Plasmid delivery method from Escherichia coli to Phaeodactylum tricornutum
Preparation of P. tricornutum cells
Two hundred and fifty μl of liquid grown culture adjusted to 1.0 × 108 cells ml−1 was plated on ½L1, 1% agar plates and grown for 4 days. Then 500 μl of L1 media was added to the plate and cells were scraped and counted using a hemocytometer. Next the concentration was adjusted to 5 × 108 cells ml^−1.
Preparation of E. coli cells.
Culture (50 ml) was grown at 37 °C to OD600of 0.8–1.0 then spun down for 10 min at 3,000g and resuspended in 500 μl of SOC media.
Conjugation of P. tricornutum with E. coli.
P. tricornutum cells (200 μl) was moved to a 1.5-ml microfuge tube and then 200 μl of E. coli cells were added and mixed by pipetting up and down few times. Next the cells were plated on ½ × L1, 5% LB, 1% agar plates and incubated for 90 minutes at 30 °C in the dark, then moved to 18 °C in the light and grown for 2 days. After 2 days, 1 ml of L1 media was added to plates and cells were scraped. Two hundred μl of the scraped cells was plated on ½ × L1, phleomycin 20 μg ml−1, 1% agar plate and incubated at 18 °C in the light. Colonies appeared after 10–14 days."
Taken from: Designer diatom episomes delivered by bacterial conjugation. (2015) https://doi.org/10.1038/ncomms7925
Biolistic transformation of TALEN or Cas9 and gRNA vectors to Phaeodactylum tricornutum
Biolistic transformation of TALEN or Cas9 and gRNA vectors.
Cells (1.5 × 10^8 total) were collected from exponentially growing cultures and spread onto 1% agar plates containing F/2 medium with 20 g L^−1 sea salt. Transformations were carried out 24 h later using the microparticle bombardment method adapted from Apt et al. 32, with minor modifications. Gold particles (0.6 μm diameter, BioRad) were coated with DNA using 1.25 M CaCl2 and 20 mM spermidine.
As a negative control, beads were coated with 3 μg NAT selection plasmid and 3 μg empty vector. For the CRISPR-Cas9 experiment, the DNA mixture contained 3 μg Cas9 expression vector (pKSdiaCas9)10, 3 μg U6-gRNA encoding plasmid, and 3 μg NAT selection plasmid. As a negative control, beads were coated with a DNA mixture consisting of 3 μg NAT selection plasmid, 3 μg Cas9 expression vector, and 3 μg empty vector. A burst pressure of 1550 psi and a vacuum of 25 Hg were used.
DNA or plasmid transfer to P. tricornutum
P. tricornutum was grown to a cell density of 4–5 × 106 cells·ml− 1. A total of 2 × 108 cells were harvested by centrifugation at 1500 g for 10 min at 4 °C, and washed three times with 1 ml 375 mM sorbitol(sterile and ice cold), then resuspended in 100 μl 375 mM sorbitol to a final density of 2 × 109 cells·ml− 1.
Under standard conditions, a suspension aliquot of 100 μl was mixed with 4 μg (0.2 μg·μl− 1) of plasmid DNA (linearized by ScaI digestion) and 40 μg (10 μg·μl− 1) of salmon sperm DNA (denatured by boiling for 1 min), and incubated on ice for 10 min, then transferred into a 2-mm electroporation cuvettes.
Electroporation was performed with a Bio-Rad Gene Pulser Xcell Electroporation System. The Electroporation System was adjusted to exponential decay, 0.5 kV field strength, 25 μF capacitance, and 400 Ohm shunt resistance.
After electroporation, cells were immediately transferred to 15-ml conical Falcon tubes containing 10 ml f/2 medium and incubated in low light (~ 30 μmol photons m− 2·s− 1) overnight without shaking.
Cells were then collected by centrifugation at 1500 g for 10 min and resuspended in 0.6 ml f/2 medium, and 0.2 ml of this suspension was plated onto solid medium containing 75 μg·ml− 1 zeocin. Colonies appeared after 10–12 d and could be further processed after 2 weeks. For cotransformation, two linearized plasmids were used and both were 2 μg.
Adapted from: High efficiency nuclear transformation of the diatom Phaeodactylum tricornutum by electroporation. (2014) https://doi.org/10.1016/j.margen.2013.10.003