The transformation was performed with pLit_chlL_chlor transformation plasmid containing chloramphenicol resistance cassette to delete the endogenous chlL gene (chloroplast genome) of Nannochlropsis oceanica.
Cell culture
The cells of N. oceanica CCMP1779 were cultivated in the Cell-Hi CN medium at 23°C at constant light intensity of 100 µE m-2 s-1 and continuous rotational shaking. After reaching the exponential growth phase concentration (2-4×106 cells/mL), the Nannochloropsis cells were harvested using centrifugation at 3000 × g for 10 min at 22°C. Various cell concentrations were spread in a circle with a ~5 cm diameter on agar-solidified Cell-Hi CN medium 24 h prior to the biolistic transformation. Two plates contained 5×107 cells, while two other plates contained 1×108 cells. All four plates were incubated under the same growth conditions.
Preparation of microcarriers
100 mg of tungsten M-10 Microcarriers (0.7 µm) (Bio Rad, Norway) were weighed out and placed into a sterile 1.5 mL eppendorf tube and washed with 1 mL of 96% ethanol. Three times of vortexing for 2 min were included in between the washing steps. The microcarriers were centrifuged at maximum speed for 1 min. The supernatant was discarded, and the microparticles were resuspended in 1 mL of sterile water. Aliquots of 50 µL were prepared in sterile eppendorf tubes. During the preparation of the aliquots, the sedimentation of the tungsten microparticles was avoided by frequent vortexing. The aliquots were stored at -20°C for subsequent use.
Preparation of microcarriers (microparticles) for biolistic transformation
The aliquots of the tungsten microparticles were thawed and resuspended by vortexing. 50 µL of the transformation plasmid pLit_chlL_chlor (120 ng/µL), 50 µL of 2.5 M CaCl2, and 20 µL of 1 M sperimidine were added to a 50 µL aliquot of tungsten microparticles under constant low-speed vortexing. The microparticles were centrifuged at 2000 × g for 5 s. The supernatant was discarded. The microparticles were resuspended in 140 µL of 70% ethanol and vortexed at lower speed for washing. The microparticles were centrifugation at 2000 × g for 2 min, and the supernatant was discarded. The microparticles were resuspended in 140 µL of 96% ethanol, vortexed, and centrifuged at 2000 × g for 2 min. After the supernatant was discarded, the microparticles were finally resuspended in 50 µL of 96% ethanol. The DNA-coated tungsten microcarriers were subsequently used during 1 h after preparation.
Preparation of Particle Delivery System
The Bio Rad® PSD-1000/He Particle Delivery System (Bio Rad, Munchen, Germany) was used for the biolistic transformation of the N. oceanica CCMP1779 cells with PvuI digested pLit_chlL_chlor transformation plasmid. The helium was supplied with a pressure of 1700 psi. Both the rupture disks (1550 psi rupture pressure; Bio Rad), the metallic stopping screens (Bio Rad) and the macrocarriers (Bio Rad) were sterilized with 70% ethanol and left to allow complete evaporation of the ethanol. The biolistic transformation was performed in a laminar floor hood, with constant sterilization of the Delivery System and its components with 70% ethanol in between the DNA-coated microparticle shots onto the target cells of N. oceanica CCMP1779.
Microparticles bombardment
The sterile metallic stopping screen was placed in the microcarrier launch assembly. The microcarriers were vortexed thoroughly, and 10 µL of the particles containing 1 µg of the PvuI digested transformation plasmid pLit_chlL_chlor was applied on the sterile macrocarrier and left to allow complete evaporation of the ethanol. The macrocarrier was installed on top of the stopping screen, while a rapture disk was installed in the disk retaining cap. The agar plate with the N. oceanica CCMP1779 cells was placed at the second level from the bottom creating a travel distance for DNA-coated microcarriers of 6 cm. The air was deflated by opening the control valve for building a vacuum of 25 psi as measured using the vacuum gauge. The helium button (fire switch), was held until the pressure reached a value of 1700 psi and the rapture disk burst, causing the DNA-coated microparticles to accelerate into the target N. oceanica cells. The vacuum was released by opening another control valve. Each of the plates containing 5×107 and 1×108 cells were shot 2 and 3 times, respectively.