Genomes to Life "Center for Molecular and Cellular Systems": a Research Program for Identification and Characterization of Protein Complexes

Omics : a Journal of Integrative Biology. 2002  |  Pubmed ID: 12630408

Goal 1 of Department of Energy's Genomes to Life (GTL) program seeks to identify and characterize the complete set of protein complexes within a cell. Goal 1 forms the foundation necessary to accomplish the other objectives of the GTL program, which focus on gene regulatory networks and molecular level characterization of interactions in microbial communities. Together this information would allow cells and their components to be understood in sufficient detail to predict, test and understand the responses of a biological system to its environment. The Center for Molecular and Cellular Systems has been established to identify and characterize protein complexes using high through-put analytical technologies.A dynamic research program is being developed that supports the goals of the Center by focusing on the development new capabilities for sample preparation and complex separations, molecular level identification of the protein complexes by mass spectrometry, characterization of the complexes in living cells by imaging techniques, and bioinformatics and computational tools for the collection and interpretation of data and formation of databases and tools to allow the data to be shared by the biological community.

Fingerprinting of Prokaryotic 16S RRNA Genes Using Oligodeoxyribonucleotide Microarrays and Virtual Hybridization

Nucleic Acids Research. Jan, 2003  |  Pubmed ID: 12527788

An oligonucleotide microarray hybridization system to differentiate microbial species was designed and tested. Seven microbial species were studied, including one Bacillus and six Pseudomonas strains. DNA sequences near the 5' end of 16S rRNA genes were aligned and two contiguous regions of high variability, flanked by highly conserved sequences, were found. The conserved sequences were used to design PCR primers which efficiently amplified these polymorphic regions from all seven species. The amplicon sequences were used to design 88 9mer hybridization probes which were arrayed onto glass slides. Single-stranded, fluorescence-tagged PCR products were hybridized to the microarrays at 15 degrees C. The experimental results were compared with the DeltaG(0) values for all matched and mismatched duplexes possible between the synthetic probes and the 16S target sequences of the seven test species, calculated using a 'virtual hybridization' software program. Although the observed hybridization patterns differed significantly from patterns predicted solely on the basis of perfect sequence matches, a unique hybridization fingerprint was obtained for each of the species, including closely related Pseudomonas species, and there was a reasonable correlation between the intensity of observed hybridization signals and the calculated DeltaG(0) values. The results suggest that both perfect and mismatched pairings can contribute to microbial identification by hybridization fingerprinting.

Optimized Beadmilling of Tissues for High-throughput RNA Production and Microarray-based Analyses

Analytical Biochemistry. Sep, 2004  |  Pubmed ID: 15301954

The preparation of RNA samples has become the rate-limiting step when performing genome-scale analyses by DNA microarrays. Methods to improve throughput of RNA isolation from tissues are needed. The effects of bead size and composition for disrupting mouse tissues have been evaluated in small centrifuge tubes and optimized for RNA production. The resulting process is inexpensive, resistant to cross-contamination, and amenable to robotic processing. After optimization, very-high-quality RNA can be produced. Comparisons between RNAs isolated by beadmilling (followed by solid-phase purification) and those by conventional isolation processes show that RNA produced by beadmilling is suitable for microarray analyses. Parallel implementation of beadmilling will enable a high-throughput tissue-to-RNA processing system for large-scale microarray analyses.

Surface Patterning of Silica Nanostructures Using Bio-inspired Templates and Directed Synthesis

Langmuir : the ACS Journal of Surfaces and Colloids. Sep, 2004  |  Pubmed ID: 15379457

Natural systems excel in directing the synthesis of inorganic materials for various functional purposes. One of the best-studied systems is silica synthesis, as occurs in diatoms and marine sponges. Various biological and synthetic polymers have been shown to template and catalyze silica formation from silicic acid precursors. Here, we describe the use of poly-L-lysine to promote the synthesis of silica in neutral, aqueous solution and when immobilized onto a silicon support structure under similar conditions. Either reagent jetting or conventional photolithography techniques can be used to pattern the templating polymer. Spots created by reagent jetting led to the creation of silica structures in the shape of a ring that may be a result of the spotting process. Photolithographically defined poly-L-lysine spots led to thin laminate structures after exposure to a dilute aqueous silicic acid solution. The laminate structures were nanostructured and highly interconnected. Photolithographic patterning of (3-aminopropyl)trimethoxysilane, a reagent that mimics the lysine functional group, led to similar silica coatings even though low-molecular-weight materials do not rapidly promote silica synthesis in solution. This result highlights the importance of functional-group arrangement for templating and promoting the synthesis of inorganic materials. The described surface-patterning techniques offer a route to integrate conventional silicon-patterning technologies with biologically based material synthesis. Such combined fabrication techniques enable controlled assembly over multiple length scales and an approach to understanding interfacial silica synthesis, as occurs in natural systems.

Automated Image Analysis of Fluorescence Microscopic Images to Identify Protein-protein Interactions

Conference Proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference. 2005  |  Pubmed ID: 17281221

The identification of protein-protein interactions along with their spatial and temporal localization is vital data for assigning functional information to proteins. Historically, these data sets obtained from fluorescence microscopy, have been analyzed manually, a process that is both time consuming and tedious. The development of an automated system that can measure the location dynamics of the interaction between two proteins inside a live cell is a high priority. This paper describes an automated image analysis system used to identify the interactions between two proteins of interest fused to either GFP or DIV IVA, a bacterial cell division protein that localizes to the cell poles [1]. Upon the induction of DIV IVA fusion protein expression, the GFP-fusion protein will be recruited to the cell poles if a positive interaction occurs. Advanced image processing and feature extraction algorithms are discussed in detail and a statistical feature set used to quantify the image-based information is developed.

Automated Image Analysis of Fluorescence Microscopic Images to Identify Protein-protein Interactions

Conference Proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference. 2005  |  Pubmed ID: 17282304

The identification of protein-protein interactions along with their spatial and temporal localization is vital data for assigning functional information to proteins. Historically, these data sets obtained from fluorescence microscopy, have been analyzed manually, a process that is both time consuming and tedious. The development of an automated system that can measure the location dynamics of the interaction between two proteins inside a live cell is a high priority. This paper describes an automated image analysis system used to identify the interactions between two proteins of interest fused to either GFP or DIV IVA, a bacterial cell division protein that localizes to the cell poles [1]. Upon the induction of DIV IVA fusion protein expression, the GFP-fusion protein will be recruited to the cell poles if a positive interaction occurs. Advanced image processing and feature extraction algorithms are discussed in detail and a statistical feature set used to quantify the image-based information is developed.

