Teaching an old ‘doc’ new tricks for algal biotechnology: Strategic filter use enables multi-scale fluorescent protein signal detection

Abstract
Fluorescent proteins (FPs) are powerful reporters with a broad range of applications in gene expression and subcellular localization. High-throughput screening is often required to identify individual transformed cell lines in organisms that favor non-homologous-end-joining integration of transgenes into genomes, like in the model green microalga Chlamydomonas reinhardtii. Strategic transgene design, including genetic fusion of transgenes to FPs, and strain domestication have aided engineering efforts in this host but have not removed the need for screening large numbers of transformants to identify those with robust transgene expression levels. FPs facilitate transformant screening by providing a visual signal indicating transgene expression. However, limited combinations of FPs have been described in alga and inherent background fluorescence from cell pigments can hinder FP detection efforts depending on available infrastructure. Here, an updated set of algal nuclear genome-domesticated plasmid parts for seven FPs and six epitope tags were generated and tested in C. reinhardtii. Strategic filter selection was found to enable detection of up to five independent FPs signals from cyan to far-red separately from inherent chlorophyll fluorescence in live algae at the agar plate-level and also in protein electrophoresis gels. This work presents technical advances for algal engineering that can assist reporter detection efforts in other photosynthetic host cells or organisms with inherent background fluorescence.

Citation
Gutiérrez, S., Wellman, G. B., & Lauersen, K. J. (2022). Teaching an old ‘doc’ new tricks for algal biotechnology: Strategic filter use enables multi-scale fluorescent protein signal detection. Frontiers in Bioengineering and Biotechnology, 10. https://doi.org/10.3389/fbioe.2022.979607

Acknowledgements
This work was supported by King Abdullah University of Science and Technology (KAUST) baseline funding awarded to KL. The authors are grateful to Dr. Andreas Wolf of Analytik Jena (Germany) and the efforts of Sean Gallagher as well as Jenise Cleary (AJ, United States) in facilitating filter acquisition and mounting. We are grateful to Paulo C. Aurelio of the KAUST Lab Equipment Maintenance (LEM) team for efforts in the installation and upgrade of the NxT Flow Cytometer. We are grateful to Dr. Hubert Fiumelli for enabling and facilitating NxT and filter selection experiments. The authors thank Prof. Dr. Ralph Bock and Dr. Juliane Neupert for strain UVM4 and Malak Abdallah for strain UPN22.

Publisher
Frontiers Media SA

Journal
Frontiers in Bioengineering and Biotechnology

DOI
10.3389/fbioe.2022.979607

Additional Links
https://www.frontiersin.org/articles/10.3389/fbioe.2022.979607/full

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