Browsing by Subject "Biofilm"
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Item Open Access Atomic force microscopy for the investigation of molecular and cellular behavior(Elsevier, 2016-10) Ozkan A.D.; Topal, A. E.; Dana, A.; Güler, Mustafa O.; Tekinay, A. B.The present review details the methods used for the measurement of cells and their exudates using atomic force microscopy (AFM) and outlines the general conclusions drawn by the mechanical characterization of biological materials through this method. AFM is a material characterization technique that can be operated in liquid conditions, allowing its use for the investigation of the mechanical properties of biological materials in their native environments. AFM has been used for the mechanical investigation of proteins, nucleic acids, biofilms, secretions, membrane bilayers, tissues and bacterial or eukaryotic cells; however, comparison between studies is difficult due to variances between tip sizes and morphologies, sample fixation and immobilization strategies, conditions of measurement and the mechanical parameters used for the quantification of biomaterial response. Although standard protocols for the AFM investigation of biological materials are limited and minor differences in measurement conditions may create large discrepancies, the method is nonetheless highly effective for comparatively evaluating the mechanical integrity of biomaterials and can be used for the real-time acquisition of elasticity data following the introduction of a chemical or mechanical stimulus. While it is currently of limited diagnostic value, the technique is also useful for basic research in cancer biology and the characterization of disease progression and wound healing processes.Item Open Access Bacteria-immobilized electrospun fibrous polymeric webs for hexavalent chromium remediation in water(Springer Berlin Heidelberg, 2016) Sarioglu, O.F.; Celebioglu A.; Tekinay, T.; Uyar, TamerThe development of hexavalent chromium remediating fibrous biocomposite mats through the immobilization of a hexavalent chromium reducing bacterial strain, Morganella morganiiSTB5, on the surfaces of electrospun polystyrene and polysulfone webs is described. The bacteria-immobilized biocomposite webs have shown removal yields of 93.60 and 93.79 % for 10 mg/L, 99.47 and 90.78 % for 15 mg/L and 70.41 and 68.27 % for 25 mg/L of initial hexavalent chromium within 72 h, respectively, and could be reused for at least five cycles. Storage test results indicate that the biocomposite mats can be stored without losing their bioremoval capacities. Scanning electron microscopy images of the biocomposite webs demonstrate that biofilms of M. morganii STB5 adhere strongly to the fibrous polymeric surfaces and are retained after repeated cycles of use. Overall, the results suggest that reusable bacteria-immobilized fibrous biocomposite webs might be applicable for continuous hexavalent chromium remediation in water systems.Item Open Access Biological properties of extracellular vesicles and their physiological functions(Taylor & Francis, 2015) Yáñez-Mó, M.; Siljander, P. R. M.; Andreu, Z.; Zavec, A. B.; Borràs, F. E.; Buzas, E. I.; Buzas, K.; Casal, E.; Cappello, F.; Carvalho, J.; Colás, E.; Cordeiro-Da, S. A.; Fais, S.; Falcon-Perez, J. M.; Ghobrial, I. M.; Giebel, B.; Gimona, M.; Graner, M.; Gursel, I.; Gursel, M.; Heegaard, N. H. H.; Hendrix, A.; Kierulf, P.; Kokubun, K.; Kosanovic, M.; Kralj-Iglic, V.; Krämer-Albers, E. M.; Laitinen, S.; Lässer, C.; Lener, T.; Ligeti, E.; Line, A.; Lipps, G.; Llorente, A.; Lötvall, J.; Manček-Keber, M.; Marcilla, A.; Mittelbrunn, M.; Nazarenko, I.; Nolte-'t Hoen, E. N. M.; Nyman, T. A.; O'Driscoll, L.; Olivan, M.; Oliveira, C.; Pállinger, E.; Del Portillo, H. A.; Reventós, J.; Rigau, M.; Rohde, E.; Sammar, M.; Sánchez-Madrid, F.; Santarém, N.; Schallmoser, K.; Ostenfeld, M. S.; Stoorvogel, W.; Stukelj, R.; Grein V. D. S.G.; Helena,ü V. M.; Wauben, M. H. M.; De Wever, O.In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells.While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.Item Open Access Glycosylation: a tool for engineering protein based materials(2019-09) Işılak, Musa EfeBiofilms are bacterial communities formed by adhesion of cells to each other via extracellular matrix. Proteins, one of the components of biofilm matrix, can form fibers and have properties enabling their use as materials. Bacillus subtilis biofilm major protein TasA is a fiber forming non-amyloidogenic protein providing biofilm rigidity. Glycosylation is a post-translational modification observed in all domains of life where sugar groups are added on proteins covalently. Campylobacter jejuni glycosylation is the first bacterial N-linked glycosylation