase chain reaction (PCR) assays. Over a 13-month sampling period, RNA viruses (astroviruses and enteroviruses) were more frequently detected compared to DNA viruses (adenoviruses, particularly type 41 and BK polyomavirus). Overall, many more viruses were shed during the winter months (December-February) compared to the summer months. Exploration of the multiple types of enteric viruses particularly in winter months identified much more significant prevalence of key viral pathogens associated with sewage pollution of the water environment than previously realized and seasonal disinfection used in some parts of the world may lead to a seeding of ambient waters. Molecular characterization of pathogenic viruses in community wastewater will improve the understanding of the potential risk of waterborne disease transmission of viral pathogens.

A conjugated polymer-peptide hybrid system for prostate-specific antigen (PSA) detection.

We developed fast and readily applicable microarray chips to detect PSA by designing a novel conjugated polymer (energy donor) and combining it with on-chip peptide synthesis. The selective cleavage of a probing peptide labelled with a dye or a quencher (energy acceptor) produced a fluorescence sensory signal via fluorescent energy resonance transfer (FRET).

From design to screening: a new antimicrobial peptide discovery pipeline.

Antimicrobial peptides (AMPs) belong to a class of natural microbicidal molecules that have been receiving great attention for their lower propensity for inducing drug resistance, hence, their potential as alternative drugs to conventional antibiotics. By generating AMP libraries, one can study a large number of candidates for their activities simultaneously in a timely manner. Here, we describe a novel methodology where in silico designed AMP-encoding oligonucleotide libraries are cloned and expressed in a cellular host for rapid screening of active molecules. The combination of parallel oligonucleotide synthesis with microbial expression systems not only offers complete flexibility for sequence design but also allows for economical construction of very large peptide libraries. An application of this approach to discovery of novel AMPs has been demonstrated by constructing and screening a custom library of twelve thousand plantaricin-423 mutants in Escherichia coli. Analysis of selected clones by both Sanger-sequencing and 454 high-throughput sequencing produced a significant amount of data for positionally important residues of plantaricin-423 responsible for antimicrobial activity and, moreover, resulted in identification of many novel variants with enhanced specific activities against Listeria innocua. This approach allows for generation of fully tailored peptide collections in a very cost effective way and will have countless applications from discovery of novel AMPs to gaining fundamental understanding of their biological function and characteristics.

Identification of non-specific hybridization using an empirical equation fitted to non-equilibrium dissociation curves.

Non-equilibrium dissociation curves (NEDCs) have the potential to identify non-specific hybridizations on high throughput, diagnostic microarrays. We report a simple method for the identification of non-specific signals by using a new parameter that does not rely on comparison of perfect match and mismatch dissociations. The parameter is the ratio of specific dissociation temperature (T(d-w)) to theoretical melting temperature (T(m)) and can be obtained by automated fitting of a four-parameter, sigmoid, empirical equation to the thousands of curves generated in a typical experiment. The curves fit perfect match NEDCs from an initial experiment with an R(2) of 0.998±0.006 and root mean square of 108±91 fluorescent units. Receiver operating characteristic curve analysis showed low temperature hybridization signals (20-48°C) to be as effective as area under the curve as primary data filters. Evaluation of three datasets that target 16S rRNA and functional genes with varying degrees of target sequence similarity showed that filtering out hybridizations with T(d-w)/T(m)<0.78 greatly reduced false positive results. In conclusion, T(d-w)/T(m) successfully screened many non-specific hybridizations that could not be identified using single temperature signal intensities alone, while the empirical modeling allowed a simplified approach to the high throughput analysis of thousands of NEDCs.

Gene-Z: a device for point of care genetic testing using a smartphone.

By 2012, point of care (POC) testing will constitute roughly one third of the $59 billion in vitro diagnostics market. The ability to carry out multiplexed genetic testing and wireless connectivity are emerging as key attributes of future POC devices. In this study, an inexpensive, user-friendly and compact device (termed Gene-Z) is presented for rapid quantitative detection of multiple genetic markers with high sensitivity and specificity. Using a disposable valve-less polymer microfluidic chip containing four arrays of 15 reaction wells each with dehydrated primers for isothermal amplification, the Gene-Z enables simultaneous analysis of four samples, each for multiple genetic markers in parallel, requiring only a single pipetting step per sample for dispensing. To drastically reduce the cost and size of the real-time detector necessary for quantification, loop-mediated isothermal amplification (LAMP) was performed with a high concentration of SYTO-81, a non-inhibiting fluorescent DNA binding dye. The Gene-Z is operated using an iPod Touch, which also receives data and carries out automated analysis and reporting via a WiFi interface. This study presents data pertaining to performance of the device including sensitivity and reproducibility using genomic DNA from Escherichia coli and Staphylococcus aureus. Overall, the Gene-Z represents a significant step toward truly inexpensive and compact tools for POC genetic testing.

Evolution combined with genomic study elucidates genetic bases of isobutanol tolerance in Escherichia coli.

