N. Niamsiri, S. Delamarre, M. Bergkvist, N. Cady, S. Stelick, G. Coates, C. Ober, C. Batt
Bionanofabrication is a novel fabrication process that takes advantage of the specificity and catalytic efficiency of biological systems to create novel nanoscale structures. Polyhydroxyalkanoates (PHAs) are a family of aliphatic polyesters produced by a variety of microorganisms as a reserve of carbon and energy. PHAs can be combined from more than 100 different monomers to give materials with widely different physical properties. PHAs are biocompatible, biodegradable and demonstrate piezo electric and non-linear optical properties making them potential useful for tissue engineering, drug delivery, degradable packaging and smart materials. The enzymes involved in the synthesis of PHAs have been harnessed in our laboratory to produce novel polymers in vitro both in bulk and on solid surfaces. Site-specific attachment of the key catalytic enzyme, PHA synthase, on nanofabricated surfaces and subsequent addition of 3-(R)-hydroxybutyryl-CoA substrates (HB-CoA), allows us to create spatially ordered polyhydroxybutyrate (PHB) polymeric structures via in situ enzymatic surface-initiated polymerization (ESIP). By varying the reaction conditions we have optimized the PHB polymer growth at the interface and the resulting material characterized by fluorescence microscopy and atomic force microscopy. In the absence of additives such as bovine serum albumin, the PHB polymer synthesized on the surfaces formed very distinct and uniform granular structures on Au patterned surfaces. The average size of PHB granules was measured to be approximately 0.5 to 1 μm in diameter and 100 nm in height from the Au surfaces. In the presence of bovine serum albumin, the average size of PHB granules and PHB film thickness sinificantly increased to be approximately 1 to 5 μm in diameter and 500 nm to 1 μm in height, respectively, uniformly covering patterned surfaces. We believe that the use of this novel enzymatic approach offers many practical applications in different areas. For example, it can be employed to generate biocompatible PHAs coated solid surfaces for tissue engineering, promoting cell attachment and growth. As a result, one of our goals is to employ ESIP for in situ solid-phase synthesis of novel functionalized PHAs micro-/nanostructures with a wide range of mechanical, thermal, and biocompatible properties. In addition to biocompatible surface coatings, we envision that the novel polymeric micro-/nano-structures can be built in spaces that cannot be accessed by convention lithographic tools or other fabrication process. For example, PHB structures can be formed in situ inside microfluidic channels to produce rapid microfluidic mixing. Currently, we are investigating the use of in situ synthesized PHB polymer on specific Au patterned surfaces such as straight ridges and staggered herringbone patterns to act as passive micromixers inside microfluidic channels.
{"title":"Bionanofabrication polyhydroxyalkanoates (PHAS) micro-/nanostructures on solid surfaces and its applications in nanobiotechnology","authors":"N. Niamsiri, S. Delamarre, M. Bergkvist, N. Cady, S. Stelick, G. Coates, C. Ober, C. Batt","doi":"10.1109/BMN.2006.330943","DOIUrl":"https://doi.org/10.1109/BMN.2006.330943","url":null,"abstract":"Bionanofabrication is a novel fabrication process that takes advantage of the specificity and catalytic efficiency of biological systems to create novel nanoscale structures. Polyhydroxyalkanoates (PHAs) are a family of aliphatic polyesters produced by a variety of microorganisms as a reserve of carbon and energy. PHAs can be combined from more than 100 different monomers to give materials with widely different physical properties. PHAs are biocompatible, biodegradable and demonstrate piezo electric and non-linear optical properties making them potential useful for tissue engineering, drug delivery, degradable packaging and smart materials. The enzymes involved in the synthesis of PHAs have been harnessed in our laboratory to produce novel polymers in vitro both in bulk and on solid surfaces. Site-specific attachment of the key catalytic enzyme, PHA synthase, on nanofabricated surfaces and subsequent addition of 3-(R)-hydroxybutyryl-CoA substrates (HB-CoA), allows us to create spatially ordered polyhydroxybutyrate (PHB) polymeric structures via in situ enzymatic surface-initiated polymerization (ESIP). By varying the reaction conditions we have optimized the PHB polymer growth at the interface and the resulting material characterized by fluorescence microscopy and atomic force microscopy. In the absence of additives such as bovine serum albumin, the PHB polymer synthesized on the surfaces formed very distinct and uniform granular structures on Au patterned surfaces. The average size of PHB granules was measured to be approximately 0.5 to 1 μm in diameter and 100 nm in height from the Au surfaces. In the presence of bovine serum albumin, the average size of PHB granules and PHB film thickness sinificantly increased to be approximately 1 to 5 μm in diameter and 500 nm to 1 μm in