Browsing by Author "Ahan, Recep Erdem"
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Item Open Access Biomineralization of calcium phosphate crystals controlled by protein-protein interactions(American Chemical Society, 2019) Duman, Elif; Şahin-Kehribar, Ebru; Ahan, Recep Erdem; Yuca, Esra; Şeker, Urartu Özgür ŞafakHydroxyapatite (HAP) is the major biomineral of bone. Despite the large number of studies addressing HAP formation, a fundamental understanding of the critical roles of HAP-forming proteins in vitro is needed. Effects of two HAP-interacting proteins, osteocalcin (OCN) and osteopontin (OPN), on HAP formation was investigated via in vitro biomineralization experiments, and their outcomes on the crystal structure of calcium phosphate (CaP) was revealed. Our data suggest that OCN concentration is negatively correlated with crystal formation rate and crystal size, yet the presence of OCN leads to a more ordered HAP crystal formation. On the other hand, OPN protein promotes faster formation of CaP crystals potentially working as a growth site for mineral formation, and it decreases the Ca:P ratio. This effect results in a shift from HAP-type minerals to less ordered crystals. The crystal size, shape, and Ca:P ratio can be tuned to design improved mammalian hard tissue environment-mimicking matrices by taking advantage of the OCN and OPN proteins on crystal formation. We believe our current findings will lead to innovative approaches for bone biomineralization in regenerative medicine.Item Embargo Biotechnological drug platforms(Bilkent University, 2023-11) Ahan, Recep ErdemBiopharmaceuticals, also known as biotechnological drugs, have revolutionized the treatment of many diseases by providing access to new mechanisms of action that can target the underlying biological processes behind the diseases. Technological advancement in biological sciences opens new paths to uncover new biopharmaceutical modalities in nature as well as to augment the existing modalities new functions. The implementation of engineering principles i.e., synthetic biology approaches have been transforming biopharmaceutical research wherein “smart” therapeutics are developed and deployed for treatment of previously intractable diseases. However, there are still unmet clinical needs that require novel and advanced biopharmaceuticals. In this thesis, I explored different biopharmaceuticals to characterize and/or advance their capabilities for diverse indications. Firstly, we have developed a prophylactic agent from the lectin protein, griffithsin, as for ancestral and the emerged strains of SARS-CoV-2. Secondly, we have advanced genetic technologies to engineer the probiotic Escherichia coli (E. coli) strain, Nissle 1917 (EcN), for therapeutical purposes. We developed a stable recombinant DNA transfer system based on cryptic plasmids of EcN. Furthermore, a synthetic protein secretion system was envisioned and functionally validated in EcN to shuttle therapeutical proteins to diseases site. Finally, peptide tags for extracellular protein secretion as well as a cell surface protein display system were developed for Lachnospiraceae species which are parts of the healthy human gut microbiome. The technologies and methodologies described herein will pay the way for inventing and/or discovering novel biopharmaceuticals to treat current and future diseases.Item Open Access Cellular biocatalysts using synthetic genetic circuits for prolonged and durable enzymatic activity(Wiley‐VCH Verlag GmbH & Co. KGaA, Weinheim, 2019) Ahan, Recep Erdem; Şaltepe, Behide; Apaydın, Onur; Şeker, Urartu Özgür ŞafakCellular biocatalysts hold great promise for the synthesis of difficult to achieve compounds, such as complex active molecules. Whole‐cell biocatalysts can be programmed through genetic circuits to be more efficient, but they suffer from low stability. The catalytic activity of whole cells decays under stressful conditions, such as prolonged incubation times or high temperatures. In nature, microbial communities cope with these conditions by forming biofilm structures. In this study, it is shown that the use of biofilm structures can enhance the stability of whole‐cell biocatalysts. We employed two different strategies to increase the stability of whole‐cell catalysts and decrease their susceptibility to high temperature. In the first approach, the formation of a biofilm structure is induced by controlling the expression of one of the curli component, CsgA. The alkaline phosphatase (ALP) enzyme was used to monitor the catalytic activity of cells in the biofilm structure. In the second approach, the ALP enzyme was fused to the CsgA curli fiber subunit to utilize the protective properties of the biofilm on enzyme biofilms. Furthermore, an AND logic gate is introduced between the expression of CsgA and ALP by toehold RNA switches and recombinases to enable logical programming of the whole‐cell catalyst for biofilm formation and catalytic action with different tools. The study presents viable approaches to engineer a platform for biocatalysis processes.Item Open Access Genetic logic gates enable patterning of amyloid nanofibers(WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, 2019) Kalyoncu, Ebuzer; Ahan, Recep Erdem; Özçelik, Cemile Elif; Şeker, Urartu Özgür ŞafakDistinct spatial patterning