Method for automated extraction and purification of nucleic acids and its implementation in microfluidic system

A method and a microfluidic device for automated extraction and purification of nucleic acids from biological samples have been developed. The method involves disruption of bacterial cells and/or viral particles by combining enzymatic and chemical lysis procedures followed by solid-phase sorbent extraction and purification of nucleic acids. The procedure is carried out in an automated mode in a microfluidic module isolated from the outside environment, which minimizes contact of the researcher with potentially infectious samples and, consequently, decreases the risk of laboratory-acquired infections. The module includes reservoirs with lyophilized components for lysis and washing buffers; a microcolumn with a solid-phase sorbent; reservoirs containing water, ethanol, and water-ethanol buffer solutions for dissolving freeze-dried buffer components, rinsing the microcolumn, and eluting of nucleic acids; and microchannels and valves needed for directing fluids inside the module. The microfluidic module is placed into the control unit that delivers pressure, heats, mixes reagents, and flows solutions within the microfluidic module. The microfluidic system performs extraction and purification of nucleic acids with high efficiency in 40 min, and nucleic acids extracted can be directly used in PCR reaction and microarray assays.     

On-line cell lysis of bacteria and its spores using a microfluidic biochip

Optimal detection of pathogens by molecular methods in water samples depends on the ability to extract DNA rapidly and efficiently. In this study, an innovative method was developed using a microfluidic biochip, produced by microelectrochemical system technology, and capable of performing online cell lysis and DNA extraction during a continuous flow process. On-chip cell lysis based on chemical/physical methods was performed by employing a sufficient blend of water with the lysing buffer. The efficiency of lysis with microfluidic biochip was compared with thermal lysis in Eppendorf tubes and with two commercial DNA extraction kits: Power Water DNA isolation kit and ForensicGEM Saliva isolation kit in parallel tests. Two lysing buffers containing 1% Triton X-100 or 5% Chelex were assessed for their lysis effectiveness on a microfluidic biochip. SYBR Green real-time PCR analysis revealed that cell lysis on a microfluidic biochip using 5% Chelex buffer provided better or comparable recovery of DNA than commercial isolation kits. The system yielded better results for Gram-positive bacteria than for Gram-negative bacteria and spores of Gram-positive bacteria, within the limits of detection at 10³ CFU/ml. During the continuous flow process in the system, rapid cells lysis with PCR-amplifiable genomic DNA were achieved within 20 minutes.     

可用于微生物群落分子生态学研究的活性污泥总DNA提取方法研究

活性污泥样品经液氮速冻、沸水浴融化、溶菌酶处理和 SDS裂解后 ,99%以上细胞裂解。所提取的 DNA经琼脂糖凝胶电泳检测和荧光法浓度测定 ,其片断大小在 2 0 kb左右 ,产量可达 1 .75 6± 0 .1 mg/g MLSS。样品 ABS2 6 0 nm/ABS2 80 nm的比值为 1 .96± 0 .2。以提取的总 DNA为模板 ,进行细菌核糖体小亚基 1 6Sr DNA基因 V3区和多组分苯酚羟化酶大亚基基因 (Lm PHs)的 PCR扩增 ,均获得成功 ,为活性污泥中微生物群落的分子生态学研究提供了一种简便、可靠的 DNA提取方法。     

A microfluidic flow-through device for high throughput electrical lysis of bacterial cells based on continuous dc voltage

Interest in electrical lysis of biological cells on a microfludic platform has increased because it allows for the rapid recovery of intracellular contents without introducing lytic agents. In this study we demonstrated a simple microfluidic flow-through device which lysed Escherichia coli cells under a continuous dc voltage. The E. coli cells had previously been modified to express green fluorescent protein (GFP). In our design, the cell lysis only happened in a defined section of a microfluidic channel due to the local field amplification by geometric modification. The geometric modification also effectively decreased the required voltage for lysis by several folds. We found that local field strength of 1000-1500 V/cm was required for nearly 100% cell death. This threshold field strength was considerably lower than the value reported in the literature, possibly due to the longer duration of the field [Lee, S.W., Tai, Y.C., 1999. Sens. Actuators A: Phys. 73, 74-79]. Cell lysis was detected by both plate count and fluorescence spectroscopy. The cell membrane was completely disintegrated in the lysis section of the microfluidic device, when the field strength was higher than 2000 V/cm. The devices were fabricated using low-cost soft lithography with channel widths considerably larger than the cell size to avoid clogging and ensure stable performance. Our tool will be ideal for high throughput processing of bacterial cells for chemical analysis of intracellular contents such as DNA and proteins. The application of continuous dc voltage greatly simplified the instrumentation compared to devices using electrical pulses for similar purposes. In principle, the same approach can also be applied for lysis of mammalian cells and electroporative transfection.     

