title: Optogenetic Regulation of Tunable Gene Expression in Yeast Using Photo-Labile Caged Methionine.
abstract: Light-mediated gene expression enables the noninvasive regulation of cellular functions. Apart from their classical application of regulating single cells with high spatiotemporal resolution, we highlight the potential of light-mediated gene expression for biotechnological issues. Here, we demonstrate the first light-mediated gene regulation in Saccharomyces cerevisiae using the repressible pMET17 promoter and the photolabile NVOC methionine that releases methionine upon irradiation with UVA light. In this system, the expression can be repressed upon irradiation and is reactivated due to consumption of methionine. The photolytic release allows precise control over the methionine concentration and therefore over the repression duration. Using this light regulation mechanism, we were able to apply an in-house constructed 48-well cultivation system which allows parallelized and automated irradiation programs as well as online detection of fluorescence and growth. This system enables screening of multiple combinations of several repression/derepression intervals to realize complex expression programs (e.g., a stepwise increase of temporally constant expression levels, linear expression rates with variable slopes, and accurate control over the expression induction, although we used a repressible promoter.) Thus, we were able to control all general parameters of a gene expression experiment precisely, namely start, pause, and stop at desired time points, as well as the ongoing expression rate. Furthermore, we gained detailed insights into single-cell expression dynamics with spatiotemporal resolution by applying microfluidics cultivation technology combined with fluorescence time-lapse microscopy.
authors: Institute for Bioorganic Chemistry, Heinrich Heine University Düsseldorf at the Forschungszentrum Jülich , 52426 Jülich, Germany.
authors: Peter M
authors: PM
authors: Kusen
authors: AVT - Biochemical Engineering, RWTH Aachen University , Worringer Weg 1, 52074 Aachen, Germany.
authors: Georg
authors: G
authors: Wandrey
authors: Institute of Bio- and Geosciences (IBG-1: Biotechnology), Forschungszentrum Jülich GmbH , 52426 Jülich, Germany.
authors: Christopher
authors: C
authors: Probst
authors: Institute of Bio- and Geosciences (IBG-1: Biotechnology), Forschungszentrum Jülich GmbH , 52426 Jülich, Germany.
authors: Alexander
authors: A
authors: Grünberger
authors: Institute for Bioorganic Chemistry, Heinrich Heine University Düsseldorf at the Forschungszentrum Jülich , 52426 Jülich, Germany.
authors: Martina
authors: M
authors: Holz
authors: Institute for Bioorganic Chemistry, Heinrich Heine University Düsseldorf at the Forschungszentrum Jülich , 52426 Jülich, Germany.
authors: Sonja
authors: S
authors: Meyer Zu Berstenhorst
authors: Institute of Bio- and Geosciences (IBG-1: Biotechnology), Forschungszentrum Jülich GmbH , 52426 Jülich, Germany.
authors: Dietrich
authors: D
authors: Kohlheyer
authors: AVT - Biochemical Engineering, RWTH Aachen University , Worringer Weg 1, 52074 Aachen, Germany.
authors: Jochen
authors: J
authors: Büchs
authors: Institute for Bioorganic Chemistry, Heinrich Heine University Düsseldorf at the Forschungszentrum Jülich , 52426 Jülich, Germany.|Institute of Bio- and Geosciences (IBG-1: Biotechnology), Forschungszentrum Jülich GmbH , 52426 Jülich, Germany.
authors: Jörg
authors: J
authors: Pietruszka
mesh_terms: D005453:Fluorescence; D015971:Gene Expression Regulation, Enzymologic; D005800:Genes, Fungal; D008715:Methionine / Q000378:metabolism*; D044085:Microfluidics; D062308:Optogenetics; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014466:Ultraviolet Rays
chemical_list: D008715:Methionine