Improved and versatile viral 2A platforms for dependable and inducible high‐level expression of dicistronic nuclear genes in Chlamydomonas reinhardtii

Abstract

SUMMARYA significantly improved viral 2A peptide system for dependable high-level expression of dicistronic genes in Chlamydomonas reinhardtii has been developed. Data are presented demonstrating that use of an especially proficient 'extended FMDV 2A' coding region allows production of two independent protein products from a dicistronic gene with almost complete efficiency. Importantly, results are also presented that demonstrate the utility of this 2A system for efficient high-level expression of foreign genes in C. reinhardtii, which has not previously been reliably achievable in this algal model system. To expand the versatility of the 2A expression system, a number of commonly used selectable marker proteins were assessed for their compatibility with the extended FMDV 2A peptide. Additional experiments demonstrate the feasibility and utility of 2A-containing dicistronic systems that rely on a strong conditional promoter for transcriptional control and a low-expression marker gene for selection. This strategy allows easy and efficient delivery of genes of interest whose expression levels require regulation either to mitigate potential toxicity or allow differential expression under controlled experimental conditions. Finally, as an additional practical demonstration of the utility of the extended FMDV 2A system, confocal fluorescence microscopy is used to demonstrate that native and foreign proteins of interest bearing post-translational remnants of the extended FMDV 2A peptide localize correctly to various cellular compartments, including a striking demonstration of the almost exclusive localization of the Rubisco small subunit protein to the pyrenoid of the C. reinhardtii chloroplast in cells maintained under ambient CO 2 concentrations.

A toolkit for Nannochloropsis oceanica CCMP1779 enables gene stacking and genetic engineering of the eicosapentaenoic acid pathway for enhanced long‐chain polyunsaturated fatty acid production

“…A variety of 2A peptides have been identified and exploited with differing levels of efficiency in a number of eukaryotes, including yeast (Doronina et al . ), animals (Kim et al ., ; Szymczak et al ., ), plants (Burén et al ., ), insects (Wang et al ., ) and algae (Plucinak et al ., ; Rasala et al ., ). Applying this approach to stramenopiles, we have successfully developed an extended P2A sequence that is efficient for peptide bond skipping in N. oceanica CCMP1779.…”

A multidomain enzyme, with glycerol‐3‐phosphate dehydrogenase and phosphatase activities, is involved in a chloroplastic pathway for glycerol synthesis in Chlamydomonas reinhardtii

“…The PCR fragments were inserted into the pQU vector, to generate a translational fusion with the mCherry CDS expressed from the PsaD promoter. This plasmid also contains the Ble r gene for zeocin resistance (Plucinak et al, 2015). The transgenes were transformed into CC-124 by electroporation, as previously described (Plucinak et al, 2015), and colonies selected on TAP-agar plates containing 16 lg mL À1 zeocin.…”

“…This plasmid also contains the Ble r gene for zeocin resistance (Plucinak et al, 2015). The transgenes were transformed into CC-124 by electroporation, as previously described (Plucinak et al, 2015), and colonies selected on TAP-agar plates containing 16 lg mL À1 zeocin. To examine the subcellular localization of the fusion proteins, images of Chlamydomonas cells were captured using a Nikon A1 confocal imaging system mounted on a Nikon Eclipse 90i microscope with a 9100 objective.…”

The Synthetic Biology Toolkit for Photosynthetic Microorganisms

“…Coordinated transcription of multiple genes can be achieved through use of the foot-and-mouth-diseasevirus 2A peptide, which allows cistron-like gene expression in eukaryotes and was found to be functional in Chlamydomonas (Rasala et al, 2012;Plucinak et al, 2015;López-Paz et al, 2017). To control gene expression posttranscriptionally, two other versatile genetic parts were characterized: riboswitches and artificial microRNA (miRNA).…”