The transformation of the Nannochloropsis oceanica cells by electroporation was performed using the pSELECT100 transformation plasmid containing the hygromycin resistance cassette.
Nannochloropsis oceanica CCMP1779 grew to a cell density of 3-8×106 cells/mL (competent cells). A total of 1-5×106 cells were harvested by centrifugation (3700 × g) for 10-15 min at 4°C. Collected cells were washed 2-3 times with 375 mM solution of sorbitol. The washed cells were distributed into aliquots in 2 mm electroporation cuvettes. An aliquot of 100 µL cells (5×107-108 cells) was incubated with plasmid amount ranging between 1.2–4.0 µg. All the washing steps, concentrating and distribution of the cells were conducted on ice.
Electroporation was performed with Bio-Rad Gene Pulser II Electroporation System, which was adjusted to 500 Ohm shunt resistance, 25 µF capacitance and field strength of various voltage ranging from 1100 to 2200 V. After electroporation, the cells were immediately collected from the electroporation cuvettes and transferred to 10 mL Cell-Hi CN medium. Subsequent incubation of the transformed cells was performed overnight at 23°C, constant light intensity of 100 µE m−2 s−1 and constant rotational shaking. Afterwards, the cells were collected by centrifugation (3700 × g) for 10 min at room temperature and resuspended in 1 mL of the left Cell-Hi CN medium. 70 µL of the cell resuspension were plated out on the agar-solidified Cell-Hi CN medium containing 50 µg/mL of hygromycin.
Result: The most efficient transformation was achieved when applying 1800 V and adding low amounts of the transformation plasmid (1-2 µg).