Breaking the cell wall for efficient DNA delivery to diatoms

breaking-the-cell-wall-for-efficient-dna-delivery-to-diatoms
Breaking the cell wall for efficient DNA delivery to diatoms

References

  1. Spoehr, H. A. Chlorella as a Source of Food. Proc. Am. Philos. Soc. 95, 62–67 (1951).

    Google Scholar 

  2. Malviya, S. et al. Insights into global diatom distribution and diversity in the world’s ocean. Proc. Natl. Acad. Sci. 113, E1516–E1525 (2016).

    Google Scholar 

  3. Field, C. B., Behrenfeld, M. J., Randerson, J. T. & Falkowski, P. Primary production of the biosphere: integrating terrestrial and oceanic components. Science 281, 237–240 (1998).

    Google Scholar 

  4. Giguere, D. J. et al. Telomere-to-telomere genome assembly of Phaeodactylum tricornutum. PeerJ 10, e13607 (2022).

    Google Scholar 

  5. Zhang, C. & Hu, H. High-efficiency nuclear transformation of the diatom Phaeodactylum tricornutum by electroporation. Mar. Genom. 16, 63–66 (2014).

    Google Scholar 

  6. Karas, B. J. et al. Designer diatom episomes delivered by bacterial conjugation. Nat. Commun. 6, 6925 (2015).

    Google Scholar 

  7. Miyahara, M., Aoi, M., Inoue-Kashino, N., Kashino, Y. & Ifuku, K. Highly efficient transformation of the diatom Phaeodactylum tricornutum by multi-pulse electroporation. Biosci. Biotechnol. Biochem. 77, 874–876 (2013).

    Google Scholar 

  8. Apt, K. E., Grossman, A. R. & Kroth-Pancic, P. G. Stable nuclear transformation of the diatomPhaeodactylum tricornutum. Mol. Gen. Genet. 252, 572–579 (1996).

    Google Scholar 

  9. Slattery, S. S. et al. An expanded plasmid-based genetic Toolbox Enables Cas9 genome editing and stable maintenance of synthetic pathways in Phaeodactylum tricornutum. ACS Synth. Biol. 7, 328–338 (2018).

    Google Scholar 

  10. Slattery, S. S. et al. Phosphate-regulated expression of the SARS-CoV-2 receptor-binding domain in the diatom Phaeodactylum tricornutum for pandemic diagnostics. Sci. Rep. 12, 7010 (2022).

    Google Scholar 

  11. Cochrane, R. R. et al. Rapid method for generating designer algal mitochondrial genomes. Algal Res. 50, 102014 (2020).

    Google Scholar 

  12. Walker, E. J. L., Pampuch, M., Chang, N., Cochrane, R. R. & Karas, B. J. Design and assembly of the 117-kb Phaeodactylum tricornutum chloroplast genome. Plant Physiol. 194, 2217–2228 (2024).

    Google Scholar 

  13. Russo, M. T., Rogato, A., Jaubert, M., Karas, B. J. & Falciatore, A. Phaeodactylum tricornutum: An established model species for diatom molecular research and an emerging chassis for algal synthetic biology. J. Phycol. 59, 1114–1122 (2023).

    Google Scholar 

  14. Pampuch, M., Walker, E. J. L. & Karas, B. J. Towards synthetic diatoms: The Phaeodactylum tricornutum Pt-syn 1.0 project. Curr. Opin. Green. Sustain. Chem. 35, 100611 (2022).

    Google Scholar 

  15. Taparia, Y. et al. A novel endogenous selection marker for the diatom Phaeodactylum tricornutum based on a unique mutation in phytoene desaturase 1. Sci. Rep. 9, 8217 (2019).

    Google Scholar 

  16. Walker, E. J. L., Jaafar, T., Kaneshan, A. & Karas, B. J. Resolving replication incompatibility between chloroplast and conjugative plasmids in E. coli. 2025.10.16.682956 Preprint at https://doi.org/10.1101/2025.10.16.682956 (2025).

  17. Kindle, K. L. High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA (1990).

  18. Kassaw, T. K., Paton, A. J. & Peers, G. Episome-based gene expression modulation platform in the model diatom Phaeodactylum tricornutum. ACS Synth. Biol. 11, 191–204 (2022).

    Google Scholar 

  19. Martino, A. D., Meichenin, A., Shi, J., Pan, K. & Bowler, C. Genetic and phenotypic characterization of Phaeodactylum tricornutum (Bacillariophyceae) accessions. J. Phycol. 43, 992–1009 (2007).

    Google Scholar 

  20. Song, Z., Lye, G. J. & Parker, B. M. Morphological and biochemical changes in Phaeodactylum tricornutum triggered by culture media: Implications for industrial exploitation. Algal Res. 47, 101822 (2020).

