Simplified Transformation of Ostreococcus tauri Using Polyethylene Glycol

Abstract

Ostreococcus tauri is an easily cultured representative of unicellular algae (class Mamiellophyceae) that abound in oceans worldwide. Eight complete 13–22 Mb genomes of phylogenetically divergent species within this class are available, and their DNA sequences are nearly always present in metagenomic data produced from marine samples. Here we describe a simplified and robust transformation protocol for the smallest of these algae (O. tauri). Polyethylene glycol (PEG) treatment was much more efficient than the previously described electroporation protocol. Short (2 min or less) incubation times in PEG gave >104 transformants per microgram DNA. The time of cell recovery after transformation could be reduced to a few hours, permitting the experiment to be done in a day rather than overnight as used in previous protocols. DNA was randomly inserted in the O. tauri genome. In our hands PEG was 20–40-fold more efficient than electroporation for the transformation of O. tauri, and this improvement will facilitate mutagenesis of all of the dispensable genes present in the tiny O. tauri genome.

Combining Nanopore and Illumina Sequencing Permits Detailed Analysis of Insertion Mutations and Structural Variations Produced by PEG-Mediated Transformation in Ostreococcus tauri

“…To determine the integrity of the transformed DNA, we measured the coverage of the Illumina read pairs that mapped to the whole pOLK4 vector ( Figure 1 C). Note that the pOLK4 construct used for this study contained sequences from O. tauri that were expected to recruit genomic reads ( Figure 1 ): a ubiquitin promoter sequence found on RCC1115 chromosome 12 (position 173,766–175,000 bp), as well as α-tubulin promoter and terminator sequences (position 119,519–120,967 bp) from the same chromosome [ 15 ]. Reads from both NT1 and NT10 untransformed lines mapped only to the regions of the vector-encoding promoter and terminator sequences, confirming the control lines did not contain the vector sequence.…”

“…Insertional mutagenesis was performed by transformation using polyethylene glycol (PEG), as described in [ 15 ]. For the transformation experiments in this study, 50 mL of culture at a density of 50 × 10 6 cells·mL −1 was used, in order to obtain at least 2000 transformed clones.…”

“…The culture was centrifuged for 10 min at 6000× g at 20 °C, and the pellet was resuspended and gently mixed with 500 µL PEG 4000 (30% PEG final concentration), and 2 µg carrier tRNA. Then, 10 µg of ScaI-digested pOLK4 DNA [ 15 ] was added, followed by incubation of the mixture for 2 min at 20 °C. The cells were then diluted into 40 mL of fresh L1 medium, and transferred to a growth chamber for 6 h to maximize the frequency of transformation.…”

“…O. tauri was shown to stably integrate an overexpression plasmid [ 13 ] and was amenable to targeted insertion by homologous recombination by electroporation-mediated transformation [ 14 ]. Recently, a simplified method to transform O. tauri using polyethylene glycol (PEG) has been described [ 15 ]. This approach was first presented in the tobacco plant [ 16 ], and in yeast and bacterial models [ 17 , 18 , 19 ], as well as in protoplasts [ 20 , 21 , 22 ].…”

A marineChlamydomonassp. emerging as an algal model

“…2014, Sanchez et al. 2019), as well as the extreme halotolerant cell wall‐less green alga Dunaliella salina , which lives in sea salt fields (Song et al. 2019); and the green macroalga Ulva mutabilis , which relies on biotic interactions with bacteria to shape its structure (Oertel et al.…”