of naturally produced materials is observed in many cellular structures and even among communities of microorganisms. Reoccurrence of spatially organized materials in all branches of life is clear proof that organization is beneficial for survival. Indeed, organisms can trick the evolutionary process by using organized materials in ways that can help the organism to avoid unexpected conditions. To expand the toolbox for synthesizing patterned living materials, Boolean type “AND” and “OR” control of curli fibers expression is demonstrated using recombinases. Logic gates are designed to activate the production of curli fibers. The gates can be used to record the presence of input molecules and give output as CsgA expression. Two different curli fibers (CsgA and CsgA‐His‐tag) production are then selectively activated to explore distribution of monomers upon coexpression. To keep track of the composition of fibers, CsgA‐His‐tag proteins are labeled with nickel–nitrilotriacetic acid (Ni–NTA‐) conjugated gold nanoparticles. It is observed that an organized living material can be obtained upon inducing the coexpression of different CsgA fibers. It is foreseen that living materials with user‐defined curli composition hold great potential for the development of living materials for many biomedical applications.Item Open Access A genetically engineered biofilm material for SARS-CoV-2 capturing and isolation(John Wiley & Sons, Ltd, 2022-09-13) Özkul, Gökçe; Kehribar, Ebru Şahin; Ahan, Recep Erdem; Köksaldı, İlkay Çisil; Özkul, A.; Dinç, B.; Aydoğan, S.; Şeker, Urartu Özgür ŞafakThe novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is continuously infecting people all around the world since its outbreak in 2019. Studies for numerous infection detection strategies are continuing. The sensitivity of detection methods is crucial to separate people with mild infections from people who are asymptomatic. In this sense, a strategy that would help to capture and isolate the SARS-CoV-2 virus prior to tests can be effective and beneficial. To this extent, genetically engineered biomaterials grounding from the biofilm protein of Escherichia coli are beneficial due to their robustness and adaptability to various application areas. Through functionalizing the E. coli biofilm protein, diverse properties can be attained such as enzyme display, nanoparticle production, and medical implant structures. Here, E. coli species are employed to express major curli protein CsgA and Griffithsin (GRFT) as fusion proteins, through a complex formation using SpyTag and SpyCatcher domains. In this study, a complex system with a CsgA scaffold harboring the affinity of GRFT against Spike protein to capture and isolate SARS-CoV-2 virus is successfully developed. It is shown that the hybrid recombinant protein can dramatically increase the sensitivity of currently available lateral flow assays for Sars-CoV-2 diagnostics.Item Open Access A living material platform for the biomineralization of biosilica(Elsevier B.V., 2022-12-15) Kırpat Konak, Büşra Merve; Bakar, Mehmet Emin; Ahan, Recep Erdem; Özyürek, Emel Uzunoğlu; Dökmeci, Serap; Şafak Şeker, Urartu ÖzgürNature has a vast array of biomineralization mechanisms. The commonly shared mechanism by many living organisms to form hardened tissues is the nucleation of mineral structures via proteins. Living materials, thanks to synthetic biology, are providing many opportunities to program cells for many functionalities. Here we have demonstrated a living material system for biosilicification. Silaffins are utilized to synthesize silicified cell walls by one of the most abundant organism groups called diatoms. The R5 peptide motif of the silaffins is known for its ability to precipitate silica in ambient conditions. Therefore, various studies have been conducted to implement the silicification activity of R5 in different application areas, such as regenerative medicine and tissue engineering. However, laborious protein purification steps are required prior to silica nanoparticle production in recombinant approaches. In this study, we aimed to engineer an alternative bacterial platform to achieve silicification using released and bacteria-intact forms of R5-attached fluorescent proteins (FP). Hence, we displayed R5-FP hybrids on the cell surface of E. coli via antigen 43 (Ag43) autotransporter system and managed to demonstrate heat-controllable release from the surface. We also showed that the bacteria cells displaying R5-FP can be used in silicification reactions. Lastly, considering the stimulating effect of silica on osteogenic differentiation, we treated human dental pulp stem cells (hDPSCs) with the silica aggregates formed via R5-FP hybrids. Earlier calcium crystal deposition around the hDPSCs was observed. We envision that our platform can serve as a faster and more economical alternative for biosilicification applications, including endodontics. © 2022Item Open Access Making the next generation of therapeutics: mRNA meets synthetic biology(American Chemical Society, 2023-09-15) Hınçer, Ahmet; Ahan, Recep Erdem; Aras, Ebru; Şeker, Urartu Özgür ŞafakThe development of mRNA-based therapeutics centers around the natural functioning of mRNA molecules to provide the genetic information required for protein translation. To improve the efficacy of these