Browsing by Subject "Curli fibers"

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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 Şafak
Cellular 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.
Open Access
A synthetic biology approach for nanomaterial design, synthesis and functionalization
(2017-11) Ölmez, Tolga Tarkan
Biological formation of inorganic material occurs in most organisms in nature. Various biomolecules such as polypeptides, lipids and metabolites are responsible for biomineralization in cells and tissues. Biological synthesis of biohybrid materials is a recently emerged discipline that uses these biomolecules in synthetic biological systems. Synthetic biology is one of most promising approaches for the development of biohybrid systems, and stands at the intersection of computer science, engineering and molecular genetics. Synthetic biology tools allow the design of programmable genetic toolkits that can compete with natural biosynthesis systems. The present thesis elaborates on the formation of well-controlled genetic systems that can synthesize and functionalize biological materials. Artificial peptides were fused to various genes through molecular genetics techniques, allowing the production of designer proteins. One aspect concerns the fusion of the 19 amino acid-long R5 motif of silaffin protein to three distinct fluorescent proteins. The R5 peptide motif can nucleate silica precursor ions to synthesize silica nanostructures. Therefore, fusion of fluorescent proteins with the R5 motif allows the synthesis and encapsulation of fluorescent silica nanoparticles. Due to its affinity to silica, R5 tag was also shown to be a candidate tag for silica resin-based affinity chromatography purification. Using synthetic biology tools, production of autonomously formed biotemplating platforms can be achieved. A bacterial functional amyloid fiber biosystem called curli can be utilized as a biotemplating platform for nanomaterials synthesis in this context. The major curli subunit CsgA was fused to artificial peptides that can nucleate and synthesize various nanomaterials. Inducible systems were also integrated into the genetic design system to confer temporal control over curli synthesis. These designs were improved through the incorporation of material-sensitive transcription factors and their cognate promoters for ions of cadmium, gold and iron. First, these material sensitive pairs were used in the development of microbial whole cell sensors that produce a fluorescence output upon induction by material precursor ions. Later, material-sensitive pairs were integrated into a modified curli nanofiber display biosystem to produce living autonomous whole cell nanomaterial synthesizers. These systems recognize precursor ions in the environment and synthesize modified curli nanofibers that can nuclate precursor ions to form functional nanomaterials.