Lichtregulierte Genexpression mittels photolabil geschützter Effektormoleküle in Saccharomyces cerevisiae

In recent decades, the optical regulation of cellular functions has become a fastdeveloping field of research. This led to the establishment of "Optogenetic" as generic term.One part of this research field is the regulation of genetic expression systems. In this thesis,light-regulated expression systems in S. cerevisiae have been developed, which are based onthe use of photolabile protected effector molecules, so-called caged compounds. Thesephotolabile compounds release upon illumination a specific effector molecule that regulatesa respective expression system. The expression systems applied were based on the pGAL1,pCUP1 and pMET17 promoters commonly used in S. cerevisiae.By means of a caged-Cu2+ compound, the induction of pCUP1-promoter-controlled genescould be gradually regulated by adjusting the UVA exposure time. In this way, the influenceof the induction time on the expression of pCUP1-controlled genes could be studied.Thereby, light-regulated gene expression was characterized by a non-invasive release of theeffector, which ensured permanent sterility, constant atmospheric conditions, constant gastransfer and a minimization of evaporation and dilution.In addition, the optical repression of pMET17 promoter-controlled genes wasdemonstrated by using a caged-methionine compound. Complex circuits up to automatedexposure regulation were generated by the combination of optical methionine release andcellular methionine degradation. They enabled the optical regulation of expression start,stop, pause, and rate. Thus, a high regulation frequency, a good dosage of the effectorrelease as well as an easy automation and a reduction of the mechanical effort could bedemonstrated.Furthermore, preliminary studies were carried out on the induction of the pGAL1 and onthe repression of the pCUP1 promoter via light-regulation. For this purpose, thecorresponding reporter strains were characterized and first caged-galactose and caged-EDTAcompounds were tested. The photolytic decay rate of caged-galactose and the masking ofthe Cu2+ affinity of caged-EDTA were identified as limiting factors and thus as starting pointsfor further work