Universal Enzyme-Based Field Workflow for Rapid and Sensitive Quantification of Water Pathogens.

A universal filtration and enzyme-based workflow has been established to allow for the rapid and sensitive quantification of leading pathogens Cryptosporidium parvum, Giardia gamblia, Campylobacter jejuni, and Escherichia coli from tap water samples with volumes up to 100 mL, and the potential to scale up to larger volumes. qPCR limits of quantification as low as four oocysts for Cryptosporidium, twelve cysts for Giardia, two cells for C. jejuni, and nineteen cells for E. coli per reaction were achieved. A polycarbonate filter-based sampling method coupled with the prepGEM enzyme-based DNA extraction system created a single-step transfer workflow that required as little as 20 min of incubation time and a 100 µL reaction mix. The quantification via qPCR was performed directly on the prepGEM extract, bypassing time-consuming, labour-intensive conventional culture-based methods. The tap water samples were shown to contain insoluble particles that inhibited detection by reducing the quantification efficiency of a representative pathogen (C. jejuni) to 30-60%. This sample inhibition was effectively removed by an on-filter treatment of 20% (v/v) phosphoric acid wash. Overall, the established workflow was able to achieve quantification efficiencies of 92% and higher for all four leading water pathogens, forming the basis of a rapid, portable, and low-cost solution to water monitoring.

Smartphone technology facilitates point-of-care nucleic acid diagnosis: a beginner's guide.

The reliable detection of nucleic acids at low concentrations in clinical samples like blood, urine and saliva, and in food can be achieved by nucleic acid amplification methods. Several portable and hand-held devices have been developed to translate these laboratory-based methods to point-of-care (POC) settings. POC diagnostic devices could potentially play an important role in environmental monitoring, health, and food safety. Use of a smartphone for nucleic acid testing has shown promising progress in endpoint as well as real-time analysis of various disease conditions. The emergence of smartphone-based POC devices together with paper-based sensors, microfluidic chips and digital droplet assays are used currently in many situations to provide quantitative detection of nucleic acid targets. State-of-the-art portable devices are commercially available and rapidly emerging smartphone-based POC devices that allow the performance of laboratory-quality colorimetric, fluorescent and electrochemical detection are described in this review. We present a comprehensive review of smartphone-based POC sensing applications, specifically on microbial diagnostics, assess their performance and propose recommendations for the future.

A novel framework for the cell-free enzymatic production of glucaric acid.

Glucaric acid (GlucA) is a valuable glucose-derived chemical with promising applications as a biodegradable and biocompatible chemical in the manufacturing of plastics, detergents and drugs. Recently, there has been a significant focus on producing GlucA microbially (in vivo) from renewable materials such as glucose, sucrose and myo-inositol. However, these in vivo GlucA production processes generally lack efficiency due to toxicity problems, metabolite competition and suboptimal enzyme ratios. Synthetic biology and accompanying cell-free biocatalysis have been proposed as a viable approach to overcome many of these limitations. However, cell-free biocatalysis faces its own limitations for industrial applications due to high enzyme costs and cofactor consumption. We have constructed a cell-free GlucA pathway and demonstrated a novel framework to overcome limitations of cell-free biocatalysis by i) the combination of both thermostable and mesophilic enzymes, ii) incorporation of a cofactor regeneration system and iii) immobilisation and recycling of the pathway enzymes. The cell-free production of GlucA was achieved from glucose-1-phosphate with a titre of 14.1 ±â€¯0.9 mM (3.0 ±â€¯0.2 g l-1) and a molar yield of 35.2 ±â€¯2.3% using non-immobilised enzymes, and a titre of 8.1 ±â€¯0.2 mM (1.70 ±â€¯0.04 g l-1) and a molar yield of 20.2 ±â€¯0.5% using immobilised enzymes with a total reaction time of 10 h. The resulting productivities (0.30 ±â€¯0.02 g/h/l for free enzymes and 0.170 ±â€¯0.004 g/h/l for immobilised enzymes) are the highest productivities so far reported for glucaric acid production using a synthetic enzyme pathway.

A portable nucleic acid detection system using natural convection combined with a smartphone.

