Spheroplasted cells: a game changer for DNA delivery to diatoms
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Abstract
Diatoms produce 20% of the world’s fixed organic carbon annually, making them vital to global carbon fixation and climate change mitigation. Their potential as cell factories for biofuels, proteins, and other high value chemicals remains underutilized due to a lack of genetic engineering tools, with DNA delivery being one of the biggest challenges. Here, we present optimized electroporation and polyethylene glycol transformation methods for delivering DNA and ribonucleoprotein complexes to Phaeodactylum tricornutum , a model diatom species and emerging chassis for algal biotechnology. It was possible to recover transformants with as little as 1 ng of DNA, and to transform linear or circular episomes as large as 55.6 kb. With the optimized electroporation protocol, episomes can be assembled in the algal cell de novo through diatom in vivo assembly (DIVA), forgoing the need for time-consuming traditional cloning steps in Escherichia coli and Saccharomyces cerevisiae . It was also possible to electroporate a Cas9 ribonucleoprotein complex in P. tricornutum , providing an alternative to biolistics for DNA free genome engineering. We have demonstrated that the PEG approach can be adapted to successfully transform Thalassiosira pseudonana , demonstrating the applicability of our methods for engineering other diatom species. These tools can be used to accelerate diatom synthetic biology projects and, therefore, the development of sustainable technologies.
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this article presents an optimized electroporation method for high efficiency transformation of P. tricornutum, an emerging diatom chassis for algal synthetic biology (Supplementary Fig. S7)
Thank you for assembling this clear guide to an efficient and easier way to do Phaeodactylum transformations! We're so excited to see that the alcalase-induced protoplast method is reproducible and being used in a really important way! Super excited to try out these methods in our lab! Thank you for sharing!
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Following electroporation, half of the total reaction was plated on ¼-salt L1 plates supplemented with 100 µg/ml nourseothricin. (d)
It seems the 1/2 salt L1 plates were previously used to allow for bacterial conjugation, but it is less clear why reduced salt (1/2 or 1/4) plates were used in these electroporation experiments?
Is it because of the protoplast's sensitivity to osmotic stress? or do you used reduced salt in the solid media plates to prevent the crashing out/precipitation that frequently occurs when making L1 agar plates?
I'm essentially wondering if the low salt is addressing a physiological concern for the cells or a technical issue in the lab.
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