height, respectively, uniformly covering patterned surfaces. We believe that the use of this novel enzymatic approach offers many practical applications in different areas. For example, it can be employed to generate biocompatible PHAs coated solid surfaces for tissue engineering, promoting cell attachment and growth. As a result, one of our goals is to employ ESIP for in situ solid-phase synthesis of novel functionalized PHAs micro-/nanostructures with a wide range of mechanical, thermal, and biocompatible properties. In addition to biocompatible surface coatings, we envision that the novel polymeric micro-/nano-structures can be built in spaces that cannot be accessed by convention lithographic tools or other fabrication process. For example, PHB structures can be formed in situ inside microfluidic channels to produce rapid microfluidic mixing. Currently, we are investigating the use of in situ synthesized PHB polymer on specific Au patterned surfaces such as straight ridges and staggered herringbone patterns to act as passive micromixers inside microfluidic channels.","PeriodicalId":271407,"journal":{"name":"2006 Bio Micro and Nanosystems Conference","volume":"21 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2007-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"115959802","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
H. Cho, A. Groisman, J. Campbell, S. Flores, A. Levchenko
Summary form only given. Escherichia coli survive in the hostile environment of host organ and cause rigorous infections. They generally form high-density colony and sustain high resistance to drug treatment as well as immune systems. Elucidating the exact mechanisms of Escherichia coli infections have been intriguing question in the field of pathogenic microbiology. Recently, we developed a new microfluidic chemostat device for bacterial study overcoming the limitations of the conventional experimental tools. The architecture of the microfluidic chemostat is notably similar to bacterial biofilms, where cells grow in high-density, with channels for supplying nutrients and removing wastes. The size of the microfluidic chemostat is also similar to that of biofilms found in host organ infections. Specifically, the microfluidic chemostat with deformable membranes developed in this study is designed to have a very thin PDMS layer between two layers mimicking the host cell membrane, which helps to simulate the intracellular biofilm-like structure formation in terms of confined boundary and chemical composition. It also makes it possible to monitor individual cell's behavior and gene expression of interest combined with fluorescence protein technique. Using this device, we observed considerable pressure up to 2.8 psi generated by cells expanding to extremely high density, which may explain the burst of host cell membrane occurred during uropathogenic bacteria infection. We also characterized the spatial and temporal distribution of stress response to correlate the mechanical stress to biological stress. Combining these findings with other information on biofilm formation and bacterial cell-cell communication will ultimately provide us with a better understanding of bacterial infection and potentially lead to new and improved treatment protocols
{"title":"Microfluidic chemostat with deformable membranes: intracellular biofilm-like structure model","authors":"H. Cho, A. Groisman, J. Campbell, S. Flores, A. Levchenko","doi":"10.1109/BMN.2006.330906","DOIUrl":"https://doi.org/10.1109/BMN.2006.330906","url":null,"abstract":"Summary form only given. Escherichia coli survive in the hostile environment of host organ and cause rigorous infections. They generally form high-density colony and sustain high resistance to drug treatment as well as immune systems. Elucidating the exact mechanisms of Escherichia coli infections have been intriguing question in the field of pathogenic microbiology. Recently, we developed a new microfluidic chemostat device for bacterial study overcoming the limitations of the conventional experimental tools. The architecture of the microfluidic chemostat is notably similar to bacterial biofilms, where cells grow in high-density, with channels for supplying nutrients and removing wastes. The size of the microfluidic chemostat is also similar to that of biofilms found in host organ infections. Specifically, the microfluidic chemostat with deformable membranes developed in this study is designed to have a very thin PDMS layer between two layers mimicking the host cell membrane, which helps to simulate the intracellular biofilm-like structure formation in terms of confined boundary and chemical composition. It also makes it possible to monitor individual cell's behavior and gene expression of interest combined with fluorescence protein technique. Using this device, we observed considerable pressure up to 2.8 psi generated by cells expanding to extremely high density, which may explain the burst of host cell membrane occurred during uropathogenic bacteria infection. We also characterized the spatial and temporal distribution of stress response to correlate