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bioRxiv · 10.1101/2025.09.20.677525

Transformation of plasmid DNA into Prochlorococcus via electroporation

Capovilla, G.·Castro, K. G.·Ziegler, C. A.·Chisholm, S. W.
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Abstract

Prochlorococcus is the most numerically abundant photosynthetic organism in the oceans and plays a role in global carbon cycling. Despite its ecological significance and the availability of over a thousand assembled genomes, progress in understanding gene function has been limited by the lack of genetic tools. Here, we report a reproducible electroporation-based protocol to introduce replicative plasmids into two strains of Prochlorococcus representing different ecotypes: MIT9313 (low-light adapted) and MED4 (high-light adapted). Using plasmids carrying a spectinomycin resistance cassette, we achieved transformation in ~33% of MED4 and ~10% of MIT9313 attempts, with greatest success when electroporating cells in late exponential phase. Transformed cells stably retained plasmids and expressed resistance genes, demonstrating functional uptake and gene expression. We also delivered a modified 13 kb plasmid carrying a CRISPR-Cpf1 system into MED4. While no targeted edits were observed, cpf1 and specR were expressed, indicating successful delivery of large constructs and active transcription. These findings represent a key step toward genetic manipulation of Prochlorococcus, enabling future optimization of gene editing approaches and deeper functional analysis of its vast and largely uncharacterized pangenome.

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Capovilla, G., Castro, K. G., Ziegler, C. A., Chisholm, S. W.. 2025-09-21. Transformation of plasmid DNA into Prochlorococcus via electroporation. https://doi.org/10.1101/2025.09.20.677525

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