Evaluation of a Surface-sampling Probe Electrospray Mass Spectrometry System for the Analysis of Surface-deposited and Affinity-captured Proteins

Rapid Communications in Mass Spectrometry : RCM. 2006  |  Pubmed ID: 16521171

A combined self-aspirating electrospray emitter/surfacing-sampling probe coupled with an ion trap mass spectrometer was used to sample and mass analyze proteins from surfaces. The sampling probe mass spectrometer system was used to sample and detect lysozyme that had been deposited onto a glass slide using a piezoelectric spotter or murine gamma-interferon affinity captured on a glass slide using surface-immobilized anti-gamma-interferon antibody. The detection level for surface-deposited lysozyme (spot size < or =200 microm) was approximately 1.0 fmol (approximately 100 fmol/mm2) as determined from the ability to measure accurately the protein molecular mass from the mass spectrum acquired by sampling the deposit. These detection limits may be sufficient for certain applications in which protein fractions from a separation method are collected onto a surface. Radiolabeled proteins were used to quantify the surface density of immobilized antibody and the efficiency of capture of the gamma-interferon on glass and higher surface area ceramic supports. The capture density of gamma-interferon at surface saturation ranged from about 23 to 50 fmol/mm2 depending on the capture surface. Nonetheless, mass spectrometric detection of affinity capture protein was successful in some cases, but the results were not reproducible. Thus, improvement of the sampling system, ionization efficiency and/or capture density will be necessary for practical sampling of affinity-captured proteins. The means to accomplish improved sampling system detection limits and to increase the absolute amounts of protein captured per unit area are discussed.

Global Molecular and Morphological Effects of 24-hour Chromium(VI) Exposure on Shewanella Oneidensis MR-1

Applied and Environmental Microbiology. Sep, 2006  |  Pubmed ID: 16957260

The biological impact of 24-h ("chronic") chromium(VI) [Cr(VI) or chromate] exposure on Shewanella oneidensis MR-1 was assessed by analyzing cellular morphology as well as genome-wide differential gene and protein expression profiles. Cells challenged aerobically with an initial chromate concentration of 0.3 mM in complex growth medium were compared to untreated control cells grown in the absence of chromate. At the 24-h time point at which cells were harvested for transcriptome and proteome analyses, no residual Cr(VI) was detected in the culture supernatant, thus suggesting the complete uptake and/or reduction of this metal by cells. In contrast to the untreated control cells, Cr(VI)-exposed cells formed apparently aseptate, nonmotile filaments that tended to aggregate. Transcriptome profiling and mass spectrometry-based proteomic characterization revealed that the principal molecular response to 24-h Cr(VI) exposure was the induction of prophage-related genes and their encoded products as well as a number of functionally undefined hypothetical genes that were located within the integrated phage regions of the MR-1 genome. In addition, genes with annotated functions in DNA metabolism, cell division, biosynthesis and degradation of the murein (peptidoglycan) sacculus, membrane response, and general environmental stress protection were upregulated, while genes encoding chemotaxis, motility, and transport/binding proteins were largely repressed under conditions of 24-h chromate treatment.

Reversible Electrowetting of Vertically Aligned Superhydrophobic Carbon Nanofibers

Langmuir : the ACS Journal of Surfaces and Colloids. Oct, 2006  |  Pubmed ID: 17014150

Reversible electrostatically induced wetting (electrowetting) of vertically aligned superhydrophobic carbon nanofibers has been investigated. Carbon nanofibers on a 5 x 5 microm pitch were grown on Si substrates, electrically insulated with a conformal dielectric, and hydrophobized with fluoropolymer. This nanostructured scaffold exhibited superhydrophobic behavior for saline (theta approximately 160 degrees). Electrowetting induced a contact angle reduction to theta approximately 100 degrees. Competitive two-liquid (dodecane/saline) electrowetting exhibited reversibility on the same nanostructured scaffold. Without applied bias, ultra-fine-point tip (approximately 25 nm radius) nanofibers result in effectively zero capacitance with the overlying saline layer. Complete electrowetting of the substrate is confirmed as capacitance values increase by several orders of magnitude with increased wetting. These results demonstrate the applicability of reversible electrowetting on nanostructured scaffolds and use of nanofabricated structures that can be integrated with various micro- and nanoelectronic technologies.

The Near-naked Hairless (Hr(N)) Mutation Disrupts Hair Formation but is Not Due to a Mutation in the Hairless Coding Region

The Journal of Investigative Dermatology. Jul, 2007  |  Pubmed ID: 17330134

Near-naked hairless (Hr(N)) is a semi-dominant, spontaneous mutation that was suggested by allelism testing to be allelic with mouse Hairless (Hr). Hr(N) mice differ from other Hr mutants in that hair loss appears as the postnatal coat begins to emerge, rather than as an inability to regrow hair after the first catagen and that the mutation displays semi-dominant inheritance. We sequenced the Hr cDNA in Hr(N)/Hr(N) mice and characterized the pathological and molecular phenotypes to identify the basis for hair loss in this model. Hr(N)/Hr(N) mice exhibit dystrophic hairs that are unable to emerge consistently from the hair follicle, whereas Hr(N)/+ mice display a sparse coat of hair and a milder degree of follicular dystrophy than their homozygous littermates. DNA microarray analysis of cutaneous gene expression demonstrates that numerous genes are downregulated in Hr(N)/Hr(N) mice, primarily genes important for hair structure. By contrast, Hr expression is significantly increased. Sequencing the Hr-coding region, intron-exon boundaries, 5'- and 3'-untranslated region, and immediate upstream region did not reveal the underlying mutation. Therefore, Hr(N) does not appear to be an allele of Hr but may result from a mutation in a closely linked gene or from a regulatory mutation in Hr.