The development of portable nucleic acid diagnostic devices has the potential to expand the availability of molecular diagnostics into low-resource settings. One of the promising solutions for rapid and simple DNA amplification is the use of Rayleigh-Bernard natural convection which is caused by a buoyancy-driven thermal gradient of liquid when heated from below. This natural convection avoids the use of the complex and sophisticated hardware that is required for precise maintenance of temperature cycles in conventional PCR. We have developed a stand-alone convective PCR (cPCR) device linked to a smartphone for rapid detection of nucleic acids using natural convection heating. The device amplifies multiple DNA samples simultaneously using a custom-made heat block controlled by Bluetooth wireless communication. The entire device is highly portable, user-friendly, battery-operated and can provide target DNA amplification in less than 30 min. A detection limit of 2.8 × 103 copies of a segment of lambda DNA was obtained when the two different fluorescently-tagged amplicons were collected magnetically and detected using the smartphone fluorescence reader. Thus, the combination of cPCR and multiplex fluorescence-based detection on a smartphone provides new opportunities for the development of affordable and portable molecular diagnostic devices for point-of-care situations or remote clinical settings.

Mixed-mode liquid chromatography for the rapid analysis of biocatalytic glucaric acid reaction pathways.

Glucaric acid (GlucA) has been identified as one of the top 10 potential bio-based chemicals for replacement of oil-based chemicals. Several synthetic enzyme pathways have been engineered in bacteria and yeast to produce GlucA from glucose and myo-inositol. However, the yields and titres achieved with these systems remain too low for the requirements of a bio-based GlucA industry. A major limitation for the optimisation of GlucA production via synthetic enzymatic pathways are the laborious analytical procedures required to detect the final product (GlucA) and pathway intermediates. We have developed a novel method for the simple and simultaneous analysis of GlucA and pathway intermediates to address this limitation using mixed mode (MM) HILIC and weak anion exchange chromatography (WAX), referred to as MM HILIC/WAX, coupled with RID. Isocratic mobile phase conditions and the sample solvent were optimised for the separation of GlucA, glucose-1-phosphate (G1P), glucose-6-phosphate (G6P), inositol-1-phosphate (I1P), myo-inositol and glucuronic acid (GA). The method showed good repeatability, precision and excellent accuracy with detection and quantitation limits (LOD and LOQ) of 1.5-2 and 577 mM, respectively. The method developed was used for monitoring the enzymatic synthesis of the final step in the GlucA pathway, and showed that GlucA was produced from GA with near 100% conversion and a titre of 9.2 g L-1.

Tools and strategies for constructing cell-free enzyme pathways.

Single enzyme systems or engineered microbial hosts have been used for decades but the notion of assembling multiple enzymes into cell-free synthetic pathways is a relatively new development. The extensive possibilities that stem from this synthetic concept makes it a fast growing and potentially high impact field for biomanufacturing fine and platform chemicals, pharmaceuticals and biofuels. However, the translation of individual single enzymatic reactions into cell-free multi-enzyme pathways is not trivial. In reality, the kinetics of an enzyme pathway can be very inadequate and the production of multiple enzymes can impose a great burden on the economics of the process. We examine here strategies for designing synthetic pathways and draw attention to the requirements of substrates, enzymes and cofactor regeneration systems for improving the effectiveness and sustainability of cell-free biocatalysis. In addition, we comment on methods for the immobilisation of members of a multi-enzyme pathway to enhance the viability of the system. Finally, we focus on the recent development of integrative tools such as in silico pathway modelling and high throughput flux analysis with the aim of reinforcing their indispensable role in the future of cell-free biocatalytic pathways for biomanufacturing.

Solid-Binding Peptides in Biomedicine.

Some peptides are able to bind to inorganic materials such as silica and gold. Over the past decade, Solid-binding peptides (SBPs) have been used increasingly as molecular building blocks in nanobiotechnology. These peptides show selectivity and bind with high affinity to a diverse range of inorganic surfaces e.g. metals, metal oxides, metal compounds, magnetic materials, semiconductors, carbon materials, polymers and minerals. They can be used in applications such as protein purification and synthesis, assembly and the functionalization of nanomaterials. They offer simple and versatile bioconjugation methods that can increase biocompatibility and also direct the immobilization and orientation of nanoscale entities onto solid supports without impeding their functionality. SBPs have been employed in numerous nanobiotechnological applications such as the controlled synthesis of nanomaterials and nanostructures, formation of hybrid biomaterials, immobilization of functional proteins and improved nanomaterial biocompatibility. With advances in nanotechnology, a multitude of novel nanomaterials have been designed and synthesized for diagnostic and therapeutic applications. New approaches have been developed recently to exert a greater control over bioconjugation and eventually, over the optimal and functional display of biomolecules on the surfaces of many types of solid materials. In this chapter we describe SBPs and highlight some selected examples of their potential applications in biomedicine.

