Browsing by Subject "Microbial Engineering"
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Item Item Electrical current generation by wild type and mutant Shewanella strains(2009-12) Baron, Daniel BenjaminThe genus Shewanella has been reported to have the capacity to couple the transfer of electrons to insoluble metal oxides and solid carbon electrodes with cellular growth. While this process may be useful as an energy generation strategy or biotechnological tool, the electron transfer pathway by which this process occurs is not completely understood, and better techniques for studying the transfer mechanism are needed. This project used single chamber electrochemical cells to show that the current generation capabilities of Shewanella oneidensis are dependent on the ability of cells comprising a biofilm to shuttle soluble electron carriers between an electrode and its outer membrane cytochromes. The extracellular electron transfer capabilities of S. oneidensis mutant strains containing deleted or transposon-interrupted copies of genes known to be involved in extracellular electron transfer were also studied and compared to wild type. Amperometry was utilized to monitor real-time electron flow between attached anaerobic wild type and mutant cells and a poised carbon working electrode. Differential pulse voltammetry and cyclic voltammetry performed on electrode attached S. oneidensis MR-1 wild type cells detected both mediated and direct electron transfer reactions at the electrode surface. Ion exchange HPLC verified the presence of endogenously produced flavin compounds in S. oneidensis liquid cultures and confirmed the most common flavin in S. oneidensis MR-1 electrochemical cell analytes is riboflavin (vitamin B2). It was also discovered that removing the medium surrounding an electrode biofilm caused current production from the electrochemical cell to decrease. Returning the filtered original medium, or adding anaerobic riboflavin resulted in the restoration of current production. The amount of current produced at the carbon working electrode increased with biofilm development and accumulation of soluble electron mediator. A correlation was observed between the concentration of the redox shuttle in potentiostat-controlled electrochemical cells and the maximum sustainable current, as well as maximum electrode biofilm thickness. For example, wild type cultures with twice the natural amount of riboflavin approximately doubled their electrode current production and also attached to the electrode in larger numbers. Electrode phenotypes of Shewanella oneidensis MR-1 mutant strains were also observed and compared to wild type. The deletion of several genes, such as for the outer membrane cytochrome MtrC, the periplasmic cytochrome MtrA, or the membrane beta barrel protein MtrB severely impaired MR-1 cells from attaching to the carbon electrode. As a result, the deletion mutant strains were incapable of producing significant anodic current and were deficient in electrode attached biomass. However, deletion of the outer membrane cytochrome OmcA, or genes related to the formation of mature biofilms resulted in a percentage of the current production being retained. This data supports the theory that MtrC is a key component in the terminal electron transfer step for S. oneidensis MR-1 cells interacting with solid surfaces. Measurements of current production from MR-1 electrode biofilms revealed that the extracellular electron transfer process involves both cell associated enzymes and flavins acting as soluble electron transfer agents. However, these separate pathways most likely utilize many of the same, membrane proteins to accomplish their function. This study indicates it is likely that the deposition of electrons by S. oneidensis MR-1 to a poised electrode can be done both directly by MtrC while stabilized or otherwise assisted by other outer membrane elements such as OmcA or MtrB, as wells as through cycling of redox active shuttles such as flavins between MtrC and the electrode surface. As a result, for electrode attached Shewanella oneidensis MR-1 cells, a complex relationship exists between soluble flavin concentration, biofilm thickness, and electrical current production. These factors pertain greatly to the capabilities and limitations of S. oneidensis, especially while functioning as part of a electrochemical device, and must be taken into account when utilizing this organism for research or other applications.Item Electrochemical analysis of Shewanella oneidensis engineered to bind gold electrodes.