the mechanical stress to biological stress. Combining these findings with other information on biofilm formation and bacterial cell-cell communication will ultimately provide us with a better understanding of bacterial infection and potentially lead to new and improved treatment protocols","PeriodicalId":271407,"journal":{"name":"2006 Bio Micro and Nanosystems Conference","volume":"5 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2007-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"121226682","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Bio-nano robots are nano-scaled robots made from biological components like proteins and DNA structures. Their nano-scaled size, ready availability (in nature), and high efficiency make them perfect tools for diagnosis and therapeutic treatments in nano-medicine. Due to their nano-scaled size, the intelligence of each individual nano robot is small when compared to that of the collection of nano robots acting together to accomplish the given task. This group intelligence, called swarm intelligence, helps the nano robots do their task more effectively, more quickly, and with fewer other resources. The coordination to accomplish the given task can be achieved by these nano robots through quorum sensing. Quorum sensing is the ability of nano robots to communicate and coordinate behavior via signaling molecules. The whole scenario of communication and coordination can be done using these nano-scaled robots and the results are studied using simulation at a high level of abstraction
{"title":"Swarm intelligence for cooperation of bio-nano robots using quorum sensing","authors":"S. Chandrasekaran, Dean Frederick Hougen","doi":"10.1109/BMN.2006.330912","DOIUrl":"https://doi.org/10.1109/BMN.2006.330912","url":null,"abstract":"Bio-nano robots are nano-scaled robots made from biological components like proteins and DNA structures. Their nano-scaled size, ready availability (in nature), and high efficiency make them perfect tools for diagnosis and therapeutic treatments in nano-medicine. Due to their nano-scaled size, the intelligence of each individual nano robot is small when compared to that of the collection of nano robots acting together to accomplish the given task. This group intelligence, called swarm intelligence, helps the nano robots do their task more effectively, more quickly, and with fewer other resources. The coordination to accomplish the given task can be achieved by these nano robots through quorum sensing. Quorum sensing is the ability of nano robots to communicate and coordinate behavior via signaling molecules. The whole scenario of communication and coordination can be done using these nano-scaled robots and the results are studied using simulation at a high level of abstraction","PeriodicalId":271407,"journal":{"name":"2006 Bio Micro and Nanosystems Conference","volume":"1979 9","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2007-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"120847208","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
J. Adamo, Jodi B. Luland-richards, Eric N. Antonelli, Eugene F. Garritt, M. Gealt
In our studies we have used three different free-living nematodes: Rhabditis, Caenorhabditis elegans and Turbatrix aceti. Rhabditis, a microscopic nematode, has been reported worldwide in most soils and a wide variety of environments (air, water and land). It has been isolated in or on many invertebrates and higher forms of life i.e. earthworms, insects, plants, birds and other animals including man. Experiments were designed and showed that this worm could survive and was very resistant to chlorine bleach treatment with no ill effect. This allowed us to control bacterial and viral activity on the surface of the nematode. Further, we demonstrated that the organism gathered, concentrated, protected, hid and only digested about 70% of the bacteria it consumed, defecating 30% viable. We determined that the worm, on average could carry 1.6times106 bacteria. By feeding alternate identifiable strains of bacteria we were able to demonstrate internal bacterial conjugation, resulting in DNA transfer, transconjugants. The same studies done with C. elegans showed very similar results as those for Rhabditis. Here we demonstrated that the nematode could vector active virus (PhiX-174, 20nm). We were also able to purge the nematode of bacteria by adding the virus to the culture. Work with T. aceti using bent glass capillary tubes connected to wells in phenol red dextrose agar (PRDA) petri plates showed statistically significant pH preferential migratory behavior. The small size of the nematodes, their ability to vector bacteria, virus and to dismember bio-films suggests possible usefulness as nanotechnology delivery systems
