Expanding the spectral palette of fluorescent proteins for the green microalga Chlamydomonas reinhardtii

Abstract

SUMMARYFluorescent proteins (FPs) have become essential tools for a growing number of fields in biology. However, such tools have not been widely adopted for use in microalgal research. The aim of this study was to express and compare six FPs (blue mTagBFP, cyan mCerulean, green CrGFP, yellow Venus, orange tdTomato and red mCherry) in the popular model microalga Chlamydomonas reinhardtii. To circumvent the transgene silencing that often occurs in C. reinhardtii, the FPs were expressed from the nuclear genome as transcriptional fusions with the sh-ble antibiotic resistance gene, with the foot and mouth disease virus 2A self-cleaving sequence placed between the coding sequences. All ble-2A-FPs tested are well-expressed and efficiently processed to yield mature, unfused FPs that localize throughout the cytoplasm. The fluorescence signals of each FP were detectable in whole cells by fluorescence microplate reader analysis, live-cell fluorescence microscopy, and flow cytometry. Furthermore, we report a comparative analysis of fluorescence levels relative to auto-fluorescence for the chosen FPs. Finally, we demonstrate that the ble-2A expression vector may be used to fluorescently label an endogenous protein (a-tubulin). We show that the mCerulean-a-tubulin fusion protein localizes to the cytoskeleton and flagella, as expected, and that cells containing this fusion protein had normal cellular function. Overall, our results indicate that, by use of the ble-2A nuclear expression construct, a wide array of FP tools and technologies may be applied to microalgal research, opening up many possibilities for microalgal biology and biotechnology.

SUMOylation by a Stress-Specific Small Ubiquitin-Like Modifier E2 Conjugase Is Essential for Survival ofChlamydomonas reinhardtiiunder Stress Conditions

“…mCherry fluorescent signal was acquired with 561.5-nm excitation and 570-to 620-nm emission, and chlorophyll autofluorescence signal was acquired at 641-nm excitation and 662-to 737-nm emission and pseudocolored green for visualization. Control cells expressing free mCherry were generated as previously described (Rasala et al, 2013).…”

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“…Plasmid construction mCherry, Venus, mCerulean, and mTagBFP were codonoptimized for expression from the nuclear genome of C. reinhardtii, as previously described [24]. The organelle transit sequences from bip1, psaD, and atpA were PCR-amplified from genomic DNA Figure 2.…”

“…Enabling Complex Genetic Engineering in Microalgae PLOS ONE | www.plosone.orgisolated from cc1690, using the oligonucleotides described in Table S1, and fused between ble2A and mCherry in the pBR9 vector [24] using the GeneArt Seamless Cloning Kit (Life Technologies, Carlsbad, CA). The 2x SV40 NLS was codonoptimized for C. reinhardtii nuclear expression, synthesized as sense and antisense single stranded oligonucleotides, annealed, and cloned into pBR25 [24] that had been digested with BamHI and EcoRI.…”

“…The 2x SV40 NLS was codonoptimized for C. reinhardtii nuclear expression, synthesized as sense and antisense single stranded oligonucleotides, annealed, and cloned into pBR25 [24] that had been digested with BamHI and EcoRI. His-Asp-Glu-Lys (HDEL) was fused to the end of mCherry by PCR using a reverse oligonucleotide encoding for the ER retention sequence.…”

“…Previously, we described a nuclear expression strategy that overcomes transgene silencing by using the foot-and-mouthdisease-virus (FMDV) 2A ''self-cleaving'' peptide to transcriptionally fuse transgene expression to the antibiotic resistance gene ble in the green microalga Chlamydomonas reinhardtii [23,24]. It is believed that the FMDV 2A sequence ''self-cleaves'' through ribosome-skipping during translation rather than a proteolytic reaction, and has been termed CHYSEL (cis-acting hydrolase element) [25,26].…”