discovered and the most studied system where a heptasaccharide is attached to protein. C. jejuni glycosylation has variety of functions among adhesion, protease resistance and thermal stability. Glycosylation may become an additional method for engineering biofilm proteins as materials with unique properties. For this purpose, we examined effect of glycosylation on structure of natively non-glycosylated proteins by glycosylating alkaline phosphatase enzyme (ALP) at different locations on the protein. Interestingly, phosphatase activity assay showed slight increase in ALP activity on pure proteins glycosylated at the C-terminus. Next, TasA protein was glycosylated at the C-terminus. Glycosylation had no significant effect on fibrillation of TasA in vitro. Secondary structure analysis using circular dichroism data revealed shift from antiparallel to helix structure with glycosylation. Quartz crystal microbalance experiments indicated increased adhesive properties on glycosylated TasA protein on gold. Its application possibility as cell adhesive was assessed by visualizing surface coverage of polystyrene cell culture plate under scanning electron microscope. As a consequence, glycosylation was used as an engineering method for protein-based material development for the first time.Item Open Access Monitoring molecular assembly of biofilms using quartz crystal microbalance with dissipation(Springer, 2022) Yuca, E.; Şeker, Urartu Özgür Şafak; Arluison, Véronique; Wien, Frank; Marcoleta, AndrésThe structure and the functionality of biofilm proteins, the main components of the extracellular matrix, can be tuned by protein engineering. The use of binding kinetics data has been demonstrated in the characterization of recombinantly produced biofilm proteins to control their behavior on certain surfaces or under certain conditions. Quartz crystal microbalance with dissipation monitoring (QCM-D) allows measuring the change in resonance frequency and the energy loss and distribution upon the interaction of molecules with the surface. The characterization of the molecular assembly of curli biofilm proteins on different surfaces using QCM-D is presented here as a detailed protocol. The experimental procedure detailed in this chapter can be applied and modified for other biofilm proteins or subunits to determine their surface adsorption and kinetic binding characteristics.Item Open Access The role of bcsE gene in the pathogenicity of Salmonella(Oxford University Press, 2021-07-19) Özdemir, Caner; Akçelik, N.; Özdemir, F. N.; Evcili, İrem; Kahraman, Tamer; Gürsel, İhsan; Akçelik, M.The effects of the bcsE gene and BcsE protein on bacterial physiology and pathogenicity in SalmonellaTyphimurium and Salmonella Group C1 were investigated. It was observed that biofilm and pellicle formation did not occur in the bcsE gene mutants of wild-type strains. Besides, the ‘rdar’ (red, dry, rough) biofilm morphotype in wild-type strains changed significantly in the mutants. In terms of the bcsE gene, the swimming and swarming motility in mutant strains showed a dramatic increase compared to the wild-type strains. The Salmonella bcsE gene was cloned into Escherichia coli BL21, and the his-tagged protein produced in this strain was purified to obtain polyclonal antibodies in BALB/c mice. The antibodies were showed labeled antigen specificity to the BscE protein. As a result of immunization and systemic persistence tests carried out with BALB/c mice, BscE protein was determined to trigger high levels of humoral and cellular responses (Th1 cytokine production, IgG2a/IgG1 > 1). Systemic persistence in the liver and spleen samples decreased by 99.99% and 100% in the bcsE mutant strains. Finally, invasion abilities on HT-29 epithelial cells of wild-type strains were utterly disappeared in their bcsE gene mutant strains.Item Open Access Synthetic biogenesis of bacterial amyloid nanomaterials with tunable inorganic-organic interfaces and electrical conductivity(American Chemical Society, 2017) Seker U.O.S.; Chen, A. Y.; Citorik, R. J.; Lu, T. K.Amyloids are highly ordered, hierarchal protein nanoassemblies. Functional amyloids in bacterial biofilms, such as Escherichia coli curli fibers, are formed by the polymerization of monomeric proteins secreted into the extracellular space. Curli is synthesized by living cells, is primarily composed of the major curlin subunit CsgA, and forms biological nanofibers with high aspect ratios. Here, we explore the application of curli fibers for nanotechnology by engineering curli to mediate tunable biological interfaces with inorganic materials and to controllably form gold nanoparticles and gold nanowires. Specifically, we used cell-synthesized curli fibers as templates for nucleating and growing