Nano-enabled Synthetic Biology

Molecular Systems Biology. 2007  |  Pubmed ID: 17625513

Biological systems display a functional diversity, density and efficiency that make them a paradigm for synthetic systems. In natural systems, the cell is the elemental unit and efforts to emulate cells, their components, and organization have relied primarily on the use of bioorganic materials. Impressive advances have been made towards assembling simple genetic systems within cellular scale containers. These biological system assembly efforts are particularly instructive, as we gain command over the directed synthesis and assembly of synthetic nanoscale structures. Advances in nanoscale fabrication, assembly, and characterization are providing the tools and materials for characterizing and emulating the smallest scale features of biology. Further, they are revealing unique physical properties that emerge at the nanoscale. Realizing these properties in useful ways will require attention to the assembly of these nanoscale components. Attention to systems biology principles can lead to the practical development of nanoscale technologies with possible realization of synthetic systems with cell-like complexity. In turn, useful tools for interpreting biological complexity and for interfacing to biological processes will result.

Controlling the Dimensions of Carbon Nanofiber Structures Through the Electropolymerization of Pyrrole

Synthetic Metals. Apr, 2007  |  Pubmed ID: 18431459

Electrically conductive polymers, such as polypyrrole (PPy), show promise for modifying the dimensions and properties of micro- and nanoscale structures. Mechanisms for controlling the formation of PPy films of nanoscale thickness were evaluated by electrochemically synthesizing and examining PPy films on planar gold electrodes under a variety of growth conditions. Tunable PPy coatings were then deposited by electropolymerization on the sidewalls of individual, electrically addressable carbon nanofibers (CNFs). The ability to modify the physical size of specific nanofibers in controllable fashion is demonstrated. The biocompatibility, potential for chemical functionalization, and ability to effect volume changes of this nanocomposite can lead to advanced functionality, such as specific, nanoscale valving of materials and morphological control at the nanoscale.

A General System for Studying Protein-protein Interactions in Gram-negative Bacteria

Journal of Proteome Research. Aug, 2008  |  Pubmed ID: 18590317

One of the most promising methods for large-scale studies of protein interactions is isolation of an affinity-tagged protein with its in vivo interaction partners, followed by mass spectrometric identification of the copurified proteins. Previous studies have generated affinity-tagged proteins using genetic tools or cloning systems that are specific to a particular organism. To enable protein-protein interaction studies across a wider range of Gram-negative bacteria, we have developed a methodology based on expression of affinity-tagged "bait" proteins from a medium copy-number plasmid. This construct is based on a broad-host-range vector backbone (pBBR1MCS5). The vector has been modified to incorporate the Gateway DEST vector recombination region, to facilitate cloning and expression of fusion proteins bearing a variety of affinity, fluorescent, or other tags. We demonstrate this methodology by characterizing interactions among subunits of the DNA-dependent RNA polymerase complex in two metabolically versatile Gram-negative microbial species of environmental interest, Rhodopseudomonas palustris CGA010 and Shewanella oneidensis MR-1. Results compared favorably with those for both plasmid and chromosomally encoded affinity-tagged fusion proteins expressed in a model organism, Escherichia coli.

Biomineralization at the Nanoscale Learning from Diatoms

Methods in Cell Biology. 2008  |  Pubmed ID: 19195546

Actuatable Membranes Based on Polypyrrole-coated Vertically Aligned Carbon Nanofibers

ACS Nano. Feb, 2008  |  Pubmed ID: 19206624

Nanoporous membranes are applicable to a variety of research fields due to their ability to selectively separate molecules with high efficiency. Of particular interest are methods for controlling membrane selectivity through externally applied stimuli and integrating such membrane structures within multiscale systems. Membranes comprised of deterministically grown, vertically aligned carbon nanofibers (VACNFs) are compatible with these needs. VACNF membranes can regulate molecular transport by physically selecting species as they pass between the fibers. Defined interfiber spacing allows for nanoscale control of membrane pore structure and resultant size selectivity. Subsequent physical or chemical modification of VACNF structures enables the tuning of physical pore size and chemical specificity allowing further control of membrane permeability. In this work, the dynamic physical modulation of membrane permeability that results when VACNFs are coated with an electrically actuatable polymer, polypyrrole, is demonstrated. Electrochemical reduction of polypyrrole on the VACNFs results in controlled swelling of the diameter of the nanofibers that in turn decreases the pore size. Dynamic control of membrane pore size enables selective transport and gating of nanoscale pores.

Application of AFM in Understanding Biomineral Formation in Diatoms

Pflügers Archiv : European Journal of Physiology. Apr, 2008  |  Pubmed ID: 18058126

We review previous work and present new data on the application of atomic force microscopy (AFM) to study biomineral formation in diatoms, unicellular algae that make cell walls of silica. Previous studies examined a small subset of mostly larger diatom species, identifying a prevalence of large particulate silica on the nanoscale. We survey different structures including valves, girdle bands, and elongated spines called setae, in a variety of species, and show a diversity of nano- and meso-scale silica morphologies, even on different portions of the same structure. A general trend of highly organized mesoscale silica structure on the proximal face of cell wall components was observed, with less organized structure occurring on the distal face. The highly organized structures have features suggestive of an underlying linear template, which defines the area of initial silica polymerization. Such features have not been imaged with such clarity previously, demonstrating the advantages of AFM to image small differences in surface morphology and providing new insights and confirming evidence for models of diatom silica structure formation. In addition to its imaging capability, more developed application of AFM to map locations of organic template components on the nanoscale will greatly aid in elucidating mechanisms of diatom biosilica synthesis.

A Comparison of Hybridization Efficiency Between Flat Glass and Channel Glass Solid Supports

Molecular Biotechnology. Jan, 2008  |  Pubmed ID: 18095192

Two different solid supports, channel glass and flat glass, were compared for their affect on the sensitivity and efficiency of DNA hybridization reactions. Both solid supports were tested using a set of arrayed, synthetic oligonucleotides that are designed to detect short insertion/deletion polymorphisms (SIDPs). A total of 13 different human SIDPs were chosen for analysis. Capture probes, designed for this test set, were covalently immobilized on substrates. Hybridization efficiency was assessed using fluorescently labeled stacking probes which were preannealed to the target and then hybridized to the support-bound oligonucleotide array; the hybridization pattern was detected by fluorescence imaging. It was found that structural features of nucleic acid capture probes tethered to a solid support and the molecular basis of their interaction with targets in solution have direct implications on the hybridization process. Our results demonstrate that channel glass has a number of practical advantages over flat glass including higher sensitivity and a faster hybridization rate.