Solid-binding peptides for immobilisation of thermostable enzymes to hydrolyse biomass polysaccharides.

BACKGROUND: Solid-binding peptides (SBPs) bind strongly to a diverse range of solid materials without the need for any chemical reactions. They have been used mainly for the functionalisation of nanomaterials but little is known about their use for the immobilisation of thermostable enzymes and their feasibility in industrial-scale biocatalysis. RESULTS: A silica-binding SBP sequence was fused genetically to three thermostable hemicellulases. The resulting enzymes were active after fusion and exhibited identical pH and temperature optima but differing thermostabilities when compared to their corresponding unmodified enzymes. The silica-binding peptide mediated the efficient immobilisation of each enzyme onto zeolite, demonstrating the construction of single enzyme biocatalytic modules. Cross-linked enzyme aggregates (CLEAs) of enzyme preparations either with or without zeolite immobilisation displayed greater activity retention during enzyme recycling than those of free enzymes (without silica-binding peptide) or zeolite-bound enzymes without any crosslinking. CLEA preparations comprising all three enzymes simultaneously immobilised onto zeolite enabled the formation of multiple enzyme biocatalytic modules which were shown to degrade several hemicellulosic substrates. CONCLUSIONS: The current work introduced the construction of functional biocatalytic modules for the hydrolysis of simple and complex polysaccharides. This technology exploited a silica-binding SBP to mediate effectively the rapid and simple immobilisation of thermostable enzymes onto readily-available and inexpensive silica-based matrices. A conceptual application of biocatalytic modules consisting of single or multiple enzymes was validated by hydrolysing various hemicellulosic polysaccharides.

A Novel Universal Detection Agent for Time-Gated Luminescence Bioimaging.

Luminescent lanthanide chelates have been used to label antibodies in time-gated luminescence (TGL) bioimaging. However, it is a challenging task to label directly an antibody with lanthanide-binding ligands and achieve control of the target ligand/protein ratios whilst ensuring that affinity and avidity of the antibody remain uncompromised. We report the development of a new indirect detection reagent to label antibodies with detectable luminescence that circumvents this problem by labelling available lysine residues in the linker portion of the recombinant fusion protein Linker-Protein G (LPG). Succinimide-activated lanthanide chelating ligands were attached to lysine residues in LPG and Protein G (without Linker) and the resulting Luminescence-Activating (LA-) conjugates were compared for total incorporation and conjugation efficiency. A higher and more efficient incorporation of ligands at three different molar ratios was observed for LPG and this effect was attributed to the presence of eight readily available lysine residues in the linker region of LPG. These Luminescence-Activating (LA-) complexes were subsequently shown to impart luminescence (upon formation of europium(III) complexes) to cell-specific antibodies within seconds and without the need for any complicated bioconjugation procedures. The potential of this technology was demonstrated by direct labelling of Giardia cysts and Cryptosporidium oocysts in TGL bioimaging.

Solid-binding peptides: smart tools for nanobiotechnology.

Over the past decade, solid-binding peptides (SBPs) have been used increasingly as molecular building blocks in nanobiotechnology. These peptides show selectivity and bind with high affinity to the surfaces of a diverse range of solid materials including metals, metal oxides, metal compounds, magnetic materials, semiconductors, carbon materials, polymers, and minerals. They can direct the assembly and functionalisation of materials, and have the ability to mediate the synthesis and construction of nanoparticles and complex nanostructures. As the availability of newly synthesised nanomaterials expands rapidly, so too do the potential applications for SBPs.

Effect of Trichoderma reesei proteinases on the affinity of an inorganic-binding peptide.

An inorganic-binding peptide sequence with high affinity to silica-containing materials was fused to a glycoside hydrolase GH26 mannanase, ManA, from the extremely thermophilic bacterium Dictyoglomus thermophilum. The resulting recombinant enzyme produced in Escherichia coli, ManA-Linker, displayed high binding affinity towards synthetic zeolite while retaining its catalytic activity at 80 °C. ManA-Linker was able to bind to the zeolite at different pH levels, indicating a true pH-independent binding. However, complete degradation of the peptide linker was observed when the recombinant ManA-Linker was exposed to the supernatant from the filamentous fungus Trichoderma reesei. This degradation was caused by extracellular proteinases produced by T. reesei during its growth phase. Several derivatives of ManA-Linker were designed and expressed in E. coli. All the derivatives carrying a single sequence of the linker were still susceptible to T. reesei proteinase degradation. Complete substitution of the linker sequence by (GGGGS)16 resulted in a proteinase-resistant ManA derivative, ManA-Linker-(GGGGS)16, which was able to bind to zeolite in a pH-dependent manner.

