Targeted expression of nuclear transgenes in Chlamydomonas reinhardtii with a versatile, modular vector toolkit
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We present a versatile vector toolkit for nuclear transgene expression in the model green microalga Chlamydomonas reinhardtii. The vector was designed in a modular fashion which allows quick replacement of regulatory elements and genes of interest. The current toolkit comprises two antibiotic resistance markers (paromomycin and hygromycin B), five codon-optimized light emission reporters, including the Gaussia princeps luciferase, as well as bright cyan, green, yellow, and red fluorescent protein variants. The system has demonstrated robust functional flexibility with signal options to target the protein of interest to the cytoplasm, the nucleus, cellular microbodies, the chloroplast, mitochondria, or via the endoplasmic reticulum-Golgi apparatus secretory pathway into the culture medium. Successful fluorescent reporter protein fusion to C. reinhardtii Rubisco small subunit 1 was accomplished with this system. Localization of the fluorescently tagged protein was observed in the chloroplast pyrenoid via live cell fluorescence microscopy, the first report of heterologous protein localization to this cellular structure. The functionalities of the vector toolkit, the individual modular elements, as well as several combinations thereof are demonstrated in this manuscript. Due to its strategic design, this vector system can quickly be adapted to individual tasks and should therefore be of great use to address specific scientific questions requiring nuclear recombinant protein expression in C. reinhardtii.
KeywordsMicroalga Chlamydomonas reinhardtii Recombinant proteins Fluorescent proteins Luciferase Protein secretion
The authors would like to acknowledge the CLIB Graduate Cluster Industrial Biotechnology (Federal Ministry of Science & Technology North Rhine Westphalia, Germany (to K.J.L.)) for financial support. The authors would also like to express thanks to Dr. Martina Lummer, Prof. Dr. Thorsten Seidel, Prof. Dr. Karsten Niehaus, Dr. Darius Widera for assistance with fluorescence protein analysis and microscopy, to Prof. Dr. Ralph Bock for strain UVM4, as well as to Jan Schwarzhans and Prof. Dr. Karl Friehs for providing the GFP standards.
Conflict of interest
The authors declare that they have no conflict of interest.
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