BACKGROUND: Isobutanol is a promising next-generation biofuel with demonstrated high yield microbial production, but the toxicity of this molecule reduces fermentation volumetric productivity and final titer. Organic solvent tolerance is a complex, multigenic phenotype that has been recalcitrant to rational engineering approaches. We apply experimental evolution followed by genome resequencing and a gene expression study to elucidate genetic bases of adaptation to exogenous isobutanol stress. RESULTS: The adaptations acquired in our evolved lineages exhibit antagonistic pleiotropy between minimal and rich medium, and appear to be specific to the effects of longer chain alcohols. By examining genotypic adaptation in multiple independent lineages, we find evidence of parallel evolution in marC, hfq, mdh, acrAB, gatYZABCD, and rph genes. Many isobutanol tolerant lineages show reduced RpoS activity, perhaps related to mutations in hfq or acrAB. Consistent with the complex, multigenic nature of solvent tolerance, we observe adaptations in a diversity of cellular processes. Many adaptations appear to involve epistasis between different mutations, implying a rugged fitness landscape for isobutanol tolerance. We observe a trend of evolution targeting post-transcriptional regulation and high centrality nodes of biochemical networks. Collectively, the genotypic adaptations we observe suggest mechanisms of adaptation to isobutanol stress based on remodeling the cell envelope and surprisingly, stress response attenuation. CONCLUSIONS: We have discovered a set of genotypic adaptations that confer increased tolerance to exogenous isobutanol stress. Our results are immediately useful to further efforts to engineer more isobutanol tolerant host strains of E. coli for isobutanol production. We suggest that rpoS and post-transcriptional regulators, such as hfq, RNA helicases, and sRNAs may be interesting mutagenesis targets for future global phenotype engineering.

Microfluidic Reactor Array Device for Massively Parallel In-situ Synthesis of Oligonucleotides.

We have designed and fabricated a microfluidic reactor array device for massively parallel in-situ synthesis of oligonucleotides (oDNA). The device is made of glass anodically bonded to silicon consisting of three level features: microreactors, microchannels and through inlet/outlet holes. Main challenges in the design of this device include preventing diffusion of photogenerated reagents upon activation and achieving uniform reagent flow through thousands of parallel reactors. The device embodies a simple and effective dynamic isolation mechanism which prevents the intermixing of active reagents between discrete microreactors. Depending on the design parameters, it is possible to achieve uniform flow and synthesis reaction in all of the reactors by proper design of the microreactors and the microchannels. We demonstrated the use of this device on a solution-based, light-directed parallel in-situ oDNA synthesis. We were able to synthesize long oDNA, up to 120 mers at stepwise yield of 98 %. The quality of our microfluidic oDNA microarray including sensitivity, signal noise, specificity, spot variation and accuracy was characterized. Our microfluidic reactor array devices show a great potential for genomics and proteomics researches.

OligoArrayDb: pangenomic oligonucleotide microarray probe sets database.

OligoArrayDb is a comprehensive database containing pangenomic oligonucleotide microarray probe sets designed for most of the sequenced genomes that are not covered by commercial catalog arrays. The availability of probe sequences, associated with custom microarray fabrication services offered by many companies and cores presents the unequalled possibility to perform microarray experiments on most of the sequenced organisms. OligoArrayDb contains more than 2.8 probes per gene in average for more than 600 organisms, mostly archaea and bacteria strains available from public database. On average, 98% of the annotated genes have at least one probe which is predicted to be specific to its intended target in >94% of the cases. OligoArrayDb is weekly updated as new sequenced genomes become available. Probe sequences, in addition to a comprehensive set of annotations can be downloaded from this database. OligoArrayDb is publicly accessible online at http://berry.engin.umich.edu/oligoarraydb.

Development and experimental validation of a predictive threshold cycle equation for quantification of virulence and marker genes by high-throughput nanoliter-volume PCR on the OpenArray platform.

Development of quantitative PCR (QPCR) assays typically requires extensive screening within and across a given species to ensure specific detection and lucid identification among various pathogenic and nonpathogenic strains and to generate standard curves. To minimize screening requirements, multiple virulence and marker genes (VMGs) were targeted simultaneously to enhance reliability, and a predictive threshold cycle (C(T)) equation was developed to calculate the number of starting copies based on an experimental C(T). The empirical equation was developed with Sybr green detection in nanoliter-volume QPCR chambers (OpenArray) and tested with 220 previously unvalidated primer pairs targeting 200 VMGs from 30 pathogens. A high correlation (R(2) = 0.816) was observed between the predicted and experimental C(T)s based on the organism's genome size, guanine and cytosine (GC) content, amplicon length, and stability of the primer's 3' end. The performance of the predictive C(T) equation was tested using 36 validation samples consisting of pathogenic organisms spiked into genomic DNA extracted from three environmental waters. In addition, the primer success rate was dependent on the GC content of the target organisms and primer sequences. Targeting multiple assays per organism and using the predictive C(T) equation are expected to reduce the extent of the validation necessary when developing QPCR arrays for a large number of pathogens or other targets.

