title: Expanding the spectral palette of fluorescent proteins for the green microalga Chlamydomonas reinhardtii.
abstract: Fluorescent 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 (α-tubulin). We show that the mCerulean-α-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.
authors.affiliation: The San Diego Center for Algae Biotechnology and Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093-0368, USA.
authors.forename: Beth A
authors.initials: BA
authors.lastname: Rasala
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Barrera
authors.forename: Jenny
authors.initials: J
authors.lastname: Ng
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Plucinak
authors.forename: Julian N
authors.initials: JN
authors.lastname: Rosenberg
authors.forename: Donald P
authors.initials: DP
authors.lastname: Weeks
authors.forename: George A
authors.initials: GA
authors.lastname: Oyler
authors.forename: Todd C
authors.initials: TC
authors.lastname: Peterson
authors.forename: Farzad
authors.initials: F
authors.lastname: Haerizadeh
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Mayfield
mesh_terms: D020418:Algal Proteins / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D016825:Chlamydomonas reinhardtii / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D005407:Flagella / Q000378:metabolism; D005434:Flow Cytometry; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics*; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins; D014170:Transformation, Genetic; D019076:Transgenes; D014404:Tubulin / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D020418:Algal Proteins; D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D014764:Viral Proteins; C099353:phleomycin resistance protein, Streptoalloteichus hindustanus; C057421:virus protein 2A