Delivery Methods

Conjugation

  • Plasmid delivery method from Escherichia coli to Thalassiosira pseudonana.

Preparation of T. pseudonana cells.

 

Five hundred ml of liquid grown culture were spun for 5 min at 4,000g at 10 °C, then most of the media were removed, cells were counted using a haemocytometer and the concentration was adjusted to 2 × 108 cells ml−1.

 

Preparation of E. coli cells.

 

Culture (150 ml) grown at 37 °C to OD600 of 0.3 was spun down for 10 min at 3,000g and resuspended in 800 μl of SOC media.

 

Conjugation of T. pseudonana with E. coli. 

 

T. pseudonana cells (200 μl) was moved to 1.5 ml microfuge tube and then 200 μl of E. coli cells were added and mixed by pipetting up and down a few times. Next the cells were plated on ½ × T. pseudonana medium, 5% LB, 1% agar plates and incubated for 90 min at 30 °C in the dark, then moved to 18 °C in the light and grown for 4 h. Then 1 ml of T. pseudonana medium was added to the plate and cells were scraped. Two hundred μl of the scraped cells was plated on ½ × T. pseudonana medium, Nourseothricin 50 μg ml−1, 1% agar plate and incubated at 18 °C in the light. Colonies appeared after 7–14 days.

 

Taken from: Designer diatom episomes delivered by bacterial conjugation. (2015) https://doi.org/10.1038/ncomms7925 

Biolistic/Microparticle Bombardment

  • Biolistic transformation of TALEN or Cas9 and gRNA vectors to Thalassiosira pseudonana

Biolistic transformation of circular plasmid DNA 

 

The DNA was introduced into T. pseudonana cells using the Biolistic PDS‐1000/He particle delivery system (BIORAD, Hercules, CA, USA). M5, M10, or M17 tungsten particles (3 mg) were coated with 5 μg of circular plasmid DNA using the CaCl2‐spermidine method according to the manufacturer's instructions. For co‐transformations, 5 μg of circular plasmid DNA of each plasmid was used for coating of 3 mg tungsten particles. T. pseudonana was grown to a cell density of 106 cells·mL−1, concentrated by centrifugation for 10 min at 3000g and then 108 cells were plated into a 5 cm diameter circle in the center of an ESAW agar plate (1.5% agar). The cells were allowed to air dry and were then placed into the Biolistic chamber at a distance of 7 cm from the stopping screen. The cells were then bombarded with 600 ng of DNA‐coated tungsten particles using three different rupture disks (1100, 1350, and 1500 psi). After bombarding the cells were immediately scraped from the agar plates, resuspended in ESAW liquid medium to a final density of 106 cells·mL−1 and incubated for 24 h under constant illumination. Five million cells were then plated onto ESAW agar plates supplemented with the appropriate antibiotic. The antibiotic Zeocin® (Invitrogen) was used at a final concentration of 1 μg/mL, and the antibiotic nourseothricin (Werner Bioagent, Jena, Germany) was used at a final concentration of 100 μg/mL. The plates were incubated under normal cultivation conditions for 8 days. Independent colonies were collected from plates and inoculated into 1.5 mL EASW supplemented with the same antibiotic and concentration used for selection of colonies on agar plates.

Taken from: Molecular genetic manipulation of the diatom Thalassiosira pseudonana. (2006). https://doi.org/10.1111/j.1529-8817.2006.00269.x

 

Electroporation

Grow cultures of T. psuedonana on f/2 supplemented sterile seawater to a density of approximately 1.1 x 10^6 cells mL^-1

 

Cell Collection Option 1

 

All procedures are carried out at 4 ^oC and cells were kept on ice. 

Collect cells from 500 mL of culture (see step 1) by centrifugation for 10 m in at 3000 x g in 10 x 50 mL conical centrifuge tubes. Discard supernatant and resuspend pellet in 1 mL of 375 mM sorbitol (filter sterilized)

 

Combine resuspended cells into one 50 mL conical centrifuge tube and centrifuge at 3000 x g for 10 min. Discard supernatant and resuspend pellet in 800 uL of 375 mM sorbitol (filter sterilized). Transfer to 1.5 mL tube and store on ice.

 

Cell Collection Option 2

 

All procedures are carried out at 4 ^oC and cells were kept on ice.

Concentrate cells by vacuum filtration to approximately 300 mL. Collect cells by centrifugation in 50 mL conical centrifugation tubes at 3000 x g for 10 min. Discard supernatant and resuspend pellet in in 2 mL of 375 mM sorbitol (filter sterilized). 

 

Combine cells into new tube and centrifuge at 3000 x g for 10 min. Discard supernatant and resuspend in 1 mM of 375 mM sorbitol.

 

Electroporation

 

On ice, add linearized plasmid (in 250 mM sorbitol) to a final concentration of 0.15 µg mL^-1 to the concentrated cells from above. 

 

Transfer 100 µL of cells + plasmid to a pre-chilled 2 mm gap electroporation cuvette.

 

Wipe cuvette dry with kimwipe and place into Biorad Gene Pulser electroporator set to 25 µF and 400 ohm resistance. In our hands, the highest transformation efficiencies were obtained using a field strength of 2.50 kv/cm.

 

Transfer electroporated cells to 10 mL of sterile, f/2 supplemented artificial seawater and incubate overnight at 17^oC under constant light (170 µE m^-2 s^-1).

 

The following day, plate ca. 5 x 10^8 cells on selective NEPC agar plates (in our experiment we used 100 µg mL^-1 NAT). Controls should include no-plasmid and no electroporation treatments.

 

Grow plates under constant light (170 µE m^-2 s^-1). Colonies appear approximately 10 days after plating.

 

Transfer colonies to 300 µL of f/2 supplemented artificial seawater in 96 well plates. Optical density (600 or 680 nm) can be used as a proxy for growth using a plate reader.

Taken from: Electroporation of Thalassiosira pseudonana​ (2019). dx.doi.org/10.17504/protocols.io.yvqfw5w