therapeutics and minimize side effects, researchers can focus on the features of mRNA itself or the properties of the delivery agent to achieve the desired response. The tools considered for mRNA manipulation can be improved in terms of targetability, tunability, and translatability to medicine. While ongoing studies are dedicated to improving conventional approaches, innovative approaches can also be considered to unleash the full potential of mRNA-based therapeutics. Here, we discuss the opportunities that emerged from introducing synthetic biology to mRNA therapeutics. It includes a discussion of modular self-assembled mRNA nanoparticles, logic gates on a single mRNA molecule, and other possibilities.Item Embargo Multiplexed cell-based diagnostic devices for detection of renal biomarkers(Elsevier, 2022-12-24) Köse, Sıla; Ahan, Recep Erdem; Köksaldı, İlkay Çisil; Olgaç, A.; Kasapkara, Çiğdem S.; Şeker, Urartu Özgür ŞafakThe number of synthetic biology-based solutions employed in the medical industry is growing every year. The whole cell biosensors being one of them, have been proven valuable tools for developing low-cost, portable, personalized medicine alternatives to conventional techniques. Based on this concept, we targeted one of the major health problems in the world, Chronic Kidney Disease (CKD). To do so, we developed two novel biosensors for the detection of two important renal biomarkers: urea and uric acid. Using advanced gene expression control strategies, we improved the operational range and the response profiles of each biosensor to meet clinical specifications. We further engineered these systems to enable multiplexed detection as well as an AND-logic gate operating system. Finally, we tested the applicability of these systems and optimized their working dynamics inside complex medium human blood serum. This study could help the efforts to transition from labor-intensive and expensive laboratory techniques to widely available, portable, low-cost diagnostic options.Item Open Access SARS-CoV 2 detection with de novo-designed synthetic riboregulators(American Chemical Society, 2021-06-30) Köksaldı, İlkay Çisil; Köse, Sıla; Ahan, Recep Erdem; Hacıosmanoğlu, Nedim; Şahin Kehribar, Ebru; Güngen, Murat Alp; Baştuğ, A.; Dinç, B.; Bodur, H.; Özkul, A.; Şeker, Urartu Özgür ŞafakSARS-CoV-2 is a human pathogen and the main cause of COVID-19 disease, announced as a global pandemic by the World Health Organization. COVID-19 is characterized by severe conditions, and early diagnosis can make dramatic changes for both personal and public health. Low-cost, easy-to-use diagnostic capabilities can have a very critical role in controlling the transmission of the disease. Here, we are reporting a state-of-the-art diagnostic tool developed with an in vitro synthetic biology approach by employing engineered de novo riboregulators. Our design coupled with a home-made point-of-care device can detect and report the presence of SARS-CoV-2-specific genes. The presence of SARS-CoV-2-related genes triggers the translation of sfGFP mRNAs, resulting in a green fluorescence output. The approach proposed here has the potential of being a game changer in SARS-CoV-2 diagnostics by providing an easy-to-run, low-cost diagnostic capability.Item Open Access A self-actuated cellular protein delivery machine(American Chemical Society, 2019) Ahan, Recep Erdem; Kırpat, Büşra Merve; Saltepe, Behide; Şeker, Urartu Özgür ŞafakEngineered bacterial cells have great promise to solve global problems, yet they are hampered by a lack of convenient strategy for controlled protein release. A well-controlled protein translocation through cellular membranes is essential for cell-based protein delivery. Here we have developed a controlled protein release system by programming a bacterial autotransporter system named Ag43. Ag43 protein is engineered by adding a protease digestion site between its translocation and cargo domains. Once it is displayed on the cell surface, we managed to release the cargo proteins in defined conditions by processing environmental signals. The protein release in terms of time and quantity can be controlled through changing the inducer conditions. We thought that the release system can be adopted for complex genetic circuitries due to its simplicity. We implemented the protein release system to develop a cellular device that is able to release proteins in a sequence response to ordered chemical signals. We envision that development of genetically controlled protein release systems will improve the applications of synthetic organisms in cell based therapies, especially for cases with a need for controlled protein release using the cues from the biological environment.Item Open Access A self-actuated cellular protein delivery machinery(Bilkent University, 2018-08) Ahan, Recep ErdemOwing to increase the knowledge on biology and available tools for genetic manipulations, biological systems are engineered to perform complex tasks. They can be designed to degrade toxic molecules in environment, produce and deliver complex biological drugs, or process and synthesize valuable materials. Hence, the cellular machines hold great promises to solve world problems such as global warming, world hunger, cancer and so forth. However, most of the complex tasks require protein release to