Channel Glass-based Detection of Human Short Insertion/deletion Polymorphisms by Tandem Hybridization

Molecular Biotechnology. Feb, 2008  |  Pubmed ID: 18219594

The development and critical evaluation of new technologies for identifying genetic polymorphisms will rapidly accelerate the discovery and diagnosis of disease-related genes. We report a novel way for distinguishing a new class of human DNA polymorphisms, short insertion/deletion polymorphisms (indels). A sensor with cylindrical pores named channel glass in combination with tandem hybridization, which uses a 5'-fluorescent labeled stacking probe and microarray-based short allele-specific oligonucleotide (capture probe) was investigated. This methodology allows indels to be detected individually and rapidly with small quantities of target DNA. This establishes a reliable quantitative test. Approaches for simultaneously hybridizing different targets to arrayed probes, designed to detect various indels in parallel, were examined. Five markers were consistently detected in a single hybridization. Possible factors impeding the hybridization reaction process are discussed.

Positional Control of Catalyst Nanoparticles for the Synthesis of High Density Carbon Nanofiber Arrays

Carbon. 2008  |  Pubmed ID: 19448842

Precise arrangement of nanoscale elements within larger systems, is essential to controlling higher order functionality and tailoring nanophase material properties. Here, we present findings on growth conditions for vertically aligned carbon nanofibers that enable synthesis of high density arrays and individual rows of nanofibers, which could be used to form barriers for restricting molecular transport, that have regular spacings and few defects. Growth through plasma-enhanced chemical vapor deposition was initiated from precisely formed nickel catalyst dots of varying diameter and spacing that were patterned through electron beam lithography. Nanofiber growth conditions, including power, precursor gas ratio, growth temperature and pressure were varied to optimize fiber uniformity and minimize defects that result from formation and migration of catalyst particles prior to growth. It was determined that both catalyst dot diameter and initial plasma power have a considerable influence on the number and severity of defects, while growth temperature, gas ratio (C(2)H(2):NH(3)) and pressure can be varied within a considerable range to fine-tune nanofiber morphology.

Effects of Ultramicroelectrode Dimensions on the Electropolymerization of Polypyrrole

Journal of Applied Physics. Jun, 2009  |  Pubmed ID: 19657404

Anode geometry can significantly affect the electrochemical synthesis of conductive polymers. Here, the effects of anode dimensions on the electropolymerization of pyrrole are investigated. Band microelectrodes were prepared with widths ranging from 2 to 500 mum. The anode dimension has a significant effect on the resulting thickness of polymer film. The electropolymerization process deviates significantly from that predicted by simple mass transfer considerations when electrode dimensions are less than approximately 20 mum. Polymer film thickness is thinner than expected when electrode dimensions become less than approximately 10 mum. A simple mathematical model was derived to explain the observed effects of anode dimensions on the polymerization process. Simulation results confirm that diffusive loss of reaction intermediates accounts for the observed experimental trends. The described simulation facilitates understanding of the electropolymerization processes and approaches to the controlled deposition of polypyrrole, particularly at the submicron scale, for microelectromechanical systems and biomedical applications.

An in Vivo Imaging-based Assay for Detecting Protein Interactions over a Wide Range of Binding Affinities

Analytical Biochemistry. Dec, 2009  |  Pubmed ID: 19698693

Identifying and characterizing protein interactions are fundamental steps toward understanding and modeling biological networks. Methods that detect protein interactions in intact cells rather than buffered solutions are likely more relevant to natural systems since molecular crowding events in the cytosol can influence the diffusion and reactivity of individual proteins. One in vivo, imaging-based method relies on the colocalization of two proteins of interest fused to DivIVA, a cell division protein from Bacillus subtilis, and green fluorescent protein (GFP). We have modified this imaging-based assay to facilitate rapid cloning by constructing new vectors encoding N- and C-terminal DivIVA or GFP molecular tag fusions based on site-specific recombination technology. The sensitivity of the assay was defined using a well-characterized protein interaction system involving the eukaryotic nuclear import receptor subunit, Importin alpha (Imp alpha), and variant nuclear localization signals (NLS) representing a range of binding affinities. These data demonstrate that the modified colocalization assay is sensitive enough to detect protein interactions with K(d) values that span over four orders of magnitude (1 nM to 15 microM). Lastly, this assay was used to confirm numerous protein interactions identified from mass spectrometry-based analyses of affinity isolates as part of an interactome mapping project in Rhodopseudomonas palustris.

Effects of Colistin on Surface Ultrastructure and Nanomechanics of Pseudomonas Aeruginosa Cells

Langmuir : the ACS Journal of Surfaces and Colloids. Apr, 2009  |  Pubmed ID: 19227989

Chronic lung infections in cystic fibrosis patients are primarily caused by Pseudomonas aeruginosa. Though difficult to counteract effectively, colistin, an antimicrobial peptide, is proving useful. However, the exact mechanism of action of colistin is not fully understood. In this study, atomic force microscopy (AFM) was used to evaluate, in a liquid environment, the changes in P. aeruginosa morphology and nanomechanical properties due to exposure to colistin. The results of this work revealed that after 1 h of colistin exposure the ratio of individual bacteria to those found to be arrested in the process of division changed from 1.9 to 0.4 and the length of the cells decreased significantly. Morphologically, it was observed that the bacterial surface changed from a smooth to a wrinkled phenotype after 3 h exposure to colistin. Nanomechanically, in untreated bacteria, the cantilever indented the bacterial surface significantly more than it did after 1 h of colistin treatment (P-value = 0.015). Concurrently, after 2 h of exposure to colistin, a significant increase in the bacterial spring constant was also observed. These results indicate that the antimicrobial peptide colistin prevents bacterial proliferation by repressing cell division. We also found that treatment with colistin caused an increase in the rigidity of the bacterial cell wall while morphologically the cell surface changed from smooth to wrinkled, perhaps due to loss of lipopolysaccharides (LPS) or surface proteins.

