Hi all,
Did somebody experience bacterial contamination problems when inducing scFv display on the EBY100 with galactose containing media (SG-CAA)? Before induction, yeast is thawed and subcultured twice in the SD-CAA (pH 6) selection media. At this point it is free of this contamination (confirmed on SD-CAA agar plates and under the microscope). After 38 hours of induction in SG-CAA (pH 6) at 20 degrees the contamination is extensive. We have tried to FACS the scFv expressing yeast and remove the contamination with different antibiotics (ampicillin, kanamycin, pen/strep, gentamicin, tetracycline), slow centrifugation, SD-CAA with pH 4.5. Nothing completely removes the bacteria. Do you have some tricks how to avoid the contamination before/during the induction? Thank you for your answers.
Barbora
Hi there,
The best is to work sterile from A to Z (ie. working sterile with sterile glassware and reagents). In your case something is obviously going wrong from the time you culture yeast on galactose... Is it due to non sterile media or non sterile manipulation, one can't tell.
I think you need to isolate and identify the contaminated bacteria, and add inhibitors after determining the species. And also, I argee with Dominique Liger , you may need work sterile from A to Z.
As the previous answer I confirm thta you need to work sterile until FACS analysis.
If your contamination is exploding during the induction I could advise you to reduce this induction time, 16 hours is enough for most of the scFv. (I observed better expression after 16h than 36h for my scFv).
You can also use Pen/Strep at 1/100 in both SD-CAA and SG-CAA. I put antibotics directly into the bottle of media as most of the time the bacteria grow up directly from the bottle.
I will just re-iterate what previous people have already mentioned, everything you use to manipulate yeast must be sterile. However, sometimes you may still get contamination, and I am convinced it has to do with the air quality in the lab. Have you noticed that you get more contamination when the weather is warmer or more humid? I used to get way more contamination in the summer months than during winter time; actually no contamination during winter. The air filtration system in our lab's building wasn't great. Ever since I moved to a new building, I haven't gotten any contamination at all, even when doing all the work on the bench. If you have access to a culture hood, I would try working in there at all times; only open the media and cultures in the tubes.
The contamination I used to get would not away with pen-strep, kanamycin, ampicillin, low pH, 10% ethanol, nothing at all. It would grow more slowly in SGCAA, making you think that the cultures where not contaminated, and then expanding aggressively when you tried to induce. The only thing that worked for me was a protocol I made up. I would spin the SD culture at 400g for 2 minutes to pellet the yeast but not the contamination; remove the supernatant carefully, resuspend in clean media, and then centrifuge again. I would do this 10 times. If you look at the supernatant after every spin, you will notice that the contamination doesn't pellet and it diminishes with every wash. It worked for me.
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