Functional analysis of C-type lysozyme in penaeid shrimp

Lysozyme is an enzyme that cleaves the β-1,4-glycosidic linkages between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan, leading to bacterial lysis. Recently, lysozyme has been found to have anti-HIV and anti-cancer properties in mammals. However, most functional analyses were done in vitro using purified or recombinant lysozyme protein. Here, we used RNA interference to silence c-type lysozyme expression in penaeid shrimp, Marsupenaeus japonicus, to analyze the function of lysozyme in vivo. Silencing of lysozyme expression by dsRNA lysozyme (dsLYZ) led to 100% mortality without any artificial bacterial infection in 5 days. Lysozyme deficiency caused the number of hemocytes in hemolymph to decrease from 1.3 × 10(7) to 2.3 × 10(6) cells/ml and caused the number of bacteria to increase from 78 to 764 colony-forming units/ml. Suppression of bacterial growth using oxytetracycline and kanamycin showed improvement in mortality, suggesting that shrimp mortality post- dsLYZ injection can be attributed to bacterial growth in the shrimp hemolymph. The majority of the bacteria, identified by 16 S rRNA analysis, were Gram-negative species such as Vibrio and Pseudomonas. Furthermore, PKH26 staining showed that the dsLYZ-injected shrimp were unable to eliminate non pathogenic Escherichia coli or Staphylococcus aureus in 24 h. These data suggest that c-type lysozyme in shrimp serves to regulate the growth of bacterial communities, particularly Gram-negative bacteria, in the hemolymph.     

Synergistic action of Galleria mellonella anionic peptide 2 and lysozyme against Gram-negative bacteria

Lysozyme and antimicrobial peptides are key factors of the humoral immune response in insects. In the present work lysozyme and anionic defense peptide (GMAP2) were isolated from the hemolymph of the greater wax moth Galleria mellonella and their antibacterial activity was investigated. Adsorption of G. mellonella lysozyme on the cell surface of Gram-positive and Gram-negative bacteria was demonstrated using immunoblotting with anti-G. mellonella lysozyme antibodies. Lysozyme effectively inhibited the growth of selected Gram-positive bacteria, which was accompanied by serious alterations of the cell surface, as revealed by atomic force microscopy (AFM) imaging. G. mellonella lysozyme used in concentrations found in the hemolymph of naive and immunized larvae, perforated also the Escherichia coli cell membrane and the level of such perforation was considerably increased by GMAP2. GMAP2 used alone did not perforate E. coli cells nor influence lysozyme muramidase activity. However, the peptide induced a decrease in the turgor pressure of the bacterial cell. Moreover, in the samples of bacteria treated with a mixture of lysozyme and GMAP2 the sodium chloride crystals were found, suggesting disturbance of ion transport across the membrane leading to cell disruption. These results clearly indicated the synergistic action of G. mellonella lysozyme and anionic peptide 2 against Gram-negative bacteria. The reported results suggested that, thanks to immune factors constitutively present in hemolymph, G. mellonella larvae are to some extent protected against infection caused by Gram-negative bacteria.     

A nanoliter-scale nucleic acid processor with parallel architecture

The purification of nucleic acids from microbial and mammalian cells is a crucial step in many biological and medical applications. We have developed microfluidic chips for automated nucleic acid purification from small numbers of bacterial or mammalian cells. All processes, such as cell isolation, cell lysis, DNA or mRNA purification, and recovery, were carried out on a single microfluidic chip in nanoliter volumes without any pre- or postsample treatment. Measurable amounts of mRNA were extracted in an automated fashion from as little as a single mammalian cell and recovered from the chip. These microfluidic chips are capable of processing different samples in parallel, thereby illustrating how highly parallel microfluidic architectures can be constructed to perform integrated batch-processing functionalities for biological and medical applications.     