Cell Free Translation in Engineered Picoliter Volume Containers

Annual ORNL Biomedical Science and Engineering Center Conference ORNL Biomedical Science and Engineering Center Conference. Jun, 2009  |  Pubmed ID: 21278819

Engineers seek to use biological design principles to manipulate information and import new functionality to synthetic devices. Such devices inspired by natural systems could, in turn, play a crucial role in allowing biologists to explore the effects of physical transport and extreme conditions of temperature and pH on reaction systems. For example, engineered reaction containers can be physically and chemically defined to control the flux of molecules of different sizes and charge. The design and testing of such a container is described here. It has a volume of 19pL with defined slits of 200nm. The device successfully contained DNA and protein molecules and is evaluated for carrying out cell-free protein synthesis. The effect of DNA concentration and slit size on protein yield is discussed.

Development and Fabrication of Nanoporous Silicon-based Bioreactors Within a Microfluidic Chip

Lab on a Chip. May, 2010  |  Pubmed ID: 20390137

Multi-scale lithography and cryogenic deep reactive ion etching techniques were used to create ensembles of nanoporous, picolitre volume, reaction vessels within a microfluidic system. The fabrication of these vessels is described and how this process can be used to tailor vessel porosity by controlling the width of slits that constitute the vessel pores is demonstrated. Control of pore size allows the containment of nucleic acids and enzymes that are the foundation of biochemical reaction systems, while allowing smaller reaction constituents to traverse the container membrane and continuously supply the reaction. In this work, a 5.4 kb DNA plasmid was retained within the reaction vessels and labeled under microfluidic control with ethidium bromide as an initial proof-of-principle. Subsequently, a coupled enzyme reaction, in which glucose oxidase (GOX) and horseradish peroxidase (HRP) were contained and fed with a substrate solution of glucose and Amplex Red to produce fluorescent resorufin, was carried out under microfluidic control and monitored using fluorescent microscopy. The fabrication techniques presented are broadly applicable and can be adapted to produce devices in which a variety of high aspect ratio, nanoporous silicon structures can be integrated within a microfluidic network. The devices shown here are amenable to being scaled in number and organized to implement more complex reaction systems for applications in sensing and actuation as well as fundamental studies of biological reaction systems.

Silver Nanocrystallites: Biofabrication Using Shewanella Oneidensis, and an Evaluation of Their Comparative Toxicity on Gram-negative and Gram-positive Bacteria

Environmental Science & Technology. Jul, 2010  |  Pubmed ID: 20509652

Microorganisms have long been known to develop resistance to metal ions either by sequestering metals inside the cell or by effluxing them into the extracellular media. Here we report the biosynthesis of extracellular silver-based single nanocrystallites of well-defined composition and homogeneous morphology utilizing the gamma-proteobacterium, Shewanella oneidensis MR-1, upon incubation with aqueous silver nitrate solution. Further characterization of these particles revealed that the crystals consist of small, reasonably monodispersed spheres in the 2-11 nm size range (average of 4 +/- 1.5 nm). The bactericidal effect of these nanoparticles (biogenic-Ag) is compared to chemically synthesized silver nanoparticles (colloidal-Ag and oleate capped silver nanoparticles, oleate-Ag) and assessed using Gram-negative (E. coli and S. oneidensis) and Gram-positive (B. subtilis) bacteria. Relative toxicity was based on the diameter of inhibition zone in disk diffusion tests, minimum inhibitory concentrations, live/dead assays, and atomic force microscopy. From a toxicity perspective, strain-dependent inhibition depended on the synthesis procedure and the surface coat. Biogenic-Ag was found to be of higher toxicity compared to colloidal-Ag for all three strains tested, whereas E. coli and S. oneidensis were found to be more resistant to either of these nanoparticles than B. subtilis. In contrast, oleate-Ag was not toxic to any of the bacteria. These findings have implications for the potential uses of Ag nanomaterials and for their fate in biological and environmental systems.

Surface Charge- and Space-dependent Transport of Proteins in Crowded Environments of Nanotailored Posts

ACS Nano. Jun, 2010  |  Pubmed ID: 20515056

The reaction and diffusion of molecules across barriers and through crowded environments is integral to biological system function and to separation technologies. Ordered, microfabricated post arrays are a promising route to creating synthetic barriers with controlled chemical and physical characteristics. They can be used to create crowded environments, to mimic aspects of cellular membranes, and to serve as engineered replacements of polymer-based separation media. Here, the translational diffusion of fluorescein isothiocyante and various forms of green fluorescent protein (GFP), including "supercharged" variants, are examined in a silicon-based post array environment. The technique of fluorescence recovery after photobleaching (FRAP) is combined with analytical approximations and numerical simulations to assess the relative effects of reaction and diffusion on molecular transport, respectively. FRAP experiments were conducted for 64 different cases where the molecular species, the density of the posts, and the chemical surface charge of the posts were varied. In all cases, the dense packing of the posts hindered the diffusive transport of the fluorescent species. The supercharged GFPs strongly interacted with oppositely charged surfaces. With similar molecular and surface charges, transport is primarily limited by hindered diffusion. For conventional, enhanced GFP in a positively charged surface environment, transport was limited by the coupled action of hindered diffusion and surface interaction with the posts. Quantification of the size-, space-, time-, and charge-dependent translational diffusion in the post array environments can provide insight into natural processes and guide the design and development of selective membrane systems.

Atomic Force Microscopy of Biological Samples

Wiley Interdisciplinary Reviews. Nanomedicine and Nanobiotechnology. Nov-Dec, 2010  |  Pubmed ID: 20672388

The ability to evaluate structural-functional relationships in real time has allowed scanning probe microscopy (SPM) to assume a prominent role in post genomic biological research. In this mini-review, we highlight the development of imaging and ancillary techniques that have allowed SPM to permeate many key areas of contemporary research. We begin by examining the invention of the scanning tunneling microscope (STM) by Binnig and Rohrer in 1982 and discuss how it served to team biologists with physicists to integrate high-resolution microscopy into biological science. We point to the problems of imaging nonconductive biological samples with the STM and relate how this led to the evolution of the atomic force microscope (AFM) developed by Binnig, Quate, and Gerber, in 1986. Commercialization in the late 1980s established SPM as a powerful research tool in the biological research community. Contact mode AFM imaging was soon complemented by the development of non-contact imaging modes. These non-contact modes eventually became the primary focus for further new applications including the development of fast scanning methods. The extreme sensitivity of the AFM cantilever was recognized and has been developed into applications for measuring forces required for indenting biological surfaces and breaking bonds between biomolecules. Further functional augmentation to the cantilever tip allowed development of new and emerging techniques including scanning ion-conductance microscopy (SICM), scanning electrochemical microscope (SECM), Kelvin force microscopy (KFM) and scanning near field ultrasonic holography (SNFUH).