Simultaneous expression of the bacterial Dictyoglomus thermophilum xynB gene under three different Trichoderma reesei promoters.

Multiple copies of expression cassettes driven by the Trichoderma reesei xylanase 2 (xyn2) and cellobiohydrolase 2 (cbh2) promoters were introduced into the recombinant T. reesei EC-21 generated to express a thermostable Dictyoglomus thermophilum xylanase (XynB) under the egl2 promoter for further improvement of the enzyme yield. The transformants were screened based on increased XynB activity only. Multiple promoter transformant MPP-4 expressing the xynB gene under all the three promoters was found to be the highest producer of XynB, giving a 65% increase in yield compared to the parental single-promoter recombinant EC-21. The multiple-promoter transformant strains harboured six to nine copies of the xynB gene. Amongst the three promoters, egl2 seemed to have the strongest effect on XynB expression. The shotgun approach we used proved to be effective for rapid enhancement of protein expression using three promoters active at the near-neutral pH of the cultivation medium.

Expression of a bacterial xylanase in Trichoderma reesei under the egl2 and cbh2 glycosyl hydrolase gene promoters.

Expression vectors were constructed for Trichoderma reesei using the promoters, secretion signals and the modular structure of the efficiently expressed and secreted cellulase enzymes EGL2 (Cel5A) and CBH2 (Cel6A) as a prelude to establishing a platform where a gene of interest can be expressed under several promoters simultaneously. The designs featured (i) EGL2sigpro (egl2 promoter and secretion signal), (ii) EGL2cbmlin (egl2 promoter, secretion signal, EGL2 cellulose binding module and linker), (iii) CBH2sigpro (cbh2 promoter and secretion signal) and (iv) CBH2cbmlin (cbh2 promoter, secretion signal, CBH2 cellulose binding module and linker). Recombinant vectors were introduced individually into the high protein-secreting T. reesei RUT-C30 strain to generate single-promoter transformants expressing the Dictyoglomus thermophilum xynB gene that encodes a thermophilic xylanase enzyme (XynB). Ten transformants producing XynB representing each of the four different types of vectors were selected for further testing and the highest XynB production was achieved from a transformant containing 1-2copies of the EGL2cbmlin vector. Best xylanase producers did not show any particular pattern in terms of the number of gene copies and their mode of integration into the chromosomal DNA. Transformants generated with the cbmlin-type vectors produced multiple forms of XynB which were decorated with various N- and O-glycans. One of the O-glycans was identified as hexuronic acid, whose presence had not been observed previously in the glycosylation patterns of T. reesei.

Efficient capture of pathogens with a zeolite matrix.

We show that a peptide linker sequence expressed as part of a fusion with protein G from Streptococcus binds to natural zeolite giving a complex to which a suitable antibody shows affinity binding. As a consequence, addition of the CRY104 antibody specific for an external surface epitope of Cryptosporidium allows the capture of this protozoan pathogen at high efficiency with the advantage of rapid concentration from water samples. The natural zeolite with the specific antibody attached was incorporated into a single and double zeolite column system. The results reported here show that it is possible to capture Cryptosporidium oocysts with outstanding efficiency (>90 %) from water and water incorporating QC-MUD using a size-sorted natural zeolite. The natural zeolite matrix not only allows for high flow rates but also for flexibility of column design and volume of water for sample collection. The system is versatile and it is possible to prepare columns with more than one specific surface antibody to allow the capture of one or two pathogens simultaneously.

Facile production and rapid purification of functional recombinant Qß replicase heterotetramer complex.

We describe an improved method for the production of recombinant Qß replicase heterotetramer. The successful expression of the soluble Qß RNA polymerase complex depends on the EF-Ts and EF-Tu subunits being co-expressed prior to ß-subunit expression. Efficient co-expression requires two different inducible operons to co-ordinate the expression of the heterotrimer. The complete heterotetramer enzyme complex is achieved by production of the recombinant S1-subunit of Qß replicase in a separate host. This approach represents a facile way for producing and purifying large amounts of soluble and active recombinant Qß replicase tetramer without the necessity of a His-tag for purification.

