Spheroplasted cells: a game changer for DNA delivery to diatoms
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Abstract
Diatoms, vital to global carbon fixation and climate change mitigation, produce 20% of the world’s fixed organic carbon annually. 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 the biggest challenge. Here, we present an optimized, highly efficient electroporation method for delivering DNA constructs as large as 55.6 kb to Phaeodactylum tricornutum , a model diatom species and emerging chassis for algal biotechnology. We also demonstrate that with this optimized protocol, episomes can be assembled de novo , forgoing the need for time-consuming traditional cloning steps in Escherichia coli and Saccharomyces cerevisiae . By incorporating other technologies, such as CRISPR genome editing, this method will accelerate diatom-based synthetic biology projects and, therefore, the development of sustainable technologies. This method should also be applicable to other diatom species.
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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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