The aim & rationale
Unicellular algae form a powerful biotechnological warehouse for production of chemical compounds and a valuable platform for synthetic biology. However, algae commercial potential can be fully revealed only after deepening the understanding of their fundamental processes. Triacylglycerols (TAGs) are lipids that can be used as the precursors in biodiesel production. Model green alga, Chlamydomonas reinhardtii, accumulates TAGs in stress conditions, like nitrogen deprivation. TAGs accumulation is reversible and chromatin-based, features that make this process a potential subject for stress priming - a phenomenon where repeated stress exposure leads to amplification of the response comparing to the 1st/single stress exposure (conceptually similar to vaccination). Stress priming was not explored in algae, even though it offers a tool to manipulate algal biofuel production!
We proposed to establish and optimize protocols for algal growth and TAGs’ accumulation in stress priming setup using a histochemical, chromatographic and microscopic approach. The aim of the project was to decipher whether stress priming exists in simple eukaryotes and whether it can be used for biotechnological/synthetic biology purpose.
The project
We undertook the following steps: 1) Set up stress priming growth conditions, including the selection of proper harvesting timepoints and preservation of harvested material; 2) Implement and optimize simple TAGs staining protocol using Nile Red dye; 3) Acquire images using confocal microscope; 4) Quantify dye intensity, normalize to cell size and extrapolate to TAGs accumulation strength (Nile Red fluorescence is tightly correlated with TAGs content); 5) Obtain chromatographic profile of accumulated TAGs using GC-MS.
Figure 1: TAG-stained C. reinhardtii cells with Nile Red (green/yellow). Red corresponds to the autofluorescence.
After some optimization, we found out that some fiddling around with recovery time, staining protocol and extraction are needed (see the link to our Hackster page below), but overall, the plan worked. We obtained some beautifully stained TAGs in alga cells and sought to properly quantify fluorescence intensity with normalizations to account for the background noise and batch variation. Finally, we were able to properly compare stress-primed samples and single-stress controls. Long story short, we saw that… stress priming indeed occurred in the conditions used for TAGs’ accumulation in model green alga! However, to our surprise the dynamics were opposite to expected – stress priming caused TAGs’ downregulation, instead of upregulation!
We wanted to study the phenomenon further and understand lipid type composition in our samples. To this end, we employed gas chromatography. Despite quantitative difference in TAGs accumulation, stress-primed cells did not show qualitative difference in lipid type profile. However, we did observe that stress samples in general exhibited lower lipid complexity than non-stressed controls.
Future outlook
Our results show that stress priming exists in simple alga. Since stress treatment is frequently employed for biosynthesis of valuable bioproducts like biofuel, antioxidants or nutraceuticals, this phenomenon can be used for biotech & synthetic approaches.
As for direct outcomes, our Hackster page contains protocols and descriptions for procedure optimization, that can be used by DIY biotechnologists. We are also drafting a small paper for the broader community. Alga growth and staining procedures can be performed in a cost-effective manner and expensive equipment (we admit we used it) can be replaced by cheaper alternatives developed in other Biomaker projects (DIY microscopes and bioreactors). The Biomaker challenge opened us to new ideas and is worth a recommendation to all the curious people!
Acknowledgements
We would like to thank the organizers for making this experience happen and Andy Truman (John Innes Centre, Norwich) for being our kind host of the grant.
Please check out our hackster page to learn more about the project.
Pawel Mikulski and Javier Santos Aberturas, John Innes Centre.