A linker peptide with high affinity towards silica-containing materials.

A peptide sequence with affinity to silica-containing materials was fused to a truncated form of Streptococcus strain G148 Protein G. The resulting recombinant Linker-Protein G (LPG) was produced in Escherichia coli and purified to apparent homogeneity. It displayed high affinity towards two natural clinoptilolite zeolites. The LPG also displayed high binding affinity towards commercial-grade synthetic zeolite, silica and silica-containing materials. A commercial sample of the truncated Protein G and a basic protein, both without the linker, did not bind to natural or synthetic zeolites or silica. We conclude that the zeolite-binding affinity is mediated by the linker peptide sequence. As a consequence, these data may imply that the binding affinity is directed to the SiO2 component rather than to the atomic orientation on the zeolite crystal surface as previously assumed.

Flow cytometry in environmental microbiology: a rapid approach for the isolation of single cells for advanced molecular biology analysis.

The isolation and subsequent characterization of microbial cells from within environmental samples is a difficult process. Flow cytometry and cell sorting, when combined with the application of fluorescent probes, have the capability for the detection and separation of diverse microbial populations from within complex mixtures. The isolation of single cells allows for downstream investigations towards system-level characterization of unknown Bacterial Phyla to occur. We describe here the combination of fluorescent in situ hybridization and cell sorting for the detection and isolation of Candidate Division TM7 bacteria from an enriched soil sample. The result is the isolation of rare cells suitable for advanced molecular analysis including whole genome amplification and high-throughput pyrosequencing.

Molecular diversity and catalytic activity of Thermus DNA polymerases.

Thermus aquaticus DNA polymerase (Taq polymerase) made the polymerase chain reaction feasible and led to a paradigm shift in genomic analysis. Other Thermus polymerases were reported to have comparable performance in PCR and there was an analysis of their properties in the 1990s. We re-evaluated our earlier phylogeny of Thermus species on the basis of 16S rDNA sequences and concluded that the genus could be divided into eight clades. We examined 22 representative isolates and isolated their DNA polymerase I genes. The eight most diverse polymerase genes were selected to represent the eight clades and cloned into an expression vector coding for a His-tag. Six of the eight polymerases were expressed so that there was sufficient protein for purification. The proteins were purified to homogeneity and examination of the biochemical characteristics showed that although they were competent to perform PCR, none was as thermostable as commercially available Taq polymerase; all had similar error-frequencies to Taq polymerase and all showed the expected 5'-3' exonuclease activity. We conclude that the initial selection of T. aquaticus for DNA polymerase purification was a far-reaching and fortuitous choice but simple mutagenesis procedures on other Thermus-derived polymerases should provide comparable thermostability for the PCR reaction.

Applications of flow cytometry in environmental microbiology and biotechnology.

Flow cytometry (FCM) is a technique for counting, examining and sorting microscopic particles suspended in a stream of fluid. It uses the principles of light scattering, light excitation and the emission from fluorescent molecules to generate specific multiparameter data from particles and cells. The cells are hydrodynamically focussed in a sheath solution before being intercepted by a focused light source provided by a laser. FCM has been used primarily in medical applications but is being used increasingly for the examination of individual cells from environmental samples. It has found uses in the isolation of both culturable and hitherto non-culturable bacteria present infrequently in environmental samples using appropriate growth conditions. FCM lends itself to high-throughput applications in directed evolution for the analysis of single cells or cell populations carrying mutant genes. It is also suitable for encapsulation studies where individual bacteria are compartmentalised with substrate in water-in-oil-in-water emulsions or with individual genes in transcriptional/translational mixtures for the production of mutant enzymes. The sensitivity of the technique has allowed the examination of gene optimisation by a procedure known as random or neutral drift where screening and selection is based on the retention of some predetermined level of activity through multiple rounds of mutagenesis.

Potential use of quantum dots in flow cytometry.

QDs may offer significant advantages in environmental and bead-based applications where the target cells need to be discriminated above background fluorescence. We have examined the possible applications of QDs for flow cytometric measurements (FCM) by studying their excitation - emission spectra and their binding to paramagnetic beads. We labelled beads with either QDs or a commonly-used fluorochrome (FITC) and studied their fluorescence intensity by FCM. Flow cytometric comparisons indicated that the minimum fluorophore concentration required for detection of QDs above autofluorescent background was 100-fold less than for FITC.