I recently transformed BL21(DE3) pLysS cells (same batch) with three different plasmids two pET30a and one pGEX6P2, separately.
As we know, that BL21(DE3) pLysS cells are resistant to Chloramphenicol, I added it at a final concentration of 50 μg/ml to the culture along with other respective antibiotics kanamycin or ampicillin.
To my surprise, only one culture showed resistance to chloramphenicol, other two can not survive it. However, when I let them grow without chloramphenicol they appear fine and express my protein of interest (leaky expression in one case).
is my bacterial throwing away the pLysS??
Hello,
I'm greatly surprised that your BL21 pLysS didn't grow on chloramphenicol . Your concentration of Cm is high, it would be better to use chloramphenicol 30 µg/ml. As Foysal said, without Cm you will lose the vector pLysS. Currently, i'm using this strain without any problem.
If you are using 2 antibiotics in the same time, it is better to use this concentration: kanamycin, 50 µg/ml; ampicillin, 100 µg/ml; chloramphenicol 30 µg/ml.
Best
Are you using fresh, commercial BL21(DE3) pLysS cells or are they a lab stock that's been around for a while? If the latter, check that the competent cells alone still grow on cm. Otherwise get yourself a fresh batch as they have probably lost the pLysS plasmid.
Probably also worth asking yourself whether you really need pLysS at all. They don't generally grow as well as straight BL21s. If you want to avoid leakage I'd recommend Tuner cells instead (or using a defined lactose free media). pLysS are handy if you want to lyse easily via freeze thaw, but that's about it.
According to my experiment, no need to chloramphenicol, i have colonies and the target proteins were confirmed by digestion
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