Fluorescence microscopy of Chlamydomonas reinhardtii for mCherry detection: secretion peptides strains.

Overview

Life-cell imaging was performed with a confocal fluorescence microscopy to observe mCherry in the secretion vacuoles. mCherry fluorescence compartmentalization was observed by a Confocal Zeiss LSM 780-NLO, using an argon laser 543 nm to excite mCherry and a spectral detector set approximately to 610-650 nm range. For chlorophyll, we used a laser at 405 nm for excitation, and spectral detector set to 680 nm region. All pictures were taken with the same system configuration and analyzed by Fiji, an ImageJ distribution software. Cells images were acquired in bundles of 0.4 μm afar photos per channel in the z-axis. 

Files info:

Each file is the raw image obtained from fluorescent microscopy.

Organization

Construct_name.czi - Ex: "pAH04mCherry.czi"

pAH04mCherry -> construct without signal peptide

pJP22mCherry -> construct with signal peptide from arylsulfatase 1 (Chlamydomonas reinhardtii)

pJP26mCherry -> construct with signal peptide from binding protein 1 (C. reinhardtii)

pJP28mCherry -> construct with signal peptide from carbonic anhydrase 1 (C. reinhardtii)

pJP29 mCherry -> construct with signal peptide from ice-binding protein 1 (Artic Chlamydomonas sp)

pJP30-35mCherry -> construct with signal peptide from in silico identified list (DOI 10.5281/zenodo.556792).

Wildtype cc1690 -> parental strain used for transformation.

For more information on the constructs, check our paper. 

Consider citing our work. 

Molino JVD, de Carvalho JCM, Mayfield SP (2018) Comparison of secretory signal peptides for heterologous protein expression in microalgae: Expanding the secretion portfolio for Chlamydomonas reinhardtii. PLoS ONE 13(2): e0192433. https://doi.org/10.1371/journal. pone.0192433