Leonie Hergenröder (Goethe-Universität Frankfurt am Main)
Leonie Hergenröder's Master thesis was awarded with the Prize for the Best Plant Science Master Thesis, which was carried out at Frankfurt University in the year 2025 with the title:
Establishment of a novel in-gel fluorescence assay for the detection of heterologously expressed proteins in diatoms
In this master's thesis, the in-gel fluorescence assay developed by Adrian Fuchs (2023) was established for the diatom Phaeodactylum tricornutum, to detect and quantify specific proteins.
Conventional protein detection methods require considerable time and effort for quantification, making the in-gel fluorescence assay a faster, more sensitive, and more quantifiable alternative. The method is based on the selective attachment of the fluorophore Cy5.5 to target proteins by the ligase connectase, allowing both detection and relative and absolute quantification. To detect the resistance proteins Nourseothricin N-Acetyltransferase and ShBle in P. tricornutum, these were cloned into episomes and introduced into the diatom via bacterial conjugation. After verifying the integrity of the episome through genetic analysis, the mutants were subjected to various cell disruption and protein precipitation methods. By combining manual grinding cell disruption with liquid nitrogen and protein precipitation, as described by Wei Wang et al. (2006), both proteins could be detected in small quantities.
This work shows that the in-gel fluorescence assay is suitable for detecting and quantifying heterologously expressed proteins in Phaeodactylum tricornutum. However, the current methodology requires a considerable amount of time, which is why further optimisations are necessary to use it more efficiently in future experiments. The results thus underscore both the potential of the assay for diatoms and the need for optimizations to make the protocols more practical.
Fuchs, Adrian C. D. (2023): Specific, sensitive and quantitative protein detection by in-gel fluorescence. Nature Communications 14 (1), S. 2505. DOI: 10.1038/s41467-023-38147-8
Wang, Wei; Vignani, Rita; Scali, Monica; Cresti, Mauro (2006): A universal and rapid protocol for protein extraction from recalcitrant plant tissues for proteomic analysis. In: Electrophoresis 27 (13), S. 2782–2786. DOI: 10.1002/elps.200500722
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Leonie Hergenröder conducted this work at the Institute for Molecular Biosciences in the working group of Prof. Dr. Claudia Büchel.