caa a22 4500 605503958 CHVBK 20210128100613.0 cr unu---uuuuu 210128e20150401xx s 000 0 eng 10.1007/s00253-014-6354-7 doi (NATIONALLICENCE)springer-10.1007/s00253-014-6354-7 Targeted expression of nuclear transgenes in Chlamydomonas reinhardtii with a versatile, modular vector toolkit [Elektronische Daten] [Kyle Lauersen, Olaf Kruse, Jan Mussgnug] 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. Springer-Verlag Berlin Heidelberg, 2015 Microalga nationallicence Chlamydomonas reinhardtii nationallicence Recombinant proteins nationallicence Fluorescent proteins nationallicence Luciferase nationallicence Protein secretion nationallicence Lauersen Kyle Faculty of Biology, Center for Biotechnology (CeBiTec), Bielefeld University, Universitätsstrasse 27, 33615, Bielefeld, Germany aut Kruse Olaf Faculty of Biology, Center for Biotechnology (CeBiTec), Bielefeld University, Universitätsstrasse 27, 33615, Bielefeld, Germany aut Mussgnug Jan Faculty of Biology, Center for Biotechnology (CeBiTec), Bielefeld University, Universitätsstrasse 27, 33615, Bielefeld, Germany aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/8(2015-04-01), 3491-3503 0175-7598 99:8<3491 2015 99 253 https://doi.org/10.1007/s00253-014-6354-7 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-014-6354-7 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Lauersen Kyle Faculty of Biology, Center for Biotechnology (CeBiTec), Bielefeld University, Universitätsstrasse 27, 33615, Bielefeld, Germany aut NATIONALLICENCE 700 1- Kruse Olaf Faculty of Biology, Center for Biotechnology (CeBiTec), Bielefeld University, Universitätsstrasse 27, 33615, Bielefeld, Germany aut NATIONALLICENCE 700 1- Mussgnug Jan Faculty of Biology, Center for Biotechnology (CeBiTec), Bielefeld University, Universitätsstrasse 27, 33615, Bielefeld, Germany aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/8(2015-04-01), 3491-3503 0175-7598 99:8<3491 2015 99 253