The GeneArt® Chlamydomonas Protein Expression Kit is a second-generation Chlamydomonas cloning and expression system from Life Technologies. Like the first-generation GeneArt® Chlamydomonas Engineering Kit, this new system offers transgene expression from the nuclear genome of eukaryotic green alga Chlamydomonas reinhardtii 137c, but is optimized for high-level expression, provides selection against gene silencing, and offers dual protein tags for detection and/or purification of your gene of interest. It includes frozen Chlamydomonas reinhardtii 137c cells, MAX Efficiency® Transformation Reagent for Algae, expression vector, OneShot® TOP10 Competent E. coli cells, and easy-to-follow protocols. Our Gibco® TAP Growth Media, offered separately, is optimized for the growth and maintenance of Chlamydomonas.
- Express up to 1% total soluble protein of your gene of interest
- Select against gene silencing, even over multiple passages
- Detect and purify your gene of interest with 6His TEV and/or V5-His epitope tags
- Use (optional) seamless assembly to create your constructs
- Enables reliable results with exceptional strain viability and purity
Better expression, reduced silencing, and a choice of purification tags
Transgene expression from the Chlamydomonas nuclear genome via the pChlamy_4 vector offers several advantages over chloroplast expression, such as post-translational modifications and protein targeting and/or secretion.
- Endogenous and constitutive promoter, RbcS2, is combined with activator, Hsp70A, resulting in increased expression of your gene of interest
- Fusion of your gene of interest to bleomycin/zeocin resistance gene, sh-ble, circumvents silencing
- Self-cleaving sequence for the 2A peptide from foot-and-mouth disease virus (FMDV) mediates proper cleavage between resistance markers and protein of interest to yield two discrete proteins
- 3’ UTR for proper transcript termination and possible additional benefits like increased translation efficiency, mRNA stability, and polyadenylation signals
- Dual protein tags 6His TEV and V5-His epitopes can be fused to both or either ends of your gene of interest, or you can elect to have no tag at all
- Use (optional) seamless assembly to create your constructs
The gene for the hydrolytic enzyme, xylanase (xyn1), from Trichoderma reesei, was cloned into vector pChlamy_4 and transformed into Chlamydomonas reinhardtii 137c. Xylanase activity was measured using the EnzChek® Ultra Xylanase Assay Kit (Cat. No. E33650). Xylanase activity levels from the pChlamy_4 constructs ('Prot Exp') were 17-fold over that observed with our first-generation GeneArt® Algae Engineering Kit systems ('Genetic Eng').
The gene for the hydrolytic enzyme, xylanase (xyn1), from Trichoderma reesei, was cloned into vector pChlamy_4 and transformed into Chlamydomonas reinhardtii 137c. Xylanase expression was measured by western blot analysis, and the results showed xylanase protein represented approximately 1% of the total soluble protein.
Selection against silencing
In order to circumvent the transgene silencing that often occurs in Chlamydomonas reinhardtii, our new pChlamy_4 vector is designed so proteins are expressed as transcriptional fusions with the bleomycin/zeocin resistance gene sh-ble (Rasala et al., 2012). The self-cleaving sequence for the 2A peptide from the foot-and-mouth disease virus (FMDV) is placed between the antibiotic resistance gene and the gene of interest. It encodes a short ~20 amino acid sequence that mediates proper cleavage to yield two discrete proteins. With this system we have seen positive transformants maintain high-expression levels for much longer than with other systems, even after many passages with or without selection pressure (see figure below).
The gene for the hydrolytic enzyme, xylanase (xyn1), from Trichoderma reesei, was cloned into vector pChlamy_4 and transformed into Chlamydomonas reinhardtii 137c. Xylanase activity was measured daily for 2 weeks using the EnzChek® Ultra Xylanase Assay Kit (Cat. No. E33650) and was compared to expression levels when the gene was expressed in other systems. Fusion of the xylanase gene bleomycin/zeocin resistance gene, sh-ble, in pChlamy_4 vector, circumvents silencing so proteins are expressed through many cell passages with or without selection pressure.
Enhanced transfection efficiency for Chlamydomonas
One of the biggest hurdles in research and development with Chlamydomonas has been the introduction of exogenous DNA into Chlamydomonas strains due to rigid cell walls. Methods such as glass bead agitation, electroporation, and microparticle bombardment are available but often result in low transformation efficiency. MAX Efficiency® Transformation Reagent for Algae, when used to pretreat the cells prior to electroporation, enhances transformation efficiency for multiple strains of Chlamydomonas species. It increases permeability of the Chlamydomonas cell wall and facilitates increased delivery of DNA into the cell nucleus by electroporation. To date, we have seen a >200-fold increase in transformation efficiency, over previously recommended transformation conditions, in 10 different Chlamydomonas strains, including wild type and mutants, using circular or linear DNA as well as PCR fragments.
Gibco® TAP Growth Media—optimized for Chlamydomonas
Gibco® TAP Growth Media, offered separately or in some configurations of our GeneArt® kits, is optimized for the growth and maintenance of Chlamydomonas. The 1X formulation lets you avoid laborious media preparation steps. The award-winning bottle design makes manipulation in the biosafety cabinet easier, minimizes the risk of contamination, and helps you perform cell culture more consistently. Superior packaging and quality, greater reliability, and improved consistency in Chlamydomonas culture results in better overall efficiency and more robust data.
Versatile and proven algal model organism
Chlamydomonas reinhardtii is a freshwater, green microalga that has been a popular model organism for physiological, molecular, biochemical, and genetic studies and has recently gained attention as a platform for the production of therapeutic proteins and vaccines. It has served as a genetic workhorse and model organism for understanding everything from mechanisms of photosynthesis and nutrient-regulated gene expression, to the assembly and function of flagella. Green algae are used as a platform for the production of biofuel and bioproducts, due mainly to their rapid growth and ability to convert sunlight and C02 to energy. As C. reinhardtii propagates by vegetative division, the time from initial transformation to product production is significantly reduced relative to plants, requiring as little as six weeks to evaluate production at flask scale, with the potential to scale up to 64,000 liters in four to six weeks.
First-generation Chlamydomonas Engineering Kits