{"title":"Nematodes as bacterial, viral and potential nanotechnology delivery systems","authors":"J. Adamo, Jodi B. Luland-richards, Eric N. Antonelli, Eugene F. Garritt, M. Gealt","doi":"10.1109/BMN.2006.330911","DOIUrl":"https://doi.org/10.1109/BMN.2006.330911","url":null,"abstract":"In our studies we have used three different free-living nematodes: Rhabditis, Caenorhabditis elegans and Turbatrix aceti. Rhabditis, a microscopic nematode, has been reported worldwide in most soils and a wide variety of environments (air, water and land). It has been isolated in or on many invertebrates and higher forms of life i.e. earthworms, insects, plants, birds and other animals including man. Experiments were designed and showed that this worm could survive and was very resistant to chlorine bleach treatment with no ill effect. This allowed us to control bacterial and viral activity on the surface of the nematode. Further, we demonstrated that the organism gathered, concentrated, protected, hid and only digested about 70% of the bacteria it consumed, defecating 30% viable. We determined that the worm, on average could carry 1.6times106 bacteria. By feeding alternate identifiable strains of bacteria we were able to demonstrate internal bacterial conjugation, resulting in DNA transfer, transconjugants. The same studies done with C. elegans showed very similar results as those for Rhabditis. Here we demonstrated that the nematode could vector active virus (PhiX-174, 20nm). We were also able to purge the nematode of bacteria by adding the virus to the culture. Work with T. aceti using bent glass capillary tubes connected to wells in phenol red dextrose agar (PRDA) petri plates showed statistically significant pH preferential migratory behavior. The small size of the nematodes, their ability to vector bacteria, virus and to dismember bio-films suggests possible usefulness as nanotechnology delivery systems","PeriodicalId":271407,"journal":{"name":"2006 Bio Micro and Nanosystems Conference","volume":"1 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2007-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"123303915","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2006-07-31DOI: 10.1017/S1431927606069194
S. Subramaniam
Summary form only given. Emerging methods in three-dimensional biological electron microscopy provide powerful tools and great promise to bridge a critical gap in imaging in the biomedical size spectrum. This gap comprises a size range of great interest in biology and medicine that includes cellular protein machines, giant protein and nucleic acid assemblies, small subcellular organelles and small bacteria. In our laboratory at the National Cancer Institute, NIH, we are using a variety of approaches that utilize electron microscopic imaging to discover and analyze biological complexity within the size gap with linear dimensions of about 50-1000 nm. A key mission of our laboratory is to quantitatively describe the spatial and temporal architecture of key molecular machines that fall into this "nano gap". Areas of current interest include: (i) the development and application of novel technologies for three-dimensional electron microscopy of specimens ranging in size from small molecules to tissues, including automated approaches to analyze the molecular structure and sub-cellular location of a variety of nanoparticles, (ii) determination of the dynamic spatial and temporal architectures of cellular structures and molecular machines involved in fundamental process such as energy transduction, cell division and chemotaxis, and (iii) determination of molecular mechanisms underlying the neutralization and cellular entry of HIV
{"title":"Bridging the imaging gap in nanobiology with three-dimensional electron microscopy","authors":"S. Subramaniam","doi":"10.1017/S1431927606069194","DOIUrl":"https://doi.org/10.1017/S1431927606069194","url":null,"abstract":"Summary form only given. Emerging methods in three-dimensional biological electron microscopy provide powerful tools and great promise to bridge a critical gap in imaging in the biomedical size spectrum. This gap comprises a size range of great interest in biology and medicine that includes cellular protein machines, giant protein and nucleic acid assemblies, small subcellular organelles and small bacteria. In our laboratory at the National Cancer Institute, NIH, we are using a variety of approaches that utilize electron microscopic imaging to discover and analyze biological complexity within the size gap with linear dimensions of about 50-1000 nm. A key mission of our laboratory is to quantitatively describe the spatial and temporal architecture of key molecular machines that fall into this \"nano gap\". Areas of current interest include: (i) the development and application of novel technologies for three-dimensional electron microscopy of specimens ranging in size from small molecules to tissues, including automated approaches to analyze the molecular structure and sub-cellular location of a variety of nanoparticles, (ii) determination of the dynamic spatial and temporal architectures of cellular structures and molecular machines involved in fundamental process such as energy transduction, cell division and chemotaxis, and (iii) determination of molecular mechanisms underlying the neutralization and cellular entry of HIV","PeriodicalId":271407,"journal":{"name":"2006 Bio Micro and Nanosystems Conference","volume":"20 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2006-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"127235452","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