extracellular space in a controlled manner. Development of efficient cellular machines is hampered by lack of convenient strategy for controlled protein release as many of proposed secretion systems are limited in a narrow focused application. In this thesis, we are proposing a novel bifunctional self-exciting protein delivery system for broader applications. The proposed protein delivery machine harbours a genetic circuit that is able to display protein-of-interest on cell surface and to secrete to extracellular space in case of need. To do so, we engineered the autotransporter protein Ag43 to display POI with TEV protease recognition site on the cell surface of Escherichia coli (E. coli). The release of displayed POI was achieved and systematically optimized in vitro via addition of purified TEV protease. To accomplish the self-exciting and controlled release of POI by cells, TEV protease was aimed to be expressed and translocated to extracellular space to cleave the recognition site between POI and Ag43 protein. Four different secretion strategies was employed to secrete TEV protease to extracellular space. While cleavage of POI from cell surface can’t be accomplished through secretion of TEV protease by type I system, YebF fusion, and co-expressing lysis gene, codisplaying TEV protease on the cell surface can release the POI. Our data revealed that release of POI can be tuned with controlling the amount of TEV protease on the cell surface. Considering the simplicity of protein display as well as ability to express Ag43 protein in various organisms, the proposed system can be implemented in more complex genetic circuits and used in diverse applications.Item Open Access A sustainable preparation of catalytically active and antibacterial cellulose metal nanocomposites via ball milling of cellulose(Royal Society of Chemistry, 2020-01) Kwiczak-Yiğitbaşı, Joanna; Laçin, Özge; Demir, Mine; Ahan, Recep Erdem; Şeker, Urartu Özgür Şafak; Baytekin, BilgeCellulose, the most abundant polymer on Earth, and its composites have recently gained importance for the production of sustainable materials. These materials should be produced using green methods that avoid the utilization of toxic chemicals to ensure integrity for environmental sustainability. Ball milling, which gives a straightforward and (often) green synthetic access to materials, can be used to achieve this goal. Previously, it was shown that mechanochemical bond breakages in polymers generate mechanoradicals, which can be used to drive further reactions and to form polymer composites. In this study, we show that cellulose mechanoradicals generated during the ball milling of cellulose can reduce various metal ions to the corresponding metal nanoparticles (NPs) (Au, Ag, Pt, Pd, Co, and Cu), which are deposited and stabilized in the cellulose matrix. Using mechanoradicals to reduce the metal ions and form the cellulose composites, (1) the number of synthetic steps is reduced, and (2) the conventionally used, toxic reducing and stabilizing agents are avoided, which also prevents the contamination of the composites. The cellulose–metal nanoparticle composites can exhibit a wide range of properties that depend on the metal nanoparticle in the composite; e.g., Au–cellulose nanocomposites exhibit catalytic activity, and Ag–cellulose nanocomposites exhibit antibacterial properties. The ball-milling method also permits blend formation using synthetic polymers, which allows tuning the physical properties of the final material. Finally, the method shown here provides a quick access to versatile metal nanoparticle cellulose composites (and their blends), which may find applications, such as in paper-based diagnostics and catalysis.Item Open Access Ultrasonication for environmentally friendly preparation of antimicrobial and catalytically active nanocomposites of cellulosic textiles(American Chemical Society, 2020) Kwiczak-Yiǧitbaşı, Joanna; Demir, Mine; Ahan, Recep Erdem; Canlı, S.; Şafak Şeker, Urartu Özgür; Baytekin, BilgeThe global demand for sustainable and functional fibers and textile materials is increasing with the pressure to limit the synthetic petroleum-based counterparts. In this study, we use ultrasonication for the preparation of eco-friendly cellulose fabrics bearing silver or gold nanoparticles (NPs). The mechanochemistry of cellulose is based on the breakage of glycosidic bonds and the formation of mechanoradicals. These mechanoradicals can reduce Au3+ and Ag+ ions in solution, and the reduced metals can be stabilized by the cellulose chains as nanoparticles. Here, we formed the mechanoradicals in the fabrics by sonication (on the order of 1018 per gram), which is confirmed by ESR. The sizes and the metallic nature of NPs and the structural and morphological changes in the fabrics upon ultrasonication were studied by SEM, XPS, FTIR-ATR, XRD, and TEM. The displayed preparation method is shown to yield antibacterial AgNP-fabric and catalytically active AuNP-fabric composites, with up to a 14% yield of metal ion reduction. Since the method involves only the sonication of the fabric in aqueous solutions, and uses no hazardous reducing and stabilizing agents, it provides quick and environment-friendly access to fabric nanocomposites, which have applications in medical textiles, catalysis, and materials for energy.