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 .
