Gregory Dame

Molecular analysis of the Rhodobacter capsulatus chaperonin (groESL) operon: purification and characterization of Cpn60

Abstract The heat-shock protein Cpn60 (chaperonin, GroEL homologue) from the phototrophic bacterium Rhodobacter capsulatus B10 was purified to homogeneity and biochemically characterized. Native Cpn60 from R. capsulatus was shown to be a tetradecamer of 840 kDa similar to that of homologous chaperones characterized so far. Cpn60 possesses ATPase activity and promotes refolding of chaotropically denatured citrate synthase. The groESL operon of R. capsulatus was cloned using a degenerate oligonucleotide and sequenced. Two open reading frames (285 and 1,635 bp) were found; they encode Cpn10 and Cpn60, with corresponding deduced molecular masses of 10.6 and 57.6 kDa. The deduced amino acid sequences coincided perfectly with those of the amino terminus and of three tryptic peptides of purified Cpn60 from R. capsulatus. Strong evidence that R. capsulatus encodes only one copy of the groESL operon was obtained. Primer-extension analysis revealed that the groESL operon is transcribed by a –35/–10-type promoter, and that transcription was initiated from the same positions before and after heat-shock under both chemotrophic and phototrophic conditions. The major initiation site is immediately followed by the inverted repeat structure CIRCE, which has been found upstream of many bacterial heat-shock operons. A second minor transcript starts just after the CIRCE element. Although heat-shock induction of a groEL-lacZ fusion failed because of thermal inactivation of the fusion protein, Western blot analysis revealed a two- to threefold induction of cellular Cpn60 levels 45–75 min after shifting from 28° C to 39° C. Deletion mapping of the groESL promoter identified upstream of the promoter a 19-bp element that enhances groESL transcription eightfold and contains the AT-rich sequence dAAATTTTT, which is found at similar positions in heat-shock operons of other gram-negative bacteria.

Modular development of an inline monitoring system for waterborne pathogens in raw and drinking water

Abstract The state-of-the-art monitoring of drinking water hygiene is based on the cultivation and enumeration of indicator bacteria. Despite its proven reliability, this approach has the disadvantages of being (a) relatively slow and (b) limited to a small number of indicator bacteria. Ideally, alternative methods would be less time-consuming while providing information about a larger set of hygienically relevant microorganisms including viruses. In this paper, we present insights into the design of a modular concentration and detection system for bacteria, bacteriophages and viruses. Following further validation, this or similar techniques have the potential to extend and speed up the monitoring of raw and drinking water hygiene in the future. The system consists of different modules for the concentration of microorganisms, an amplification and detection unit that includes a module for the differentiation between live and dead microorganisms, and an automated system for decision support and self-diagnosis. The ongoing testing under controlled laboratory conditions and real-life conditions in the water supply industry yields further system improvements. Moreover, the increased sensitivity and broader range of microbiological parameters emphasize the need for a reconsideration of the currently used criteria for the assessment of (drinking) water hygiene.

Fenton fragmentation for faster electrophoretic on chip purification of amplifiable genomic DNA.

With a rapid and simple actuation protocol electrophoretic nucleic acid extraction is easy automatable, requires no moving parts, is easy to miniaturize and furthermore possesses a size dependent cut-off filter adjustable by the pore size of the hydrogel. However electrophoretic nucleic acid extraction from bacteria has so far been applied mainly for short RNA targets. One of the reasons is that electrophoretic processing of unfragmented genomic DNA strands is time-consuming, because of the length. Here DNA fragmentation would accelerate extraction and isolation. We introduce on-chip lysis and non-enzymatic DNA cleavage directly followed by a purifying step for receiving amplifiable DNA fragments from bacteria in less than 25 min. In contrast to restriction enzymes the Fenton reaction is known to cleave DNA without nucleotide specificity. The reaction mix contains iron(II) EDTA, sodium ascorbate, hydrogen peroxide and lysozyme. The degree of fragmentation can be adjusted by the concentration of reagents. The results enable electrophoretic extraction methods to unspecifically process long genomic DNA in a short time frame, e.g. for pathogen detection in a lab-on-a-chip format.

A microchip for automated extraction of RNA from Gram-positive bacteria

A Lab-on-a-Chip for fully automated quantitative extraction of RNA from Gram-positive bacterial cells is presented. The method combines thermo-electric lysis with electrophoretic purification. The chip is capable of RNA extraction proportional to the deployed amount of cells down to 1 colony forming units (CFU). The performance of the chip is demonstrated for transfer-messenger RNA (tmRNA) from the Gram-positive bacterium Streptococcus thermophilus (S.thermophilus). The RNA extraction lasts 10.5 minutes and the RNA yield is higher than for commercially available extraction kits. The extraction is particularly efficient for low molecular weight RNAs and may find broad application in clinical diagnostics and drug discovery.

Direct DNA and RNA detection from large volumes of whole human blood

PCR inhibitors in clinical specimens negatively affect the sensitivity of diagnostic PCR and RT-PCR or may even cause false-negative results. To overcome PCR inhibition, increase the sensitivity of the assays and simplify the detection protocols, simple methods based on quantitative nested real-time PCR and RT-PCR were developed to detect exogenous DNA and RNA directly from large volumes of whole human blood (WHB). Thermus thermophilus (Tth) polymerase is resistant to several common PCR inhibitors and exhibits reverse transcriptase activity in the presence of manganese ions. In combination with optimized concentrations of magnesium ions and manganese ions, Tth polymerase enabled efficient detection of DNA and RNA from large volumes of WHB treated with various anticoagulants. The applicability of these methods was further demonstrated by examining WHB specimens collected from different healthy individuals and those stored under a variety of conditions. The detection limit of these methods was determined by detecting exogenous DNA, RNA, and bacteria spiked in WHB. To the best of our knowledge, direct RNA detection from large volumes of WHB has not been reported. The results of the developed methods can be obtained within 4 hours, making them possible for rapid and accurate detection of disease-causing agents from WHB.