P. Dickinson, C. Campbell, S. Evans, A. Buck, C. Mountford, L. Keane, J. Terry, T. Su, A. Mount, A. Walton, J. Beattie, J. Crain, P. Ghazal
The Holliday junction (HJ) structure, consisting of four DNA double helices with a central branch point, is capable of switching between conformational states upon ion binding. The HJ nanoswitch described here comprises a long, dual labeled cloverleaf oligonucleotide and a short, unlabeled oligonucleotide. Fluorescent labeling with donor and acceptor dyes placed on the HJ arms of the cloverleaf strand allows the ion induced conformational switch to be detected optically using fluorescence resonance energy transfer (FRET). The influence of donor and acceptor dye location on the detection of conformational switching has been investigated using two distinct HJ structures. In addition, the effect of increasing HJ arm length in order to increase donor and acceptor dye separation has been evaluated. We report that a preferential HJ nanoswitch structure can be determined, capable of efficient detection of ion induced conformational switching
{"title":"Positional characteristics of fluorophores influencing signal output of a DNA nanoswitch","authors":"P. Dickinson, C. Campbell, S. Evans, A. Buck, C. Mountford, L. Keane, J. Terry, T. Su, A. Mount, A. Walton, J. Beattie, J. Crain, P. Ghazal","doi":"10.1109/BMN.2006.330892","DOIUrl":"https://doi.org/10.1109/BMN.2006.330892","url":null,"abstract":"The Holliday junction (HJ) structure, consisting of four DNA double helices with a central branch point, is capable of switching between conformational states upon ion binding. The HJ nanoswitch described here comprises a long, dual labeled cloverleaf oligonucleotide and a short, unlabeled oligonucleotide. Fluorescent labeling with donor and acceptor dyes placed on the HJ arms of the cloverleaf strand allows the ion induced conformational switch to be detected optically using fluorescence resonance energy transfer (FRET). The influence of donor and acceptor dye location on the detection of conformational switching has been investigated using two distinct HJ structures. In addition, the effect of increasing HJ arm length in order to increase donor and acceptor dye separation has been evaluated. We report that a preferential HJ nanoswitch structure can be determined, capable of efficient detection of ion induced conformational switching","PeriodicalId":271407,"journal":{"name":"2006 Bio Micro and Nanosystems Conference","volume":"141 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2006-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"115712611","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
M. Plomp, Terrance Leighton, Hoi-ying Holman, Alexander J. Malkin
Summary form only given. The elucidation of microbial surface architecture and function is critical to determining mechanisms of pathogenesis, immune response, physicochemical properties, environmental resistance and development of countermeasures against bioterrorist agents. We have utilized high-resolution in vitro AFM for studies of structure, assembly, function and environmental dynamics of several microbial systems including bacteria and bacterial spores. Lateral resolutions of ~2.0 nm were achieved on pathogens, in vitro. We have demonstrated, using various species of Bacillus and Clostridium bacterial spores, that in vitro AFM can address spatially explicit spore coat protein interactions, structural dynamics in response to environmental changes, and the life cycle of pathogens at near-molecular resolution under physiological conditions. We found that strikingly different species-dependent crystalline structures of the spore coat appear to be a consequence of nucleation and crystallization mechanisms that regulate the assembly of the outer spore coat, and we proposed a unifying mechanism for outer spore coat self-assembly. Furthermore, we revealed molecular-scale transformations of the spore coat during the germination process, which include profound, previously unrecognized changes of the spore coat. We will present data on the direct visualization of stress-induced environmental response of metal-resistant Arthrobacter oxydans bacteria to Cr(VI) exposure, resulting in the formation of a supramolecular crystalline hexagonal structure on the cell surface. At higher Cr(VI) concentrations the formation of microbial extracellular polymers, which cover microbial colony was observed. High-resolution visualization of stress-induced structures on bacterial surfaces builds a foundation for real time in vitro molecular scale studies of structural dynamics of metal-resistant bacteria in response to environmental stimuli. In the case of the bacterium Chlamedia trachomatis, we were able to identify surface exposed proteins versus proteins embedded in the outer membrane. These studies establish in vitro AFM as a powerful new tool capable of revealing pathogen architecture, structural dynamics and variability at nanometer-to-micrometer scales
