A synthetic biology approach for engineered functional biofilm
Şeker, Urartu Özgür Şafak
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Extracellular 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.