TY - THES A1 - Condratov, Alexandru T1 - Flow cytometry based directed evolution of cell surface displayed laccase N2 - Directed evolution is a technique applied in protein engineering, which consists of randomly mutating a gene, allowing the improvement of different enzyme properties, which are desired for specific industrial processes. Some of the properties include affinity, solubility, organic solvent resistance, enantioselectivity and thermostability. The probability of succeeding in directed evolution experiment depends on the size of the library generated, because the more variants are present, the higher the chances of detecting an enzyme with improved properties. The throughput represents the main bottleneck in enzyme screening, as most of the methods allow the analysis of 104 variants per round, which is not enough for a library size of 109 variants. Ultra high-throughput screening (uHTS) enables the analysis of 107 events per hour. It can reduce the time, costs and consumables needed for an applied round of directed evolution. One of its limitations is the use of a flow cytometer, which is expensive and not affordable for everyone. Also uHTS is restricted to single catalytic reactions and their coupling with synthetic fluorophores. The objective of this project is to develop an uHTS method based on fluorescence activated cell sorting (FACS). The principle of this method is to sort cells expressing the active enzyme variant, which have a fluorescent hydrogel formed on the cell membrane. Successful completion of this protocol will allow the screening of different enzyme classes, for example, laccases, alcohol dehydrogenases, organophosphorus hydrolases. In order to prove that the concept is working, laccase will be used for generating the library, which will then be displayed on the surface of E. coli. The variants which are enzymatically active, will produce the radicals that initiate the polymerization reaction of fluorescent hydrogel, allowing the flow cytometer to sort cells based on the fluorescence emitted. One round of sorting should achieve at least a 3-fold enrichment of the active enzyme population. Y1 - 2021 ER -