Paper

Transformation of plasmid DNA into Prochlorococcus via electroporation

Capovilla, Giovanna, Ziegler, Christine A., Castro, Kurt G., Chisholm, Sallie W.

2025

Overview

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.

• Publisher: Cold Spring Harbor Laboratory
• Subject: Molecular Biology