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 .