(2011-03) Kane, Aunica L.Three-electrode bioreactors can be utilized to examine the mechanisms involved in electron flow from bacteria to insoluble electron acceptors and allow these processes to be analyzed quantitatively. As an electrode, gold is an ideal surface to study the electrophysiology occurring during extracellular respiration; yet previous research has shown that Shewanella is resistant to colonization on gold surfaces. Therefore, the goal of this work was to direct adhesion of Shewanella oneidensis to gold surfaces via cell surface display of a modified E. coli outer membrane protein, LamB, and a gold-binding peptide (5rGBP) to encourage microbe-electrode interaction, improve whole-cell biocatalytic systems, and increase overall current production. Expression of LamB-5rGBP increased the affinity of Shewanella for gold surfaces, but also led to the displacement of certain outer membrane components required for extracellular electron transport. Displacement of these outer membrane proteins decreased the rate at which Shewanella was able to reduce both insoluble iron and riboflavin. Expression of LamB-5rGBP, although effectively increasing attachment to gold, did not greatly increase current production in gold-electrode bioreactors.Item Engineering Ralstonia eutropha for the production of mcl-PHAs from plant oils.(2011-05) Rouse, Daniel P.With the world’s petroleum reserves being depleted at a rapid rate, vast pollution caused by the inability of nature to break down modern day plastics, and global warming greatly affecting the Earth’s climate, green alternatives to current industrial practices are becoming more necessary now than ever before. One possible environmentally friendly alternative is the production of poly 3-hydroxyalkanoates (PHAs), biodegradable polymers naturally synthesized by many types of bacteria. PHB is the most common and easiest to produce PHA, but the polymer is quite brittle and holds little commercial value. However, research has shown that a wide variety of monomers can be incorporated into the polymer chains giving a vast array of physical and material properties to the polymer, some with the potential to be very valuable. With the advances in molecular biology techniques and DNA sequencing technology, metabolic engineering has become a quick and powerful tool for constructing recombinant bacteria with gene sets capable of performing tasks previously unattainable. The main focus of this thesis was to construct a recombinant strain of Ralstonia eutropha capable of producing medium chain length (mcl) PHAs using soybean oil as the sole carbon source. Two approaches were used to accomplish this goal. In one approach the mcl-PHA polymerase gene phaC1 from Pseudomonas oleovorans was integrated into the genome of R. eutropha in place of the native phaC PHB polymerase. Another approach tested was to construct a plasmid based expression system using homologous recombination in yeast to express an engineered mcl-PHA operon containing the P. oleovorans phaC1 in conjunction with the phaJ from P. oleovorans or the fox2 gene from S. cerevisiae on a polycistronic gene cassette in a mutated strain of R. eutropha rendered unable to synthesize PHAs naturally. Both recombinant systems were tested for mcl-PHA production in fed batch bioreactors using minimal media supplemented with soybean oil. GC-MS analysis of cell samples taken from the reactor experiments indicated the recombinant organism harboring the integrated phaC1 gene accumulated trace amounts of mcl-PHAs, mostly in the form of 3-hydroxyoctanoate, but the plasmid expression systems showed much higher accumulation of mcl-PHAs with constituents of 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydecenoate. This demonstrated the ability of the recombinant R. eutropha to accumulate mcl-PHAs when grown on fatty acids. It was also demonstrated that R. eutropha has the ability to grow to high cell densities using soybean oil as the sole carbon source, and that foreign gene expression is possible in R. eutropha using plasmid based systems and through gene integrations.Item Metabolic model design and elementary mode analysis of Shewanella oneidensis MR-1 and Derivative Strains & Plasmid construction to facilitate PHB production in Saccharomyces cerevisiae using a single vector.