Hi everyone!
Previously, I transformed a BL21(DE3) strain with a pET-15b vector carrying my gene of interest. The strain grew and I could express my protein.
Now I need to do that again, but everytime I put my culture on LB-Amp, it grows only until OD600=0.55-0.60, (37°C; 160 rpm).
I prepared new Amp solution, I transformed again a BL21 strain, and this problem is still happening.
Any suggestion?
Check for phage contamination. Also, double check the culture medium constituents -are you using a different batch? etc., things like that. Sometimes tryptones and peptones have low levels of lactose contamination and there is induction before the addition of IPTG; if the protein is toxic, premature induction could explain what you are seeing. You could try adding glucose to the medium; it might help if the latter case is true.
Hope it helps!
i'm agree with Alejandro. check for phage contamination.
in case you can try to solve it by using the bl21(de3) of neb (cos. c2527) which carry the fhuA2 mutation. that make them resistant to page T1 that is one of the most common phage.
good luck
Manuele
Thank you very much for your answers!
I solved the problem, and phage contamination was probably the cause (I lost 2 weeks....).
Yesterday (as an advice from one lab member) I grew my original transformed strain and other BL21(DE3) with other plasmid in LB from bottle "X" (a bottle containing a small amount of LB, different from that I was using for starter culture preparation). In 6.5 h the two strains grew very well (OD600 = 1.7-1.8). So I transferred 100 uL of that cultures to tubes with LB from bottle X and LB from bottle A (the one that I was using for inoculum).
In LB-X the culture reach OD=2.1 after 18 h and only OD=0.66 in LB-A.
The problem was the LB, which probably was contaminated with coliphages. I used LB-A for an experiment with Salmonella without problems.
I left a picture of my strain in LB-X and the subcultures in LB-X (the good LB) and LB-A (the bad LB).
If you have been working in the lab and generating aerosols of a phage to which BL21(DE3) is susceptible, I'd advice -just in case- cleaning all surfaces with hypochlorite, irradiating everything in the lab with UV, disassembling suspect pipettes and cleaning them and of course, autoclaving and disposing of the LB-A. Otherwise you stand to lose much more than just two weeks...
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