Adaptational changes in cellular phospholipids and fatty acid composition of the food pathogen Listeria monocytogenes as a stress response to disinfectant sanitizer benzalkonium chloride

Aims: This study provides a first approach to observing the alterations of the cell membrane lipids in the adaptation response of Listeria monocytogenes to the sanitizer benzalkonium chloride. Methods and Results: A thorough investigation of the composition of polar and neutral lipids from L. monocytogenes grown when exposed to benzalkonium chloride is compared to cells optimally grown. The adaptation mechanism of L. monocytogenes in the presence of benzalkonium chloride caused (i) an increase in saturated‐chain fatty acids (mainly C_16:0 and C_18:0) and unsaturated fatty acids (mainly C_16:1 and C_18:1) at the expense of branched‐chain fatty acids (mainly C_a‐15:0 and C_a‐17:0) mainly because of neutral fatty acids; (ii) no alteration in the percentage of neutral and polar lipid content among total lipids; (iii) a decrease in lipid phosphorus and (iv) an obvious increase in the anionic phospholipids and a decrease in the amphiphilic phosphoaminolipid. Conclusions: These lipid changes could lead to decreased membrane fluidity and also to modifications of physicochemical properties of cell surface and thus changes in bacterial adhesion to abiotic surfaces. Significance and Impact of the Study: The adaptation and resistance of L. monocytogenes to disinfectants is able to change its physiology to allow growth in food‐processing plants. Understanding microbial stress response mechanisms would improve the effective use of disinfectants.     

Finite Element Model of Oxygen Transport for the Design of Geometrically Complex Microfluidic Devices Used in Biological Studies

Red blood cells play a crucial role in the local regulation of oxygen supply in the microcirculation through the oxygen dependent release of ATP. Since red blood cells serve as an oxygen sensor for the circulatory system, the dynamics of ATP release determine the effectiveness of red blood cells to relate the oxygen levels to the vessels. Previous work has focused on the feasibility of developing a microfluidic system to measure the dynamics of ATP release. The objective was to determine if a steep oxygen gradient could be developed in the channel to cause a rapid decrease in hemoglobin oxygen saturation in order to measure the corresponding levels of ATP released from the red blood cells. In the present study, oxygen transport simulations were used to optimize the geometric design parameters for a similar system which is easier to fabricate. The system is composed of a microfluidic device stacked on top of a large, gas impermeable flow channel with a hole to allow gas exchange. The microfluidic device is fabricated using soft lithography in polydimethyl-siloxane, an oxygen permeable material. Our objective is twofold: (1) optimize the parameters of our system and (2) develop a method to assess the oxygen distribution in complex 3D microfluidic device geometries. 3D simulations of oxygen transport were performed to simulate oxygen distribution throughout the device. The simulations demonstrate that microfluidic device geometry plays a critical role in molecule exchange, for instance, changing the orientation of the short wide microfluidic channel results in a 97.17% increase in oxygen exchange. Since microfluidic devices have become a more prominent tool in biological studies, understanding the transport of oxygen and other biological molecules in microfluidic devices is critical for maintaining a physiologically relevant environment. We have also demonstrated a method to assess oxygen levels in geometrically complex microfluidic devices.     

The peptidoglycan-degrading property of lysozyme is not required for bactericidal activity in vivo

Lysozyme is an abundant, cationic antimicrobial protein that plays an important role in pulmonary host defense. Increased concentration of lysozyme in the airspaces of transgenic mice enhanced bacterial killing whereas lysozyme deficiency resulted in increased bacterial burden and morbidity. Lysozyme degrades peptidoglycan in the bacterial cell wall leading to rapid killing of Gram-positive organisms; however, this mechanism cannot account for the protective effect of lysozyme against Gram-negative bacteria. The current study was therefore designed to test the hypothesis that the catalytic activity (muramidase activity) of lysozyme is not required for bacterial killing in vivo. Substitution of serine for aspartic acid at position 53 (D53S) in mouse lysozyme M completely ablated muramidase activity. Muramidase-deficient recombinant lysozyme (LysM(D53S)) killed both Gram-positive and Gram-negative bacteria in vitro. Targeted expression of LysM(D53S) in the respiratory epithelium of wild-type (LysM(+/+)/LysM(D53S)) or lysozyme M(null) mice (LysM(-/-)/LysM(D53S)) resulted in significantly elevated lysozyme protein in the airspaces without any increase in muramidase activity. Intratracheal challenge of transgenic mice with Gram-positive or Gram-negative bacteria resulted in a significant increase in bacterial burden in LysM(-/-) mice that was completely reversed by targeted expression of LysM(D53S). These results indicate that the muramidase activity of lysozyme is not required for bacterial killing in vitro or in vivo.     

Differential Lytic Response of Enterococci Associated with Addition Order of Lysozyme and Anions

Suspensions of Streptococcus faecium, prepared by washing with and resuspending in water, were lysed slowly if sodium chloride was added prior to lysozyme; however, if brief incubation with lysozyme was followed by addition of sodium chloride, lysis was immediate and extensive. Relatively lysozyme-resistant strains of S. faecalis could be lysed readily by adding lysozyme first. The primary addition of lysozyme apparently resulted in a "sensitized" cell with a damaged wall, as evidenced by N-acetylhexosamine release. Anionic detergents could replace sodium chloride in lysing these sensitized cells. The difference in activity associated with the order of addition probably involved a competition for reactive sites on the cell surface.     