(2010-04) Flynn, Chris M.In Chapter 1, a stoichiometric model describing the central metabolism of Shewanella oneidensis MR-1 wild-type and derivative strains was developed for use in elementary mode (EM) analysis. An EM model was created and verified by comparing growth phenotypes of single- and double-knockout strains cultivated on differing carbon sources, under aerobic and anaerobic conditions. Furthermore, several single knockout growth phenotypes under specific conditions have been predicted, offering potential for model verification. This wild-type model can be easily adapted to different carbon sources or metabolic products, and allows the prediction of single- and multiple- knockout strains that are expected to operate under defined conditions with increased efficiency when compared to wild type cells. To this end, the S. oneidensis wild-type EM model was adapted by addition of both glycerol import reactions and reactions needed to facilitate ethanol production from pyruvate. EM simulations were then performed on this glycerol-ethanol model to predict that the optimal anaerobic, ethanol producing strain cultivated on glycerol would contain the following mutations: Δpta ΔaldA ΔgcvT. This strain is expected to require ethanol secretion to accumulate biomass under anaerobic conditions, and as a result to produce ethanol yields between 0.4668 mol ethanol/mol glycerol and 1 mol ethanol/mol glycerol consumed (theoretical yield is 1 mol/mol). Future work should focus on performing these gene deletions and characterizing the resultant mutant strains, as well as determining the role of formate in anaerobic shewanella metabolism. In addition, the steps in generating a single plasmid which expresses all three R. eutropha PHA synthesis genes in Saccharomyces cerevisisae is described in Chapter 2. Several methods were employed to attempt to generate this plasmid, while plasmid construction was successful using yeast-mediated ligation and inserting the TEF1-phaC1 promoter-gene into p2DPT-RK(U) to create the plasmid p2DPTT1-RKS1(U). The GAL1-10 promoter is present in the host vector, and it is likely that previous efforts were hampered by plasmid instability resulting from undesired intra-plasmid homologous recombination between these duplicate GAL1-10 promoter regions. The exact sequence of the region downstream of the phaC1 gene should be determined prior to application PHA production and other experiments. This plasmid should facilitate the direct observation of PHA accumulation in vivo using fluorescence microscopy, as well as improve PHA yields in transformed strains when compared to the two-plasmid system.Item Visualizing gene expression in geobacter sulfurreducens biofilms on graphite electrodes(2010-12) Remarcik, Clint MichaelThe Geobacteraceae are a family of deltaproteobacterial anaerobes known to play important roles in environmental Fe(III)-reduction, subsurface petroleum bioremiediation, bioprecipitation of heavy metals, and reduction of anodes in microbial fuel cells. Electron transfer by Geobacteraceae requires formation of a multicellular biofilm, as cells must attach to surfaces or insoluble particles to bring redox proteins in contact with their electron acceptor, and growth of daughter cells requires cell-cell contact to facilitate longer-range electron transfer. The primary aim of this thesis was to develop new tools for three-dimensional visualization of gene expression within these biofilms. To detect differential expression in a developing biofilm, the genetically tractable representative Geobacter sulfurreducens was utilized as a model. Promoters from two c-type cytochromes proven to be important in electron transfer were inserted upstream of the fluorescent protein mCherry, or a derivative of mCherry containing an additional peptide sequence (LVA) to accelerate protein degradation and act as a signal of more recent expression. Control plasmids containing no mCherry, or with the fluorescent protein under control of a constitutive taclac promoter were also transformed into G. sulfurreducens. The plasmid backbone used for expression was found to affect growth of cells as biofilms, and a new mobilizable plasmid was developed for use in these experiments. Cells had to be exposed to oxygen for four hours to achieve maximum fluorescence, indicating that future real-time studies with these proteins reliant on oxygen leakage into biofilms would not be reliable. In addition, as G. sulfurreducens was found to possess significant background fluorescence when grown as a biofilm in the same emission range as mCherry, future studies should investigate reporters that operate at different wavelengths. After correcting for these effects, it was demonstrated the promoter for omcZ was expressed fairly uniformly throughout the biofilm, with localized pockets of high expression. The promoter for omcS, was expressed at significantly higher levels towards the top of the biofilm when biofilms were at maximum thickness (>20 μm). These results demonstrate that promoter-reporter fusions can be used for visualization of cytochrome expression in G. sulfurreducens biofilms, and highlights numerous issues related to plasmid choice, protein oxidation, and background fluorescence which could create artifacts and confound results.