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Item Physiology of Pseudomonas aeruginosa in biofilms as revealed by transcriptome analysis(2010) Folsom, James P.; Richards, Lee A.; Roe, Frank L.; Ehrlich, Garth D.; Parker, Albert E.; Mazurie, Aurélien J.; Stewart, Philip S.BACKGROUND: Transcriptome analysis was applied to characterize the physiological activities of Pseudomonas aeruginosa grown for three days in drip-flow biofilm reactors. Conventional applications of transcriptional profiling often compare two paired data sets that differ in a single experimentally controlled variable. In contrast this study obtained the transcriptome of a single biofilm state, ranked transcript signals to make the priorities of the population manifest, and compared rankings for a priori identified physiological marker genes between the biofilm and published data sets.RESULTS: Biofilms tolerated exposure to antibiotics, harbored steep oxygen concentration gradients, and exhibited stratified and heterogeneous spatial patterns of protein synthetic activity. Transcriptional profiling was performed and the signal intensity of each transcript was ranked to gain insight into the physiological state of the biofilm population. Similar rankings were obtained from data sets published in the GEO database (www.ncbi.nlm.nih.gov/geo). By comparing the rank of genes selected as markers for particular physiological activities between the biofilm and comparator data sets, it was possible to infer qualitative features of the physiological state of the biofilm bacteria. These biofilms appeared, from their transcriptome, to be glucose nourished, iron replete, oxygen limited, and growing slowly or exhibiting stationary phase character. Genes associated with elaboration of type IV pili were strongly expressed in the biofilm. The biofilm population did not indicate oxidative stress, homoserine lactone mediated quorum sensing, or activation of efflux pumps. Using correlations with transcript ranks, the average specific growth rate of biofilm cells was estimated to be 0.08 h-1.CONCLUSIONS: Collectively these data underscore the oxygen-limited, slow-growing nature of the biofilm population and are consistent with antimicrobial tolerance due to low metabolic activity.Item Rheological Characterization of HPMCAS in Various Solvents(Montana State Univeristy, 2017-04) Young, MarkThe purpose of this study was to rheologically characterize solutions by changing the concentration of hydroxypropyl methylcellulose acetate succinate (HPMCAS) dissolved in a variety of solvents, including acetone, methanol, and an acetone and water mixture. HPMCAS is a synthetic polymer derived from cellulose, it has found high interest in the pharmaceutical industry where it heavily used in the formation of spray-dried dispersion's. The characterization of the rheological properties of these solutions is of interest to better understand how the solvent choice and the polymer concentration impact the spray-dried dispersion. Polymer solutions exhibit critical concentrations of overlap and entanglement that correlate to when the polymer chains in solution begin to overlap with other chains and when polymers chains begin to entangle with other chains in the solution. When polymer chains overlap with each other a network forms in the solution, this leads to an increased viscosity as well as viscoelastic behavior that can be observed and characterized. Steady state flow tests, strain sweeps, and frequency sweeps were performed to observe the rheological properties of these polymer solutions. Overlap and entanglement concentrations were found for solutions with the solvents of Acetone, Methanol and an acetone and water mixture. The viscoelasticity of these solutions was then analyzed at polymer concentrations between and above these critical concentrations.Item Identifying specific extracellular residues of the sensory kinase SaeS important in the recognition and response to hydrogen peroxide in Staphylococcus aureus(Montana State Univeristy, 2017-04) Robinson, TannerThe Staphylococcus aureus (S. aureus) exoprotein secretion system (SaeR/S) is a two-component protein system within Staphylococcus aureus that has been linked to this pathogen’s ability to survive within human neutrophils (polymorphonuclear leukocytes or PMNs). Prior studies have shown that an extracellular (EC) loop, consisting of nine amino acid residues on SaeS, is vital for S. aureus to sense and respond to extracellular stimuli--specifically components of human PMNs. Additionally, γ-hemolysin (hlgA) is a predominant virulence factor that targets immune and red blood cells. This toxin has been shown to be regulated by SaeR/S. New hlgA-GFP S. aureus cell strains--including point mutations of the residues on the EC loop--have been developed in order to study the role of each residue in S. aureus survival. All strains contained a plasmid on which the hlgA gene was linked with the GFP reporter. The current study sought to analyze the activity of these strains in the presence of hydrogen peroxide, a predominant reactive oxygen species produced by neutrophils. GFP fluorescence following transcription of hlgA was measured using spectrophotometry. This method was used to investigate the expression of hlgA following S. aureus incubation (up-to 6 hours) with non-lethal to lethal doses of hydrogen peroxide. Experimental findings suggest that the hlgA-GFP reporter may not be sensitive enough to definitively show differences in hlgA expression in wild-type and in SaeS EC point mutation S. aureus strains following exposure to hydrogen peroxide. While high concentrations of hydrogen peroxide did negatively impact cellular growth early on, GFP concentrations were not significant enough at those time points to distinguish differences inItem Does aquaporin 3b affect the number and or characteristics of calcium waves in the neural plate of Xenopus laevis embryos?