    Google Scholar 

  21. Avasthi, P., MacQuarrie, C. D. & Radkov, A. Inducing protoplast formation in Phaeodactylum tricornutum by silica deprivation, enzymatic treatment, or cytoskeletal inhibition. The Stacks https://doi.org/10.57844/arcadia-fh8f-xz51 (2023)

  22. Ohse, M., Takahashi, K., Kadowaki, Y. & Kusaoke, H. Effects of plasmid DNA sizes and several other factors on transformation of Bacillus subtilis ISW1214 with plasmid DNA by electroporation. Biosci. Biotechnol. Biochem 59, 1433–1437 (1995).

    Google Scholar 

  23. Szostková, M. & Horáková, D. The effect of plasmid DNA sizes and other factors on electrotransformation of Escherichia coli JM109. Bioelectrochem. Bioenerg. 47, 319–323 (1998).

    Google Scholar 

  24. Cochrane, R. R. et al. Superior conjugative plasmids delivered by bacteria to diverse fungi. BioDesign Res. 2022, 9802168 (2022).

    Google Scholar 

  25. Gibson, D. G. Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides. Nucleic Acids Res 37, 6984–6990 (2009).

    Google Scholar 

  26. Almagro Armenteros, J. J. et al. Detecting sequence signals in targeting peptides using deep learning. Life Sci. Alliance 2, e201900429 (2019).

    Google Scholar 

  27. Diamond, A. et al. Instability of extrachromosomal DNA transformed into the diatom Phaeodactylum tricornutum. Algal Res. 70, 102998 (2023).

    Google Scholar 

  28. Serif, M. et al. One-step generation of multiple gene knock-outs in the diatom Phaeodactylum tricornutum by DNA-free genome editing. Nat. Commun. 9, 3924 (2018).

    Google Scholar 

  29. Gietz, R. D., Schiestl, R. H., Willems, A. R. & Woods, R. A. Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11, 355–360 (1995).

    Google Scholar 

  30. Hwang, H.-J., Kim, Y. T., Kang, N. S. & Han, J. W. A simple method for removal of the chlamydomonas reinhardtii cell wall using a commercially available Subtilisin (Alcalase). J. Mol. Microbiol. Biotechnol. 28, 169–178 (2018).

    Google Scholar 

  31. Woo, J. W. et al. DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nat. Biotechnol. 33, 1162–1164 (2015).

    Google Scholar 

  32. Borowitzka, M. A. & Volcani, B. E. The Polymorphic Diatom Phaeodactylum Tricornutum: Ultrastructure Of Its Morphotypes. J. Phycol. 14, 10–21 (1978).

    Google Scholar 

  33. Yin, W., Zhang, Y., Huang, Q., Pan, Y. & Hu, H. Genetic transformation of the freshwater diatom Cyclotella meneghiniana via bacterial conjugation. Algal Res. 84, 103794 (2024).

    Google Scholar 

  34. Sprecher, B. N. et al. Genetic transformation methods for diatom Nitzschia captiva: New tools to better understand dinotom endosymbiosis. Algal Res. 72, 103136 (2023).

    Google Scholar 

  35. Maeda, Y., Nakamura, M., Watanabe, K., Okamoto, E. & Tanaka, T. Functional analysis of the putative centromere sequences of marine oleaginous diatom Fistulifera solaris. Algal Res. 74, 103225 (2023).

    Google Scholar 

  36. Angstenberger, M., Krischer, J., Aktaş, O. & Büchel, C. Knock-down of a ligIV homologue enables DNA integration via homologous recombination in the marine diatom Phaeodactylum tricornutum. ACS Synth. Biol. 8, 57–69 (2019).

    Google Scholar 

  37. Slattery, S. S. et al. Plasmid-based complementation of large deletions in Phaeodactylum tricornutum biosynthetic genes generated by Cas9 editing. Sci. Rep. 10, 13879 (2020).

    Google Scholar 

  38. Moosburner, M. A. et al. Multiplexed knockouts in the model diatom Phaeodactylum by episomal delivery of a selectable Cas9. Front Microbiol 11, 5 (2020).

    Google Scholar 

  39. Karas, B. J. et al. Transferring whole genomes from bacteria to yeast spheroplasts using entire bacterial cells to reduce DNA shearing. Nat. Protoc. 9, 743–750 (2014).

    Google Scholar 

  40. Rice, P., Longden, I. & Bleasby, A. EMBOSS: The European Molecular Biology Open Software Suite. Trends Genet. 16, 276–277 (2000).

    Google Scholar 

  41. Sayers, E. W. et al. Database resources of the National Center for Biotechnology Information. Nucleic Acids Res. 50, D20–D26 (2022).

    Google Scholar 

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