Effects of Engineered Cerium Oxide Nanoparticles on Bacterial Growth and Viability

Applied and Environmental Microbiology. Dec, 2010  |  Pubmed ID: 20952651

Interest in engineered nanostructures has risen in recent years due to their use in energy conservation strategies and biomedicine. To ensure prudent development and use of nanomaterials, the fate and effects of such engineered structures on the environment should be understood. Interactions of nanomaterials with environmental microorganisms are inevitable, but the general consequences of such interactions remain unclear, due to a lack of standard methods for assessing such interactions. Therefore, we have initiated a multianalytical approach to understand the interactions of synthesized nanoparticles with bacterial systems. These efforts are focused initially on cerium oxide nanoparticles and model bacteria in order to evaluate characterization procedures and the possible fate of such materials in the environment. The growth and viability of the Gram-negative species Escherichia coli and Shewanella oneidensis, a metal-reducing bacterium, and the Gram-positive species Bacillus subtilis were examined relative to cerium oxide particle size, growth media, pH, and dosage. A hydrothermal synthesis approach was used to prepare cerium oxide nanoparticles of defined sizes in order to eliminate complications originating from the use of organic solvents and surfactants. Bactericidal effects were determined from MIC and CFU measurements, disk diffusion tests, and live/dead assays. For E. coli and B. subtilis, clear strain- and size-dependent inhibition was observed, whereas S. oneidensis appeared to be unaffected by the particles. Transmission electron microscopy along with microarray-based transcriptional profiling was used to understand the response mechanism of the bacteria. Use of multiple analytical approaches adds confidence to toxicity assessments, while the use of different bacterial systems highlights the potential wide-ranging effects of nanomaterial interactions in the environment.

Characterization of Cell Surface and Extracellular Matrix Remodeling of Azospirillum Brasilense Chemotaxis-like 1 Signal Transduction Pathway Mutants by Atomic Force Microscopy

FEMS Microbiology Letters. Jan, 2011  |  Pubmed ID: 21105907

To compete in complex microbial communities, bacteria must sense environmental changes and adjust cellular functions for optimal growth. Chemotaxis-like signal transduction pathways are implicated in the regulation of multiple behaviors in response to changes in the environment, including motility patterns, exopolysaccharide production, and cell-to-cell interactions. In Azospirillum brasilense, cell surface properties, including exopolysaccharide production, are thought to play a direct role in promoting flocculation. Recently, the Che1 chemotaxis-like pathway from A. brasilense was shown to modulate flocculation, suggesting an associated modulation of cell surface properties. Using atomic force microscopy, distinct changes in the surface morphology of flocculating A. brasilense Che1 mutant strains were detected. Whereas the wild-type strain produces a smooth mucosal extracellular matrix after 24 h, the flocculating Che1 mutant strains produce distinctive extracellular fibril structures. Further analyses using flocculation inhibition, lectin-binding assays, and comparison of lipopolysaccharides profiles suggest that the extracellular matrix differs between the cheA1 and the cheY1 mutants, despite an apparent similarity in the macroscopic floc structures. Collectively, these data indicate that disruption of the Che1 pathway is correlated with distinctive changes in the extracellular matrix, which likely result from changes in surface polysaccharides structure and/or composition.

Biofabrication of Discrete Spherical Gold Nanoparticles Using the Metal-reducing Bacterium Shewanella Oneidensis

Acta Biomaterialia. May, 2011  |  Pubmed ID: 21241833

Nanocrystallites have garnered substantial interest due to their various applications, including catalysis and medical research. Consequently important aspects of synthesis related to control of shape and size through economical and non-hazardous means are desirable. Highly efficient bioreduction-based fabrication approaches that utilize microbes and/or plant extracts are poised to meet these needs. Here we show that the γ-proteobacterium Shewanella oneidensis can reduce tetrachloroaurate (III) ions to produce discrete extracellular spherical gold nanocrystallites. The particles were homogeneously shaped with multiple size distributions and produced under ambient conditions at high yield, 88% theoretical maximum. Further characterization revealed that the particles consist of spheres in the size range of ∼2-50 nm, with an average size of 12±5 nm. The nanoparticles were hydrophilic and resisted aggregation even after several months. Based on our experiments, the particles are likely fabricated by the aid of reducing agents present in the bacterial cell membrane and are capped by a detachable protein/peptide coat. Ultraviolet-visible and Fourier transform infrared spectroscopy, X-ray diffraction, energy dispersive X-ray spectra and transmission electron microscopy measurements confirmed the formation, surface characteristics and crystalline nature of the nanoparticles. The antibacterial activity of these gold nanoparticles was assessed using Gram-negative (Escherichia coli and S. oneidensis) and Gram-positive (Bacillus subtilis) bacterial species. Toxicity assessments showed that the particles were neither toxic nor inhibitory to any of these bacteria.