(Montana State University, 2017-04) Ricker, DeanDoes aquaporin 3b affect the number and or characteristics of calcium waves in the neural plate of Xenopus laevis embryos? Aquaporin 3b (Aqp3b) is a water channel protein that is expressed in two lines of cells along the edges of the neural plate. The neural plate then rolls up into the neural tube through a process called neural tube closure. The neural tube is the precursor to the entire central nervous system. When aqp3b expression is inhibited, neural tube closure does not occur. My hypothesis is that neural tube closure is orchestrated by calcium waves triggered by Aqp3b. In order to test my hypothesis, I inject three different groups of albino Xenopus laevis embryos at the four cell stage. One group, the control, is injected with a tracer (Rhodamine Dextran) and GCaMP6 mRNA that is translated into a protein that fluoresces in the presence of calcium. A second group, the test group, is injected with the tracer, GCaMP6, and aqp3b morpholino oligonucleotide (aqp3b MO), which inhibits translation of aqp3b mRNA. The final group, the second control is injected with tracer, GCaMP6, and a modified aqp3b MO that does not inhibit translation. This second control tests whether it is the presence of an MO or the inhibition of expression that affects calcium waves. The embryos are then allowed to develop to neurula stage, at which point a microscope camera is used to take a time lapse of them. Thus far, my efforts in this project have been to learn the necessary techniques and gain a qualitative understanding of the characteristics of calcium waves from the imaging I have done so far. Going forward I will be collecting more images of calcium activity in each of the three groups and developing a method for analyzing the data.Item Monitoring single-cell bacterial growth using drop-based microfluidics(Montana State University, 2017-04) Pratt, ShawnaDrop-based microfluidics is a technology by which monodisperse water-in-oil emulsions are created through the manipulation of fluids in a microfluidic device. The resulting drops act as individual, contained environments that can carry biological cargo; in the case of this study, the cargo is single Pseudomonas aeruginosa bacterial cells. Here, the growth of two strains of P. aeruginosa, wild type and a mutant hibernation promotion factor knockout strain, Δhpf, was monitored using specially developed microfluidic drop incubation technology. The Δhpf gene helps cells to successfully enter dormancy when undergoing starvation. Here we have developed an incubation technique that utilizes a uniquely engineered microfluidic device to hold drops in a set position and prevent drop evaporation for the duration of a 24 hr growth period. This technique allows for the growth of individual cells to be monitored, meaning that insights such as the heterogeneity of cell growth are lost to bulk data. During the growth period, drops are continually imaged through confocal technology to determine changes in fluorescence output, which reflects cell growth. This technique demonstrates the ability to monitor the growth of single cells to produce growth curves for each individual cell. In this study the developed microfluidic incubation technology facilitates a deeper investigation of the demographics of growth between the two strains explored, and allows for probing the heterogeneity of bacterial populations at a single cell level.Item Attempting to identify the sources of microbial methane production from coal(Montana State University, 2017-04) Platt, GeorgeAs the world begins to transition away from high-emissions fossil fuels, natural gas has become increasingly relevant. One natural gas reserve is found in subsurface coal seams, known as coalbed methane (CBM). Biogenic methane production occurs in anoxic environments where microorganisms catalyze the conversion of coal to methane through fermentation and methanogenesis. This study focuses on the upstream biogeochemical processes that promote the degradation of the coal matrix into bioavailable organic intermediates. A series of solid-liquid extractions were performed with coal from the Powder River Basin (PRB) using methanol, dichloromethane, and water in various treatments and sequences. The residual coal from the extractions was used in anaerobic bioreactors inoculated with a native microbial consortium from the PRB to assess and quantify the variation in methane production via Gas Chromatography (GC). Additionally, the liquid fraction of the extracts were analyzed using Gas Chromatography–Mass Spectrometry (GC-MS) and the bioreactor contents were analyzed using Fluorescence Excitation-Emission Matrix Spectroscopy (EEMS) to assess their chemical composition and fluorescent signatures. While the bioreactors produced limited amounts of methane compared to previous CBM studies, the EEMS analysis showed that the bioreactor contents experienced a shift in fluorescent signatures indicating potential biotic and abiotic chemical conversion of the coal. Dissolved inorganic and organic carbon measurements showed significant differences between treatments, indicating that the coal pre-treatment affected the biogeochemical processes necessary for coal conversion. Ultimately, this study provided insight into the organic intermediates that are bioavailable for coal conversion.Item Characterizing Algae Growth and Biomass Composition under Autotrophic, Mixotrophic and Heterotrophic Conditions(Montana State University, 2017-04) Peter, DanielThe algal research group at the Center for Biofilm Engineering has a library of over 100 algal cultures, for most of which the metabolic pathways are not known. By growing each culture under phototrophic, heterotrophic, and mixotrophic conditions on a small