{"title":"Probing the structure-function relationships of microbial systems by high-resolution in vitro atomic force microscopy","authors":"M. Plomp, Terrance Leighton, Hoi-ying Holman, Alexander J. Malkin","doi":"10.1109/BMN.2006.330923","DOIUrl":"https://doi.org/10.1109/BMN.2006.330923","url":null,"abstract":"Summary form only given. The elucidation of microbial surface architecture and function is critical to determining mechanisms of pathogenesis, immune response, physicochemical properties, environmental resistance and development of countermeasures against bioterrorist agents. We have utilized high-resolution in vitro AFM for studies of structure, assembly, function and environmental dynamics of several microbial systems including bacteria and bacterial spores. Lateral resolutions of ~2.0 nm were achieved on pathogens, in vitro. We have demonstrated, using various species of Bacillus and Clostridium bacterial spores, that in vitro AFM can address spatially explicit spore coat protein interactions, structural dynamics in response to environmental changes, and the life cycle of pathogens at near-molecular resolution under physiological conditions. We found that strikingly different species-dependent crystalline structures of the spore coat appear to be a consequence of nucleation and crystallization mechanisms that regulate the assembly of the outer spore coat, and we proposed a unifying mechanism for outer spore coat self-assembly. Furthermore, we revealed molecular-scale transformations of the spore coat during the germination process, which include profound, previously unrecognized changes of the spore coat. We will present data on the direct visualization of stress-induced environmental response of metal-resistant Arthrobacter oxydans bacteria to Cr(VI) exposure, resulting in the formation of a supramolecular crystalline hexagonal structure on the cell surface. At higher Cr(VI) concentrations the formation of microbial extracellular polymers, which cover microbial colony was observed. High-resolution visualization of stress-induced structures on bacterial surfaces builds a foundation for real time in vitro molecular scale studies of structural dynamics of metal-resistant bacteria in response to environmental stimuli. In the case of the bacterium Chlamedia trachomatis, we were able to identify surface exposed proteins versus proteins embedded in the outer membrane. These studies establish in vitro AFM as a powerful new tool capable of revealing pathogen architecture, structural dynamics and variability at nanometer-to-micrometer scales","PeriodicalId":271407,"journal":{"name":"2006 Bio Micro and Nanosystems Conference","volume":"54 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2006-04-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"122568379","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2006-03-14DOI: 10.1557/PROC-0944-AA02-08
M. Firestone, B. Reiss, O. Auciello, L. Ocola
Summary form only given. Combinatorial phage display methods have been used to identify a circularly constrained heptapeptide sequence, ISLLHST, that strongly associates with a perovskite ferroelectric, lead zirconium titanate, Pb(ZrxTi1-x)O3 (PZT). The affinity and selectively of binding to polycrystalline MOCVD deposited PZT thin films supported on Si/SiO2/Pt substrates were determined by titering and immunofluorescence microscopy, and the peptide was shown to selectively bind PZT in the presence of Pt, Si, Au, and several different photoresists. Ferroelectric properties were determined by measurement of the P-E hysteresis loop on unmodified and phage bound PZT thin films, and no change in the coercive field, Ec , or the saturation polarization, Ps was observed after contacting the PZT with aqueous buffer or phage binding. Since this preliminary characterization indicates that the PZT is compatible with biological chemistry, work is currently underway to develop a gated nanofluidic device using this chemistry. This is being accomplished by using electron beam lithography to etch nanochannels (~100 nm in width) in photoresists deposited on these PZT substrates. The base of these channels can be modified using the PZT-specific peptide, and by incorporating metallic electrodes into this structure, the charge of the functionalized PZT can be manipulated, forming the basis for such a device