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO₂ sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.     

Lysis of Streptococcus mutans by hen egg white lysozyme and inorganic sodium salts.

Streptococcus mutans BHT was grown in a synthetic medium containing radioactive thymidine to monitor deoxyribonucleic acid release. Kinetic experiments demonstrated that although lysozyme alone could not liberate deoxyribonucleic acid, cellular deoxyribonucleic acid was liberated from lysozyme-treated cells by addition of low concentrations of inorganic sodium salts. When the salts were tested for their ability to dislodge cell-bound tritiated lysozyme, the extent of the initial release of enzyme by individual anions correlated with the anion potency for deoxyribonucleic acid liberation (SCN- greater than ClO4- greater than I- greater than Br- greater than NO3- greater than Cl- greater than F-), although the total amount of lysozyme dislodged did not correspond directly with cell lysis. Differences in the effectiveness of anions (SCN-, HCO3-, Cl- and F-) in potentiating cell lysis could be enhanced or minimized by varying the lysozyme, anion, and bacterial cell concentrations. As the anion concentration was increased for each enzyme concentration and cell concentration, the lysis increased, in some cases markedly, until maximum levels of released deoxyribonucleic acid were attained. The maximum levels of lysis of SCN- and HCO3- were similar and were greater than those for Cl- and F-. In addition, the maximum levels were observed to increase for each of the anions as the concentration of lysozyme increased.     

Pentane Inhibition of the Egg-white Lysozyme-induced Cell Lysis of Micrococcus lysodeikticus

Pentane inhibited the cell lysis of Micrococcus lysodeikticus by egg-white lysozyme, when added to the lysozyme solution before mixing the cells. The pentane inhibition was not observed when pentane was added either before to the cell suspensions or to the cell-lysozyme mixture. The degree of pentane inhibition was proportional to the concentration of pentane, and the maximum inhibition was achieved with about 5% pentane close to the saturation point. On the other hand, pentane did not inhibit the hydrolysis of glycol chitin by the lysozyme, showing that the β-1,4-glucosaminidase activity of lysozyme remained unchanged. The inhibitory action of pentane on the lysozyme-induced cell lysis was of a competitive nature. The pentane inhibition had no ph-dependence, but it was influenced by the ionic strength of the buffer used as solvent.

When the lysozyme solution was treated with pentane, a characteristic ultraviolet spectrum of lysozyme was produced; a blue shift with a minimum at 280 nm and a trough at 291 nm. The degree of spectral change at 280 nm depended on the concentration of pentane. Pentane was, therefore, found to interact with egg-white lysozyme to affect the conformation and enzymatic activity of lysozyme. The mechanism of pentane inhibition on the lysozyme-induced cell lysis was discussed.     

Cloning and expression of bacteriophage FMV lysocyme gene in cells of yeasts <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Cloning, sequencing, and expression of the gene for soluble lysozyme of bacteriophage FMV from Gram-negative Pseudomonas aeruginosa bacteria were conducted in yeast cells. Comparable efficiency of two lysozyme expression variants (as intracellular or secreted proteins) was estimated in cells of Saccharomyces cerevisiae and Pichia pastoris. Under laboratory conditions, yeast S. cerevisiae proved to be more effective producer of phage lysozyme than P. pastoris, the yield of the enzyme in the secreted form being significantly higher than that produced in the intracellular form.     

Association between algal productivity and phycosphere composition in an outdoor Chlorella sorokiniana reactor based on multiple longitudinal analyses

Microalgae as a biofuel source are of great interest. Bacterial phycosphere inhabitants of algal cultures are hypothesized to contribute to productivity. In this study, the bacterial composition of the Chlorella sorokiniana phycosphere was determined over several production cycles in different growing seasons by 16S rRNA gene sequencing and identification. The diversity of the phycosphere increased with time during each individual reactor run, based on Faith’s phylogenetic diversity metric versus days post-inoculation (R = 0.66, P < 0.001). During summer months, Vampirovibrio chlorellavorus, an obligate predatory bacterium, was prevalent. Bacterial sequences assigned to the Rhizobiales, Betaproteobacteriales and Chitinophagales were positively associated with algal biomass productivity. Applications of the general biocide, benzalkonium chloride, to a subset of experiments intended to abate V. chlorellavorus appeared to temporarily suppress phycosphere bacterial growth, however, there was no relationship between those bacterial taxa suppressed by benzalkonium chloride and their association with algal productivity, based on multinomial model correlations. Algal health was approximated using a model-based metric, or the ‘Health Index’ that indicated a robust, positive relationship between C. sorokiniana fitness and presence of members belonging to the Burholderiaceae and Allorhizobium–Neorhizobium–Pararhizobium–Rhizobium clade. Bacterial community composition was linked to the efficiency of microalgal biomass production and algal health.     