scale, we have been able to determine which pathways each organism is capable of utilizing. Upon completion of a larger scale experiment, information about a select organism’s nutrient uptake, growth, and composition will be analyzed to gain a rigorous understanding of the growth under each metabolic condition. Due to the plentiful organic carbon and often light limited conditions present in wastewater, algae that is capable of growth using multiple metabolic pathways are uniquely suited to these conditions and are of particular value in exploitation and even remediation of this otherwise low value waste stream.Item Developing a Biocompatible Formulation for Stereolithographic 3D Printing(Montana State University, 2017-04) Owens, MadisonTo determine how factors such as biofilm structure and microbial density affect the production of specific metabolites, and to address the mass transport and material property limitations inherent in naturally-formed biofilms, we propose to use stereolithography (SLA)-based 3D printing to construct biofilms with well-defined structures and properties. In this approach, a solid object is built up layer-by-layer from a liquid resin using a focused laser spot. Polymerization occurs at the laser focal point, which is raster scanned within the resin bath. Recently we have developed a biocompatible, water-based formulation for printing viscoelastic hydrogels using a commercial SLA 3D printer (Formlabs, Form 2). The formulation relies on free-radical polymerization of a polyethylene glycol-diacrylate (PEG-DA) monomer dissolved in water, which is initiated by a photoinitiator (LAP) responsive to 405 nm wavelength light. Microbes, such as A. sarcoides, Escherichia coli, Bacillus subtilis, and Pseudomonas aeruginosa, which are suspended in the liquid are entrapped in the PEG-DA hydrogel as it polymerizes and survive for extended periods of time. Moreover, PEG-DA provides a versatile platform for varying gel mechanics because parameters such as elastic modulus and toughness can be tuned by controlling the concentrations and ratios of divalent and trivalent PEG-DA monomers. This flexibility is important as the microbe-loaded hydrogels are not naturally-formed biofilms, and effort must be taken to determine how closely microbes embedded in our hydrogel replicate biofilm behavior. To do this, we must understand how parameters such as gel mechanics and chemical modification of the gel polymers impact gene expression.Item Linking Microbial Biofilms to Nitrate Removal in Groundwater Sediments(Montana State University, 2017-04) Olson, CaitlinA fundamental goal in the field of microbial ecology is to link the activity of specific microorganisms to processes occurring within an ecosystem. This project aims to identify the drivers of community structure and succession by identifying the metabolically active fraction of microbial communities from both pure and contaminated groundwater wells. The groundwater samples include a variety of contaminants, the most important of these for this experiment being nitrate. The use of four diverse wells in conjunction with enumeration and sequencing of transnationally active microorganisms, activity assays, and geochemical measurements will allow for the explanation of the mechanisms controlling for shaping community structure and function. Multiple assay comparisons will be used to achieve an accurate characterization of the active microbial communities in the samples, and will ultimately be applied to continuous sediment cores from pure and contaminated wells. A combination of methodological approaches will be used to evaluate the active fraction of microbial communities, as well as the associated rates of activity from the pure and contaminated wells. Biolog Microbial Identification System will be used to biochemically test and identify a broad range of bacteria. The visualization and sequencing of translationally active bacterial, archaeal, and denitrifying cells was studied by applying bioorthoganol non-canonical amino acid tagging (BONCAT). For contaminated wells large proportions of the community were identified as translationally active however, specific rates of activity were low. Total cell abundances ranged from 1.11-2.07 X 105 cells/mL with 73-84% of the community being transnationally active.Item Microfluidic techniques for encapsulating gastric organoids(Montana State University, 2017-04) Oloff, EstherWe investigated the use of microfluidics, in which small volumes of fluid are precisely manipulated in a lab-on-a-chip device, to manipulate the growth of gastric organoids. Organoids are populations of stem cells grown into tissue spheroids which mimic in vivo organs and systems. The focus of our research is on human gastric organoids, which are grown from gastric epithelial cells within Corning Matrigel Basement Membrane Matrix. We use specialized microfluidic devices to encapsulate these cells within different geometries, varying between 100-450 μm in diameter. The first technique is to make a thin PDMS film with an array of holes that is used like an “ice-cube tray” to make different 3D shapes. The second technique is to use a standard drop-making microfluidic device to form drops of Matrigel that encase cells. The viability of the cells were examined within the different geometries. By isolating and supporting a single human gastric epithelial cell within a Matrigel shape that can grow into an organoid, that organoid can be more easily analyzed, specialized, and transported. A potential application is to design and bioprint an organoid with ports, allowing easier introduction of drugs or removal of waste products from the organoid. Research is done under Connie Chang in the Center for Biofilm Engineering, in collaboration with Diane Bimczok in Microbiology and Immunology at Montana State University.