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In nature, pyocyanin is produced by Pseudomonas aeruginosa and many biological roles of pyocyanin for P. aeruginosa have been described. These roles arise mainly from the intrinsic feature of pyocyanin, i.e. its reversible redox activity. Due to this activity, pyocyanin can also act as electron shuttle enabling pyocyanin to interchange electrons between different redox active substrates by cycling between its oxidation and reduction state. As a result, pyocyanin is an electro-active compound and, consequently, a possible candidate as electrical reporter molecule in a bacterial biosensor. The main scope of this thesis was therefore to develop a bacterial biosensor that produces pyocyanin upon detection of a specific input signal after which the electrical output signal of the biosensor, i.e. pyocyanin, is detected by a microchip.The bacterial biosensor is an Escherichia coli strain that is engineered in this study to produce pyocyanin in a dose-responsive manner. To this end, the pyocyanin biosynthesis genes (phz) were introduced in E. coli by a genetic construct containing the phz genes, modulated by an arabinose-inducible promoter. This construct allows controlling pyocyanin production in a dose-responsive manner, such that the concentration of produced pyocyanin by E. coli is strictly dependent on the concentration of arabinose added to the bacterial culture. This study therefore provides a first proof-of-principle that pyocyanin is an appropriate output molecule for a bacterial biosensor as its production is tightly dependent on the input signal, i.e. arabinose. Pyocyanin has also an antibiotic activity which affects cell fitness of E. coli when it is triggered to produce pyocyanin. As a consequence, performance of the bacterial biosensor is affected and further optimization of the biosensor is required in order to validate pyocyanin as an efficient and accurate output signal in the bacterial biosensor. Therefore, a directed evolution experiment was performed in this study to develop an E. coli strain with an increased tolerance towards pyocyanin. As a result, an E. coli strain was obtained that, compared to the original E. coli strain, is capable of producing pyocyanin at approximately four times higher levels with an increased growth yield. These results demonstrate that directed evolution is a promising strategy for improving the function of part of a synthetic circuit in a genetic context. Whole genome sequencing analysis of ten individual clones was performed in order to identify the genotype of this evolved E. coli strain. This revealed that at least ten genes (ompR, acrB, recA, spoT, cyaA, fre, adhE, hycC, dipZ, mhpR) are affected by mutations during the evolution experiment. Based on literature data, a hypothesis is made on how these genes and their mutations are influencing the phenotype of the evolved E. coli. First, mutations in the genes ompR and acrB are assumed to reduce the intracellular levels of pyocyanin thereby increasing the resistance of E. coli to pyocyanin. In addition, restoration of the recA1 mutation of E. coli DH10B relieves the inhibition of the SOS response of E. coli which can sustain higher levels of pyocyanin. The precise effect of the mutations in spoT and cyaA is less clear, but they will probably influence transcriptional regulation of a multitude of genes resulting in a pleiotropic effect on cellular function. Mutations in genes, fre and adhE, on the other hand, are believed to indirectly increase pyocyanin production by E. coli. This hypothesis needs, however, further validation in order to clarify which factors are important for pyocyanin production in E. coli. To this end, several follow-up experiments are suggested.Besides optimizing the biological level of the microchip-based biosensor with an evolutionary approach, the electrical component of this system, i.e. detection of pyocyanin with the microchip, was investigated in this study. Therefore, the electrochemical detection of pyocyanin by cyclic voltammetry was studied with a three-electrode system containing a microelectrode array as working electrode. As a result, it was demonstrated that this system is sensitive and specific enough for pyocyanin detection in bacterial cultures, but lacks accuracy as experiments could not be performed in a repeatable and reproducible manner which hinders the further characterization and validation of pyocyanin as electrical reporter molecule in biosensors. Therefore, it is suggested to develop a novel microchip system which is specifically designed for the electrochemical detection of pyocyanin in bacterial cultures. Although a properly working microchip-based biosensor with pyocyanin as key player could not be developed in this study, this doctoral thesis emphasizes the power of combining synthetic biology with microfluidics and microelectronics. As many techniques have been developed in the field of synthetic biology to engineer microorganisms, integrating microbiology with microdevices is a promising approach to develop new miniaturized sensing devices and other technologie

579.841.11 --- Academic collection --- Pseudomonas --- Theses --- 579.841.11 Pseudomonas

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Lait --- milk --- Produit laitier --- Milk products --- Pseudomonas --- Protéase --- Proteases --- Résistance à la température --- Temperature resistance --- Identification --- identification --- PCR --- Immunodiagnostic --- Immunodiagnosis --- 579.841.11 --- 637.12 --- 637.06 --- 577.152.34 --- Milk (fresh, unprocessed milk) --- Faults, defects, contamination, adulteration of animal produce --- Acting on peptide bonds (peptide hydrolases), proteolytic fermentation. Proteinases --- 577.152.34 Acting on peptide bonds (peptide hydrolases), proteolytic fermentation. Proteinases --- 637.06 Faults, defects, contamination, adulteration of animal produce --- 637.12 Milk (fresh, unprocessed milk) --- 579.841.11 Pseudomonas --- identification.