Effects of Sub-minimum Inhibitory Concentrations of Ciprofloxacin on Enteroaggregative Escherichia Coli and the Role of the Surface Protein Dispersin

International Journal of Antimicrobial Agents. Jul, 2011  |  Pubmed ID: 21570813

Enteroaggregative Escherichia coli (EAEC) are bacterial pathogens that cause watery diarrhoea, which is often persistent and can be inflammatory. The antibiotic ciprofloxacin is used to treat EAEC infections, but a full understanding of the antimicrobial effects of ciprofloxacin is needed for more efficient treatment of bacterial infections. In this study, it was found that sub-minimum inhibitory concentrations (sub-MICs) of ciprofloxacin had an inhibitory effect on EAEC adhesion to glass and mammalian HEp-2 cells. It was also observed that bacterial surface properties play an important role in bacterial sensitivity to ciprofloxacin. In an EAEC mutant strain where the hydrophobic positively charged surface protein dispersin was absent, sensitivity to ciprofloxacin was reduced compared with the wild-type strain. Identified here are several antimicrobial effects of ciprofloxacin at sub-MIC concentrations indicating that bacterial surface hydrophobicity affects the response to ciprofloxacin. Investigating the effects of sub-MIC doses of antibiotics on targeted bacteria could help to further our understanding of bacterial pathogenicity and elucidate future antibiotic treatment modalities.

Model for Biological Communication in a Nanofabricated Cell-mimic Driven by Stochastic Resonance

Nano Communication Networks. Mar, 2011  |  Pubmed ID: 21731597

Cells offer natural examples of highly efficient networks of nanomachines. Accordingly, both intracellular and intercellular communication mechanisms in nature are looked to as a source of inspiration and instruction for engineered nanocommunication. Harnessing biological functionality in this manner requires an interdisciplinary approach that integrates systems biology, synthetic biology, and nanofabrication. Here, we present a model system that exemplifies the synergism between these realms of research. We propose a synthetic gene network for operation in a nanofabricated cell mimic array that propagates a biomolecular signal over long distances using the phenomenon of stochastic resonance. Our system consists of a bacterial quorum sensing signal molecule, a bistable genetic switch triggered by this signal, and an array of nanofabricated cell mimic wells that contain the genetic system. An optimal level of noise in the system helps to propagate a time-varying AHL signal over long distances through the array of mimics. This noise level is determined both by the system volume and by the parameters of the genetic network. Our proposed genetically driven stochastic resonance system serves as a testbed for exploring the potential harnessing of gene expression noise to aid in the transmission of a time-varying molecular signal.

Distinct Microbial Communities Within the Endosphere and Rhizosphere of Populus Deltoides Roots Across Contrasting Soil Types

Applied and Environmental Microbiology. Sep, 2011  |  Pubmed ID: 21764952

The root-rhizosphere interface of Populus is the nexus of a variety of associations between bacteria, fungi, and the host plant and an ideal model for studying interactions between plants and microorganisms. However, such studies have generally been confined to greenhouse and plantation systems. Here we analyze microbial communities from the root endophytic and rhizospheric habitats of Populus deltoides in mature natural trees from both upland and bottomland sites in central Tennessee. Community profiling utilized 454 pyrosequencing with separate primers targeting the V4 region for bacterial 16S rRNA and the D1/D2 region for fungal 28S rRNA genes. Rhizosphere bacteria were dominated by Acidobacteria (31%) and Alphaproteobacteria (30%), whereas most endophytes were from the Gammaproteobacteria (54%) as well as Alphaproteobacteria (23%). A single Pseudomonas-like operational taxonomic unit (OTU) accounted for 34% of endophytic bacterial sequences. Endophytic bacterial richness was also highly variable and 10-fold lower than in rhizosphere samples originating from the same roots. Fungal rhizosphere and endophyte samples had approximately equal amounts of the Pezizomycotina (40%), while the Agaricomycotina were more abundant in the rhizosphere (34%) than endosphere (17%). Both fungal and bacterial rhizosphere samples were highly clustered compared to the more variable endophyte samples in a UniFrac principal coordinates analysis, regardless of upland or bottomland site origin. Hierarchical clustering of OTU relative abundance patterns also showed that the most abundant bacterial and fungal OTUs tended to be dominant in either the endophyte or rhizosphere samples but not both. Together, these findings demonstrate that root endophytic communities are distinct assemblages rather than opportunistic subsets of the rhizosphere.

Monodispersed Biocompatible Silver Sulfide Nanoparticles: Facile Extracellular Biosynthesis Using the γ-proteobacterium, Shewanella Oneidensis

Acta Biomaterialia. Dec, 2011  |  Pubmed ID: 21798382

Interest in engineered metal and semiconductor nanocrystallites continues to grow due to their unique size- and shape-dependent optoelectronic, physicochemical and biological properties. Therefore identifying novel non-hazardous nanoparticle synthesis routes that address hydrophilicity, size and shape control and production costs has become a priority. In the present article we report for the first time on the efficient generation of extracellular silver sulfide (Ag₂S) nanoparticles by the metal-reducing bacterium Shewanella oneidensis. The particles are reasonably monodispersed and homogeneously shaped. They are produced under ambient temperatures and pressures at high yield, 85% theoretical maximum. UV-visible and Fourier transform infrared spectroscopy, dynamic light scattering, X-ray diffraction, transmission electron microscopy and X-ray photoelectron spectroscopy measurements confirmed the formation, optical and surface properties, purity and crystallinity of the synthesized particles. Further characterization revealed that the particles consist of spheres with a mean diameter of 9±3.5 nm, and are capped by a detachable protein/peptide surface coat. Toxicity assessments of these biogenic Ag₂S nanoparticles on Gram-negative (Escherichia coli and S. oneidensis) and Gram-positive (Bacillus subtilis) bacterial systems, as well as eukaryotic cell lines including mouse lung epithelial (C 10) and macrophage (RAW-264.7) cells, showed that the particles were non-inhibitory and non-cytotoxic to any of these systems. Our results provide a facile, eco-friendly and economical route for the fabrication of technologically important semiconducting Ag₂S nanoparticles. These particles are dispersible and biocompatible, thus providing excellent potential for use in optical imaging, electronic devices and solar cell applications.

Continuous Protein Production in Nanoporous, Picolitre Volume Containers

Lab on a Chip. Oct, 2011  |  Pubmed ID: 21879140

The synthetic manufacture of functional proteins enables a bottom-up understanding of the workings of biological systems and opens new opportunities for the treatment of disease. Cell-free protein synthesis is a practical approach for enabling such manufacturing, however, it is typically carried out in fairly large volumes, when compared to a natural cell, leading to increases in cost and loss of efficiency. Here we demonstrate continuous cell free protein synthesis in arrays of cellular scale containers that continuously exchange energy and materials with their environment. A multiscale fabrication process allows the monolithic integration of nanoporous silicon containers within an addressable microfluidic network. Synthesis of enhanced green fluorescent protein (eGFP) in the containers continues beyond 24 h and yields more than twice the amount of protein, on a per volume basis, than conventional scale batch reactions. By mimicking the physical volume and controlled flux of a natural cell, the resulting "cell mimic" devices can enable fundamental studies of biological systems as well as serve applications related to the functional screening of proteins and the on-demand production of biologics.