{"title":"Ferroelectric-specific peptides as building blocks for bioinorganic devices","authors":"M. Firestone, B. Reiss, O. Auciello, L. Ocola","doi":"10.1557/PROC-0944-AA02-08","DOIUrl":"https://doi.org/10.1557/PROC-0944-AA02-08","url":null,"abstract":"Summary form only given. Combinatorial phage display methods have been used to identify a circularly constrained heptapeptide sequence, ISLLHST, that strongly associates with a perovskite ferroelectric, lead zirconium titanate, Pb(ZrxTi1-x)O3 (PZT). The affinity and selectively of binding to polycrystalline MOCVD deposited PZT thin films supported on Si/SiO2/Pt substrates were determined by titering and immunofluorescence microscopy, and the peptide was shown to selectively bind PZT in the presence of Pt, Si, Au, and several different photoresists. Ferroelectric properties were determined by measurement of the P-E hysteresis loop on unmodified and phage bound PZT thin films, and no change in the coercive field, Ec , or the saturation polarization, Ps was observed after contacting the PZT with aqueous buffer or phage binding. Since this preliminary characterization indicates that the PZT is compatible with biological chemistry, work is currently underway to develop a gated nanofluidic device using this chemistry. This is being accomplished by using electron beam lithography to etch nanochannels (~100 nm in width) in photoresists deposited on these PZT substrates. The base of these channels can be modified using the PZT-specific peptide, and by incorporating metallic electrodes into this structure, the charge of the functionalized PZT can be manipulated, forming the basis for such a device","PeriodicalId":271407,"journal":{"name":"2006 Bio Micro and Nanosystems Conference","volume":"19 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2006-03-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"125985273","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The ability to discriminate nucleic acid sequences is necessary for a wide variety of applications: high throughput screening, distinguishing genetically modified organisms (GMOs), molecular computing, differentiating biological markers, fingerprinting a specific sensor response for complex systems, etc. Hybridization-based target recognition and discrimination is central to the operation of nucleic acid microsensor systems. Therefore developing a quantitative correlation between mishybridization events and sensor output is critical to the accurate interpretation of results. Additionally, knowledge of such correlation can be used to design intelligent sensor systems that incorporate mishybridization noise into system design. Using experimental data produced by introducing single mutations (single nucleotide polymorphisms, SNPs) in the probe sequence of computational catalytic molecular beacons (deoxyribozyme gates) [Stojanovic & Stefanovic, 2003], we investigate correlations between free energy of the target-probe complex and the measured fluorescence of the deoxyribozyme gate. Experimental data for forty-five SNP-containing probe sequences are compiled and compared against the true probe sequence to determine the relationship between position, type of mutation, and the fluorescence level of the molecular beacon. The sequence set accounts for every possible SNP for a fifteen-base probe. Experiments are conducted using a 55 mul detection volume containing a modified YESiA(E6) deoxyribozyme molecular beacon (100 nM) [Stojanovic et al., 2001], TAMRA substrate (1 muM) and input sequences (2 muM). Using free energy as a first-approximation of the energetic interactions that occur during target-probe recognition, we generate empirical data for each target-probe pair using a nucleic acid hybridization thermodynamics server called HyTher (http://ozone2.chem.wayne.edu/). HyTher uses empirical fits of experimentally measured data to generate hybridization thermodynamic predictions for nucleic acid sequence pairs. Empirical data for all target-probe combinations are correlated with experimental fluorescence measurements to determine a quantitative link between target-probe hybridization free energy and molecular beacon fluorescence for each SNP-containing probe. We investigate Bayesian-based classification approaches as well as combinatorial design based methods for identifying and classifying mismatch patterns that produce similar fluorescence levels
{"title":"Quantitative assessment of SNP discrimination for computational molecular beacons","authors":"S. Brozik, P. Crozier, P. Dolan, E. May","doi":"10.1109/BMN.2006.330887","DOIUrl":"https://doi.org/10.1109/BMN.2006.330887","url":null,"abstract":"The ability to discriminate nucleic acid sequences is necessary for a wide variety of applications: high throughput screening, distinguishing genetically modified organisms (GMOs), molecular computing, differentiating biological markers, fingerprinting a specific sensor response for complex systems, etc. Hybridization-based target recognition and discrimination is central to the operation of nucleic acid microsensor systems. Therefore developing a quantitative correlation between mishybridization events and sensor output is critical to the accurate interpretation of results. Additionally, knowledge of such correlation can be used to design intelligent sensor systems that incorporate mishybridization noise into system design. Using experimental data produced by introducing single mutations (single nucleotide polymorphisms, SNPs) in the probe sequence of computational catalytic molecular beacons (deoxyribozyme gates) [Stojanovic & Stefanovic, 2003], we investigate correlations between free energy of the target-probe complex and the measured fluorescence of the deoxyribozyme gate. Experimental data for forty-five SNP-containing probe sequences are compiled and compared against the true probe sequence to determine