Nannochloropsis transformation methods

Electroporation

Electroporation is the most widely utilized technique to introduce DNA for genetic transformation in Nannochloropsis. Preparation of DNA usually involves linearization of plasmid constructs with restriction enzymes and concentration by column cleanup or alcohol precipitation. Fresh log phase cells work best, optimal cultures free debris are produced by diluting the cultures with fresh media daily with supernatant transfer to new vessels when debris collect. Cells must be washed to remove salts, it is unclear if using cold or room temperature sorbitol washes enhances transformation. Recovery of 12 to 48 hours is used, 12 hours for resistance develop seems to be adequate. Longer recovery may be preferred when using CRISPR to give time for mutations to arise before plating, antibiotic selection can be added to the recovery tube.

Conjugation

Conjugation between E. coli and algae has recently been conducted, with green algae, diatoms, and Nannochloropsis. Conjugation between E coli and Nannochloropsis and diatoms results in episomal expression.

Insertion versus episomal

Nannochloropsis is able to maintain episomal DNA, usually containing the S. cerevisiae CEN/ARS region. Episomal DNA is transformed circular plasmid DNA and is maintained with antibiotic selection. An episome is usually lost without selection pressure but segregation efficiencies of 97% have been reported in N. oceanica. The mechanisms of episome replication and segregation are still unknown.

Several studies have now maintained episomes in stramenopiles but green algae do not seem to have the capability.