gold nanoparticles and showed that nanoparticle size could be modulated as a function of curli fiber gold-binding affinity. Furthermore, we demonstrated that gold nanoparticles can be preseeded onto curli fibers and followed by gold enhancement to form nanowires. Using these two approaches, we created artificial cellular systems that integrate inorganic-organic materials to achieve tunable electrical conductivity. We envision that cell-synthesized amyloid nanofibers will be useful for interfacing abiotic and biotic systems to create living functional materials.Item Open Access A synthetic biology approach for engineered functional biofilm(2017-12) Kalyoncu, EbuzerExtracellular polymeric substances consist of molecules, DNAs, carbohydrates, and proteins that are secreted by microbial biofilms. These molecules assist in the synthesis of bacterial biofilms as highly ordered, complex and dynamic material systems, contribute to the adaptation of cells to their environment, and increase their flexibility and functionality under a broad range of conditions. Bacterial biofilms are promising tools for functional applications as bionanomaterials. They are synthesized by well-defined machinery, readily form fiber networks covering large areas, and can be engineered for different functionalities. One aspect of the present thesis focuses on controlling the expression of the curli proteins of Escherichia coli and functionalize the curli fibers by genetically fusing various peptide molecules. Biofilm proteins were functionalized with designed conductive aromatic aminoacids by using programmed cellular machines in order to develop electrically conductive protein nanofiber networks. It has been shown how biological conductivity can be used to control and direct metabolic activities of bacterial populations. Understanding and building conductive biological interfaces to merge living systems with electronic gadgets is a demanding subject. First time in the literature we succeeded to demonstrate living cells enabled bio-conductivity via a conductive nanofiber network formation. In E. coli, there are two proteins as backbones of the nano-fibers (CsgA and CsgB) responsible for the formation of biofilms. In this thesis, tunability of the morphology and mechanical properties of biofilm backbones were investigated by using protein engineering. The effect of minor and major proteins and their engineered form on the final mechanical properties of the biofilm structures were probed by scanning probe microscopy. The minor protein plays a crucial role in tuning the mechanical and morphological properties of the biofilm structures. Biofilm protein engineering for material science can be used through the genetically tunable biofabrication of self-assembling functional materials. Using synthetic biological tools, externally controllable biofilm patterns can be achieved. Recombinase based genetic logic gates encoding AND, and OR to control the expression of structural protein CsgA with 6x-Histaq modification were engineered with using two independent control signals. In this thesis, the opportunity to engineer bacterial biofilms using synthetic biology approaches was demonstrated.Item Open Access Synthetic cellular systems for whole cell biocatalysis(2017-09) Apaydın, OnurSynthetic biology is a field utilizing basic science and engineering approaches to create novel synthetic systems. Biocatalysis is one of those already existing processes which was reviewed intensely due to its advantages of using enzymes as catalysts. It is efficient, requires less additional reagents compared to chemical transformation methods, and it is environment friendly. Due to selectivity of enzymes it is easier to separate products. Enzymes are capable of carrying out many basic and complex reactions however some common problems occur in most strategies due to the nature of enzymes and mostly requirement of purification of the enzymes. Major issues are longevity-sustainability of the enzymes, modularity of the system, and yield of the enzymes. Thanks to the present advances in recombinant DNA technologies and discoveries in bacteria mechanisms like secretion, these pitfalls are addressable through Syntethic biology. We proposed a series of genetic circuits for the sustainability of biocatalysis systems by employing engineered bacterial biofilms. The final, biofilm proteins made nanofibers are protecting both cells and enzymes thus providing an environment fit for replenishment of the enzymes along with modularity to the system. Here we present two synthetic cellular systems utilizing engineered biofilms to address the issues of biocatalysis and we propose an RNA based synthetic regulatory component to increase the robustness of our systems.