the relationship between position, type of mutation, and the fluorescence level of the molecular beacon. The sequence set accounts for every possible SNP for a fifteen-base probe. Experiments are conducted using a 55 mul detection volume containing a modified YESiA(E6) deoxyribozyme molecular beacon (100 nM) [Stojanovic et al., 2001], TAMRA substrate (1 muM) and input sequences (2 muM). Using free energy as a first-approximation of the energetic interactions that occur during target-probe recognition, we generate empirical data for each target-probe pair using a nucleic acid hybridization thermodynamics server called HyTher (http://ozone2.chem.wayne.edu/). HyTher uses empirical fits of experimentally measured data to generate hybridization thermodynamic predictions for nucleic acid sequence pairs. Empirical data for all target-probe combinations are correlated with experimental fluorescence measurements to determine a quantitative link between target-probe hybridization free energy and molecular beacon fluorescence for each SNP-containing probe. We investigate Bayesian-based classification approaches as well as combinatorial design based methods for identifying and classifying mismatch patterns that produce similar fluorescence levels","PeriodicalId":271407,"journal":{"name":"2006 Bio Micro and Nanosystems Conference","volume":"171 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2006-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"132151274","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Bacteria, ranging from oxygenic photosynthetic cyanobacteria to heterotrophic sulfate reducing bacteria, produce electrically-conductive appendages referred to as bacterial nanowires. Dissimilatory metal reducing bacteria, including Shewanella oneidensis and Geobacter sulfurreducens, produce electrically conductive nanowires in direct response to electron acceptor limitation and facilitate electron transfer to solid phase iron oxides. Nanowires produced by S. oneidensis strain MR-1, which served as our primary model organism, are functionalized by decaheme cytochromes MtrC and OmcA that are distributed along the length of the nanowires. Mutants deficient in MtrC and OmcA produce nanowires that were poorly conductive. These mutants also differ from wild type cells in their ability to reduce solid phase iron oxides, to produce electrical current in a mediator less microbial fuel cell, and to form complex biofilms at air liquid interfaces. Although currently less completely characterized, conductive nanowires produced by other organisms reveal a strategy of energy/electron distribution that is conserved across a broad metabolic spectrum. This presentation will target the implications of bacterial nanowire for energy distribution and communication in biofilms and other natural microbial communities, bioelectrical coupling of electron donors with poorly accessible electron acceptors, and applications for alternative energy (microbial fuel cells) and nanoelectronic technologies
{"title":"Bacterial nanowires: electrically conductive filaments and their implications for energy transformation and distribution in natural and engineered systems","authors":"Y. Gorby","doi":"10.1109/BMN.2006.330941","DOIUrl":"https://doi.org/10.1109/BMN.2006.330941","url":null,"abstract":"Bacteria, ranging from oxygenic photosynthetic cyanobacteria to heterotrophic sulfate reducing bacteria, produce electrically-conductive appendages referred to as bacterial nanowires. Dissimilatory metal reducing bacteria, including Shewanella oneidensis and Geobacter sulfurreducens, produce electrically conductive nanowires in direct response to electron acceptor limitation and facilitate electron transfer to solid phase iron oxides. Nanowires produced by S. oneidensis strain MR-1, which served as our primary model organism, are functionalized by decaheme cytochromes MtrC and OmcA that are distributed along the length of the nanowires. Mutants deficient in MtrC and OmcA produce nanowires that were poorly conductive. These mutants also differ from wild type cells in their ability to reduce solid phase iron oxides, to produce electrical current in a mediator less microbial fuel cell, and to form complex biofilms at air liquid interfaces. Although currently less completely characterized, conductive nanowires produced by other organisms reveal a strategy of energy/electron distribution that is conserved across a broad metabolic spectrum. This presentation will target the implications of bacterial nanowire for energy distribution and communication in biofilms and other natural microbial communities, bioelectrical coupling of electron donors with poorly accessible electron acceptors, and applications for alternative energy (microbial fuel cells) and nanoelectronic technologies","PeriodicalId":271407,"journal":{"name":"2006 Bio Micro and Nanosystems Conference","volume":"11 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"1900-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"115480646","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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