The Chemotaxis-like Che1 Pathway Has an Indirect Role in Adhesive Cell Properties of Azospirillum Brasilense

FEMS Microbiology Letters. Oct, 2011  |  Pubmed ID: 22092709

The Azospirillum brasilense chemotaxis-like Che1 signal transduction pathway was recently shown to modulate changes in adhesive cell surface properties that, in turn, affect cell-to-cell aggregation and flocculation behaviors rather than flagellar-mediated chemotaxis. Attachment to surfaces and root colonization may be functions related to flocculation. Here, the conditions under which A. brasilense wild-type Sp7 and che1 mutant strains attach to abiotic and biotic surfaces were examined using in vitro attachment and biofilm assays combined with atomic force microscopy and confocal microscopy. The nitrogen source available for growth is found to be a major modulator of surface attachment by A. brasilense and could be promoted in vitro by lectins, suggesting that it depends on interaction with surface-exposed residues within the extracellular matrix of cells. However, Che1-dependent signaling is shown to contribute indirectly to surface attachment, indicating that distinct mechanisms are likely underlying flocculation and attachment to surfaces in A. brasilense.

Expression Optimization and Synthetic Gene Networks in Cell-free Systems

Nucleic Acids Research. Dec, 2011  |  Pubmed ID: 22180537

Synthetic biology offers great promise to a variety of applications through the forward engineering of biological function. Most efforts in this field have focused on employing living cells, yet cell-free approaches offer simpler and more flexible contexts. Here, we evaluate cell-free regulatory systems based on T7 promoter-driven expression by characterizing variants of TetR and LacI repressible T7 promoters in a cell-free context and examining sequence elements that determine expression efficiency. Using the resulting constructs, we then explore different approaches for composing regulatory systems, leading to the implementation of inducible negative feedback in Escherichia coli extracts and in the minimal PURE system, which consists of purified proteins necessary for transcription and translation. Despite the fact that negative feedback motifs are common and essential to many natural and engineered systems, this simple building block has not previously been implemented in a cell-free context. As a final step, we then demonstrate that the feedback systems developed using our cell-free approach can be implemented in live E. coli as well, illustrating the potential for using cell-free expression to fast track the development of live cell systems in synthetic biology. Our quantitative cell-free component characterizations and demonstration of negative feedback embody important steps on the path to harnessing biological function in a bottom-up fashion.

Cytotoxicity Induced by Engineered Silver Nanocrystallites is Dependent on Surface Coatings and Cell Types

Langmuir : the ACS Journal of Surfaces and Colloids. Feb, 2012  |  Pubmed ID: 22216981

Due to their unique antimicrobial properties silver nanocrystallites have garnered substantial attention and are used extensively for biomedical applications as an additive to wound dressings, surgical instruments and bone substitute materials. They are also released into unintended locations such as the environment or biosphere. Therefore it is imperative to understand the potential interactions, fate and transport of nanoparticles with environmental biotic systems. Numerous factors including the composition, size, shape, surface charge, and capping molecule of nanoparticles are known to influence cell cytotoxicity. Our results demonstrate that the physical/chemical properties of the silver nanoparticles including surface charge, differential binding and aggregation potential, which are influenced by the surface coatings, are a major determining factor in eliciting cytotoxicity and in dictating potential cellular interactions. In the present investigation, silver nanocrystallites with nearly uniform size and shape distribution but with different surface coatings, imparting overall high negativity to high positivity, were synthesized. These nanoparticles included poly(diallyldimethylammonium) chloride-Ag, biogenic-Ag, colloidal-Ag (uncoated), and oleate-Ag with zeta potentials +45 ± 5, -12 ± 2, -42 ± 5, and -45 ± 5 mV, respectively; the particles were purified and thoroughly characterized so as to avoid false cytotoxicity interpretations. A systematic investigation on the cytotoxic effects, cellular response, and membrane damage caused by these four different silver nanoparticles was carried out using multiple toxicity measurements on mouse macrophage (RAW-264.7) and lung epithelial (C-10) cell lines. Our results clearly indicate that the cytotoxicity was dependent on various factors such as surface charge and coating materials used in the synthesis, particle aggregation, and the cell-type for the different silver nanoparticles that were investigated. Poly(diallyldimethylammonium)-coated Ag nanoparticles were found to be the most toxic, followed by biogenic-Ag and oleate-Ag nanoparticles, whereas uncoated or colloidal silver nanoparticles were found to be the least toxic to both macrophage and lung epithelial cells. Also, based on our cytotoxicity interpretations, lung epithelial cells were found to be more resistant to the silver nanoparticles than the macrophage cells, regardless of the surface coating.

Enzyme Reactions in Nanoporous, Picoliter Volume Containers

Analytical Chemistry. Jan, 2012  |  Pubmed ID: 22148720

Advancements in nanoscale fabrication allow creation of small-volume reaction containers that can facilitate the screening and characterization of enzymes. A porous, ∼19 pL volume vessel has been used in this work to carry out enzyme reactions under varying substrate concentrations. Assessment of small-molecule and green fluorescent protein diffusion from the vessels indicates that pore sizes on the order of 10 nm can be obtained, allowing capture of proteins and diffusive exchange of small molecules. Glucose oxidase and horseradish peroxidase can be contained in these structures and diffusively fed with a solution containing glucose and the fluorogenic substrate amplex red through the engineered nanoscale pore structure. Fluorescent microscopy was used to monitor the reaction, which was carried out under microfluidic control. Kinetic characteristics of the enzyme (K(m) and V(max)) were evaluated and compared with results from conventional scale reactions. These picoliter, nanoporous containers can facilitate quick determination of enzyme kinetics in microfluidic systems without the requirement of surface tethering and can be used for applications in drug discovery, clinical diagnostics, and high-throughput screening.