Peptidoglycan fragments elicit antibacterial protein synthesis in larvae of Manduca sexta

In several insect species, serum lysozyme and antibacterial peptide concentration increases after injection of bacteria and other foreign substances. The purpose of this study was to characterize the specificity of this induction in the tobacco hornworm, Manduca sexta. By 48 h after injection of killed bacteria, lysozyme activity was approximately tenfold greater than in untreated insects. This maximal response was observed after injection of every bacterial species tested and after injection of purified cell walls of Micrococcus luteus. A variety of acellular particles, soluble molecules, and bacterial cell wall components were either poor lysozyme inducers or elicited no change in lysozyme concentration. The polysaccharide zymosan from yeast cell walls was a moderate lysozyme inducer. Peptidoglycan from M. luteus cell walls was found to induce lysozyme to a level as great or greater than whole cell walls. Small fragments of peptidoglycan generated by hen egg white lysozyme digestion were isolated, partially characterized, and shown to be good inducers of lysozyme as well as other antibacterial peptides. It appears that peptidoglycan provides a signal that initiates antibacterial responses in the insect.     

Lysis of Micrococcus lysodeikticus cells by lysozyme covalently immobilized on the lumen of hollow fibers

Summary A lytic enzyme reactor for microbial cell lysis is described in which lysozyme is immobilized on the lumen of hemodialyzer hollow fibers using epichlorohydrin as a coupling agent. The cell suspension flows through the lumen without any hindrance where the cells are lyzed by the immobilized lysozyme efficiently. Micrococcus lysodeikticus cells at concentrations of 0.25 g/L and 5 g/L were successfully lyzed without clogging the hollow fiber. In comparison with lysozyme immobilized on submicron particles, the activity retention was at least 8 times higher.     

Physiological consequence of expression of soluble and active hen egg white lysozyme in Escherichia coli

Hen egg white lysozyme was expressed as a protein fusion with the OmpA signal sequence and an octapeptide linker in Escherichia coli. The expression yielded soluble and enzymatically active lysozyme. Lysozyme activity was detected in the periplasmic space, in the cytosol and in the insoluble cytosolic fraction of E. coli. The results indicate that the environmental conditions in both the cytosol and the periplasmic space of E. coli were sufficient for correct protein folding and disulphide bond formation of eukaryotic recombinant lysozyme. However, the expression of active enzyme in E. coli consequently led to bacterial cell lysis due to hydrolysis of the peptidoglucan. Correspondence to: B. Fischer     

Efficient mechanical disruption of Lactobacillus helveticus,Lactococcus lactis and Propionibacterium freudenreichii by a new high-pressure homogenizer and recovery of intracellular aminotransferase activity

Microbiological studies often involve bacterial cell fractionation, which is known to be difficult for Gram-positive as compared to Gram-negative bacteria. Our purpose was to test the breaking efficiency of a new high-pressure pilot homogenizer for three Gram-positive species involved in dairy technology and to assess the activity of an intracellular aminotransferase. Varied pressures (50, 100 and 200 MPa) were applied to concentrated bacterial suspensions (1.2 mg dry weight/ml) of Lactobacillus helveticus, Lactococcus lactis and Propionibacterium freudenreichii. Breaking efficiency was estimated by decreases in optical density at 650 nm, cellular dry weight and viability. The proteins released were quantified and the residual intracellular aminotransferase activity was estimated using leucine as substrate. One run at 50 MPa was sufficient to break 80% of lactobacilli cells whereas 200 MPa were required for the same efficiency for L. lactis and P. freudenreichii. Whatever the pressure, leucine aminotransferase activity was recovered in the supernatant after cell breaking. This new high-pressure pilot homogenizer can allow rapid (20 s/run), easy, continuous and highly efficient cell breaking for intracellular enzyme recovery or other purposes. As the species tested were not phylogenetically related, and had different morphologies and cell wall compositions, we conclude that most Gram-positive bacteria may be broken efficiently by this new device. Electronic Publication