Conjugation of Thalassiosira pseudonana
Pick bacterial colonies from your Gent+Kan plates and inoculate 10 mL LB medium.
Grow overnight.
Measure OD600 and start a 150 mL LB subculture (recommended starting OD600 either 0.05 or 0.1).
Grow at 37oC until OD600 reaches 0.3-0.4.
Centrifuge at 4,000 rpm, 10oC, for 10 min.
Decant supernatant and resuspend in 800 µL SOC.
Tp was cultured in L1 medium as described in the 'Before start' section.
Spin down 2 x 108 cells at 4000 rpm, 10oC, for 10 min.
Decant supernatant and resuspend pellet in 1 mL L1 medium.
Mix 200 µL T. pseudonana cells and 200 µL E. coli cells in a 1.5 mL tube.
Pipette up and down a few times.
Plate on 1/2xL1 1% agar plates w/ 5% LB.
Incubate in dark at 30oC for 90 minutes.
Move plates to your standard Tp growth conditions - in my case 18oC and constant light - for 4 hours.
Add 1 mL L1 medium and scrape with a cell scraper or L spreader.
Expect to recover ~500 µL co-culture suspension after scraping.
Plate 200 µL of the resulting suspension on pre-dried 1/2xL1 1% agar plates w/ 100 µg/mL nourseothricin.
Leave at 18oC and constant light until colonies appear - ~2 weeks.
Here are a few ways to confirm the presence and expression of your heterologous gene in resulting colonies:
Growth under selection pressure.
Make sure colonies are able to grow in liquid L1 medium with 100 µg/mL nourseothricin (Nou100).
Pick colonies with a small tip or better a toothpick and inoculate ~0.5 mL L1 medium.
Once you observe growth subculture in larger volume.
PCR- Use 1 µL of diatom culture as a template to amplify your expression cassette.
- Confirm the absence of donor DNA by amplifying E. coli-specific genes.
NOTE:I used primers to amplify the corC gene.
Make sure you always run E. coli EPI300 posititve control.
- Confirm the absence of live donor cells by plating some diatom culture on LB plates w/o antibiotics.
NOTE: Any remaining donors cells and donor DNA are gone after a few liquid subcultures.
You can also patch colonies on fresh 1/2xL1 Nou100 plates.
RT-PCR- Purify total RNA from Nou100 diatom culture, convert it to cDNA and use it to run a PCR with heterologous gene-specific primers.
- An example of my result with controls can be found here.
Episome recovery- Perform a diatom miniprep as described by Karas et al.
(2015).
- Transform E. coli with diatom-derived DNA.
- Select on LB agar plates with 50 µg/mL kanamycin.
- Miniprep, digest and analyze on a gel.
NOTE:You will learn more about the state of your episome from this analysis.
Western blot
Protein-specific assays- enzymatic assay, microscopy etc.
I've had success with 1, 2 and 3; tried 4, 5 and 6 without success.
