Full Contact Microbiology (a.k.a Diatom Transformation via Bacterial Conjugation)
Grow the diatom culture to mid-log phase (≈ 8.0E6 cells/ml for Phaeodactylum tricornutum grown on F/2 media or 5.0 E7 cell/ml when grown on BG-11).
We have investigated transformation efficiency throughout the growth curve and found this to be the sweetspot.
Grow 1 mL of E. coli culture containing both the mobillity plasmid (Pta-MOB) and carrier plasmid, overnight (16-20 hrs) in LB+antibiotics, for each planned transformation.
(We grow them at 37°C at 270 rpm in a shaking incubator.).
On the day of transformation, use the overnight culture to inoculate 50 mL of fresh LB+antibiotic, 1:50 dilution, for each planned transformation.Grow to an OD600 of 0.8 - 1.0 (37°C with 270 rpm shaking).This takes about 3-4 hours.
During the 3-4 hours the E. coli culture is growing, measure the Phaeodactylum tricornutum cell concentration with a FlowCam or haemocytometer to calculate the required volume needed to collect 2.5E8 cells for each transformation.
For each transformation, centrifuge 50 mL of E. coli culture and the required Phaeodactylum tricornutum volume at 4000 x g for 10 minutes at 4°C.
Resuspend E. coli pellet in 500 μL of SOC medium.Resuspend P. tricornutum pellet in 500 μL of L1 medium.
Note: The diatom and E. coli cultures should be centrifuged at around the same time to minimize the amount of time they spend concentrated.
In a 1.5 mL tube mix 200 μl of E. coli cells with 200 μl of Phaeodactylum tricornutum cells.
Negative control:   In a 1.5 mL tube mix 200 μl of SOC medium with 200 μl of Phaeodactylum tricornutum cells.
Note: Incubate and treat the negative control plates identically to conjugation plates.
Spread the mixture (400 μL) on Conjugation Plates.
(0.5x BG-11 with 5% LB and 1% agar).
Incubate plates for 90 minutes at 30°C in the dark.
Move plates to light incubator (18°C and 100 μmol photons m-2 s-1) for 2 days.
Collect cells by adding 1 mL of L1 medium.
Use a cell scraper to concentrate cells and medium to one side of the plate.
Transfer resuspended cells to a 1.5 ml microcentrifuge tube with a P1000 pipette and filter tips.
Spread 200 μl of the cell suspension on a Selection Plate.
Incubate at 18°C and 100 μmol photons m-2 s-1 until colonies appear.
After a minimum of 8-12 days, untransformed Phaeodactylum tricornutum cells die off, and colonies of transformed cells begin to appear – in some cases, this can take 3-4 weeks.
Alternatively selection can be done in liquid BG-11 Selection media using eGFP as a reporter and sorted using FACS.
un-transformed Pt eGFP  expression.
This protocol was modified from the original procedure and correspondence with the authors.