A light writable microfluidic "flash memory": optically addressed actuator array with latched operation for microfluidic applications.

This paper presents a novel optically addressed microactuator array (microfluidic "flash memory") with latched operation. Analogous to the address-data bus mediated memory address protocol in electronics, the microactuator array consists of individual phase-change based actuators addressed by localized heating through focused light patterns (address bus), which can be provided by a modified projector or high power laser pointer. A common pressure manifold (data bus) for the entire array is used to generate large deflections of the phase change actuators in the molten phase. The use of phase change material as the working media enables latched operation of the actuator array. After the initial light "writing" during which the phase is temporarily changed to molten, the actuated status is self-maintained by the solid phase of the actuator without power and pressure inputs. The microfluidic flash memory can be re-configured by a new light illumination pattern and common pressure signal. The proposed approach can achieve actuation of arbitrary units in a large-scale array without the need for complex external equipment such as solenoid valves and electrical modules, which leads to significantly simplified system implementation and compact system size. The proposed work therefore provides a flexible, energy-efficient, and low cost multiplexing solution for microfluidic applications based on physical displacements. As an example, the use of the latched microactuator array as "normally closed" or "normally open" microvalves is demonstrated. The phase-change wax is fully encapsulated and thus immune from contamination issues in fluidic environments.

In situ-synthesized virulence and marker gene biochip for detection of bacterial pathogens in water.

Pathogen detection tools with high reliability are needed for various applications, including food and water safety and clinical diagnostics. In this study, we designed and validated an in situ-synthesized biochip for detection of 12 microbial pathogens, including a suite of pathogens relevant to water safety. To enhance the reliability of presence/absence calls, probes were designed for multiple virulence and marker genes (VMGs) of each pathogen, and each VMG was targeted by an average of 17 probes. Hybridization of the biochip with amplicon mixtures demonstrated that 95% of the initially designed probes behaved as predicted in terms of positive/negative signals. The probes were further validated using DNA obtained from three different types of water samples and spiked with pathogen genomic DNA at decreasing relative abundance. Excellent specificity for making presence/absence calls was observed by using a cutoff of 0.5 for the positive fraction (i.e., the fraction of probes yielding a positive signal for a given VMG). A split multiplex PCR design for simultaneous amplification of the VMGs resulted in a detection limit of between 0.1 and 0.01% relative abundance, depending on the type of pathogen and the VMG. Thermodynamic analysis of the hybridization patterns obtained with DNA from the different water samples demonstrated that probes with a hybridization Gibbs free energy of approximately -19.3 kcal/mol provided the best trade-off between sensitivity and specificity. The developed biochip may be used to detect the described bacterial pathogens in water samples when parallel and specific detection is required.

Cytophobic surface modification of microfluidic arrays for in situ parallel peptide synthesis and cell adhesion assays.

A combination of PEG-based surface passivation techniques and spatially addressable SPPS (solid-phase peptide synthesis) was used to demonstrate a highly specific cell-peptide adhesion assay on a microfluidic platform. The surface of a silicon-glass microchip was modified to form a mixed self-assembled monolayer that presented PEG moieties interspersed with reactive amino terminals. The PEG provided biomolecular inertness and the reactive amino groups were used for consequent peptide synthesis. The cytophobicity of the surface was characterized by on-chip fluorescent binding assays and was found to be resistant to nonspecific attachment of cells and proteins. An integrated system for parallel peptide synthesis on this reactive amino surface was developed using photogenerated acid chemistry and digital microlithography. A constant synthesis efficiency of >98% was observed for up to 7mer peptides. To demonstrate specific cell adhesion on these synthetic peptide arrays, variations of a 7mer cell binding peptide that binds to murine B lymphoma cells were synthesized. Sequence-specific binding was observed on incubation with fluorescently labeled, intact murine B lymphoma cells, and key residues for binding were identified by deletional analysis.

Colloids with high-definition surface structures.

Compared with the well equipped arsenal of surface modification methods for flat surfaces, techniques that are applicable to curved, colloidal surfaces are still in their infancy. This technological gap exists because spin-coating techniques used in traditional photolithographic processes are not applicable to the curved surfaces of spherical objects. By replacing spin-coated photoresist with a vapor-deposited, photodefinable polymer coating, we have now fabricated microstructured colloids with a wide range of surface patterns, including asymmetric and chiral surface structures, that so far were typically reserved for flat substrates. This high-throughput method can yield surface-structured colloidal particles at a rate of approximately 10(7) to 10(8) particles per operator per day. Equipped with spatially defined binding pockets, microstructured colloids can engage in programmable interactions, which can lead to directed self-assembly. The ability to create a wide range of colloids with both simple and complex surface patterns may contribute to the genesis of previously unknown colloidal structures and may have important technological implications in a range of different applications, including photonic and phononic materials or chemical sensors.