A lab-on-a-chip for preconcentration of bacteria and nucleic acid extraction

To improve detection sensitivity, molecular diagnostics require preconcentration of low concentrated samples followed by rapid nucleic acid extraction. This is usually achieved by multiple centrifugation, lysis and purification steps, for instance, using chemical reagents, spin columns or magnetic beads. These require extensive infrastructure as well as time consuming manual handling steps and are thus not suitable for point of care testing (POCT). To overcome these challenges, we developed a microfluidic chip combining free-flow electrophoretic (FFE) preconcentration (1 ml down to 5 μl) and thermoelectric lysis of bacteria as well as purification of nucleic acids by gel-electrophoresis. The integration of these techniques in a single chip is unique and enables fast, easy and space-saving sample pretreatment without the need for laboratory facilities, making it ideal for the integration into small POCT devices. A preconcentration efficiency of nearly 100% and a lysis/gel-electrophoresis efficiency of about 65% were achieved for the detection of E. coli. The genetic material was analyzed by RT-qPCR targeting the superfolder Green Fluorescent Protein (sfGFP) transcripts to quantify mRNA recovery and qPCR to determine DNA background.

Correction: A lab-on-a-chip for preconcentration of bacteria and nucleic acid extraction

Correction for ‘A lab-on-a-chip for preconcentration of bacteria and nucleic acid extraction’ by M. Hugle et al., RSC Adv., 2018, 8, 20124–20130.

Capacity of rTth polymerase to detect RNA in the presence of various inhibitors

The full potential of the real-time reverse transcription polymerase chain reaction (RT-PCR) as a rapid and accurate diagnostic method is limited by DNA polymerase inhibitors as well as reverse transcriptase inhibitors which are ubiquitous in clinical samples. rTth polymerase has proven to be more resistant to DNA polymerase inhibitors present in clinical samples for DNA detection and also exhibits reverse transcriptase activity in the presence of Mn2+ ions. However, the capacity of rTth polymerase, which acts as DNA polymerase and reverse transcriptase, to detect RNA in the presence of various inhibitors has not been investigated in detail. Herein, the inhibitors originating from various clinical samples such as blood, urine, feces, bodily fluids, tissues and reagents used during nucleic acid extraction were employed to evaluate the capacity of rTth polymerase to detect RNA. The results show that the inhibitors have different inhibitory effects on the real-time RT-PCR reactions by rTth polymerase, and the inhibitory effects are concentration dependent. Additionally, the capacity of rTth polymerase to detect RNA in the presence of various inhibitors is better or at least comparable with its capacity to detect DNA in the presence of various inhibitors. As a consequence, RNA may be directly detected in the presence of co-purified inhibitors or even directly from crude clinical samples by rTth polymerase.

In-Situ Electrophoretic Mobility Determination by Particle Image Velocimetry for Efficient Microfluidic Enrichment of Bacteria

We present a novel approach for the efficiency enhancement of microfluidic bacteria enrichment systems based on free-flow electrophoresis (FFE). FFE efficiency is highly dependent on the electrophoretic mobility μ of the bacteria. As μ varies strongly with the suspension medium, fast and accurate determination of μ is needed to achieve optimal enrichment performance from different suspension media. For the first time, μ is determined in-situ for multiple media during on-chip FFE by Particle Image Velocimetry (PIV) of fluorescent bacteria, obviating the need for separate measurement equipment or chemical staining of the bacteria.

Magnetron Enhanced Plasma-Polymerization for Biocompatible Sensor Coatings and Membranes on Polymeric Based Materials

One of the key questions in the application of miniaturized sensors and actuators for acute and/or chronic use in living-body environment is the biocompatibility. In case of gas sensors additionally a very fine balance between the biocompatibility of the device and the gas (e.g. O2, NO, CO) permeability of its coating must be maintained. In many sensor configurations polymeric substrate materials are used. Here, we present the application of a unique deposition technique for nano-films with thickness ranging between 10 and 200 nm on top of flexible polymeric foils used as substrates in the technology of a variety of biosensors and lab-on-chip structures. The method employs a 15 kHz magnetron-enhanced glow discharge plasma-polymerization process using methane as precursor. It is configurable for laboratory scale batch sizes but also for continuous industrial coating lines. Unsurpassed results of this processing technique have been documented with contact lenses already. Hence, we tested depositions with this process on top of PMMA, polyimide and polystyrene foils of different surface morphology. Compatibility of the process and of the coatings with these materials, adherence in dry and aqueous environment were checked. Antibacterial behaviour of the films were tested by immersing the coated samples in a bio-film reactor for 48 hours as well as for 7 days in E-coli bacteria solutions. After the inoculation time samples were rinsed and treated in an ultrasonic bath. Colonies formed on different culture media out of the rinsing water were enumerated. Number of colony forming units, depending on inoculation time and coating conditions, has been investigated. Remarkable reduction of bacterial attachment was proven with film thicknesses as low as about 15 nm, which allows a reasonable gas permeation rate. Hence, the technology provides production of antibacterial and gas permeable membranes for miniaturized sensors and sensor arrays on chip.