Chlamydomonas Engineering Kits

The GeneArt® Chlamydomonas Engineering Kits employ a eukaryotic protein expression system, based on the green alga Chlamydomonas reinhardtii, that offers a simplified approach for metabolic engineering, protein expression, and downstream applications such as production of biofuels, specialty chemicals, and nutraceuticals. Using the optimized cloning and expression vectors, frozen cells, specially formulated Gibco® medium, and easy-to-follow protocols, growth and transformation of C. reinhardtii is straightforward (Figure 1), which helps to ensure reliable results and faster scale-up.

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Characteristics of C. reinhardtii

The unicellular green alga Chlamydomonas reinhardtii has served as a genetic workhorse and model organism for understanding everything from the mechanisms of light- and nutrient-regulated gene expression to the assembly and function of flagella. Green algae are used as platforms for the production of biofuels and other bioproducts, mainly because of their rapid growth and ability to convert sunlight and CO2 to energy. In addition, C. reinhardtii is categorized as GRAS (generally recognized as safe) by the USDA. That, along with these features, makes it a particularly attractive system for the expression of recombinant proteins:

  • Fully Sequenced and Annotated Genome
  • Short time between the generation of initial transformants and scale-up to production volumes
  • Ability to grow phototrophically or heterotrophically
  • Ability to grow cultures at scales ranging from a few milliliters to 500,000 L, in a cost-effective manner
  • Well-characterized mating system, making it possible to carry out classical breeding

 

 Figure 1: Frozen C. reinhardtii cells were resuscitated in Gibco® TAP medium, in triplicate samples. Growth was monitored by OD750 measurements taken periodically during the study.

Achieve Production Targets Sooner

Compared to land plants, C. reinhardtii grows at a much faster rate, doubling cell numbers in approximately 8 hours under heterotrophic growth conditions and 12 hours under photosynthetic growth conditions.

As C. reinhardtii propagates vegetatively, the time from initial transformation to product production is significantly reduced relative to plants, requiring as little as 6 weeks to evaluate production at flask scale, with the potential to scale up to 64,000 L in 4 to 6 weeks.

C. reinhardtii Cloning Vectors

We have developed two C. reinhardtii expression vectors: pChlamy_1, which contains a standard multiple cloning site (MCS), and pChlamy_1/D-TOPO®, which allows you to carry out rapid and efficient directional cloning of a blunt-end PCR product using our proven TOPO® cloning technology.

Our C. reinhardtii vectors contain:

  • Hsp70A-RbcS2 constitutive promoter for expression of the gene of interest
  • Cop1 3´ UTR
  • Directional TOPO® Cloning site for rapid and efficient directional cloning of a blunt-end PCR product
  • Hygromycin resistance gene driven by the beta-tubulin promoter for selection in C. reinhardtii
  • Ampicillin resistance gene for selection in E. coli
  • pUC origin for maintenance in E. coli

Ethanol Production from the GeneArt® Chlamydomonas Engineering Kit

To test the expression obtained using the GeneArt® Chlamydomonas Engineering Kit, an ethanol production system was used to transform C. reinhardtii. After 5 days, ethanol production in the engineered strain was dramatically improved compared to the untransformed strain (Figure 2).

 Figure 2: Ethanol production in C. reinhardtii engineered with GeneArt® Algae Engineering Kits. The adh1 alcohol dehydrogenase gene was PCR-amplified from Saccharomyces cerevisiae, cloned into the pChlamy_1/D-TOPO® vector, and propagated in OneShot® TOP10 E. coli competent cells. The plasmid was used to transform GeneArt® C. reinhardtii cells that were grown and prepared for transformation using GeneArt® Algae Engineering Kit protocols. Transformants were selected on Gibco® TAP Medium plates containing hygromycin. Colonies were innoculated into 4 mL Gibco TAP medium containing hygromycin and grown for 5 days, as specified in the GeneArt® kit protocol. Ethanol production was measured by enzymatic assay. The data above are representative of production from a single wild type culture and a single engineered culture.
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