The problem:
I transform BL21 E.coli cells with pET5 derivative plasmids bearing genes of proteins I want to overexpress. I get transformed cells, they grow on the respective antibiotic containing solid LB perfectly well and when I pick up single colonies to check for protein overexpression, I get 1 out of 10 or 3 out of 10, 0 out of 10 (!!) etc. expressing and the rest are "idle". How can this aberrant behaviour be explained? Induction is done with IPTG.
Could the solution/problem lie in the media? I have been using Lysogeny Broth (LB) so far. Does anyone has similar experience? What kind of sorcery is that?
*note-the colonies that express the genes seem to maintain this characteristic
You are right, my omission. Corrected in original question.
in our lab lab we experienced similar problem. can you please tell me the source of plasmid.
because if you are using plasmid that is amplified by PCR for re cloning a new gene you may experience this kind of problem.
i suggest you to re clone your gene into a different batch of same plasmid which can solve the problem.
0.5mM to 1mM IPTG is enough for induction. also check how old is your IPTG. we don't use IPTG which is more than 4year old.
problem can't be the media, unless they are contaminated with inhibitors.
1. may be your isolated pET5 is contaminated with non-recombinant plasmids
2. Your antibiotic might not be working/expired. Contamination of other bacteria during overexpression from mishandling, outnumbering the e.coli cells.
BL21 cells fail to maintain foreign plasmid DNA or recombinant DNA for a longer period of time. After transformation, it is quite normal to observe such behaviour as not all the transformed cells express equally. You need to select few colonies to check for expression in small scale and then prepare glycerol stock from highly expressive colonies.
Hi there,
What I usually do is starting experiment with full transformation mix cultured overnight in batch in rich medium (2YT with selection) and used this to inoculate the culture for expression. As a result the culture is a mix of different transformed clones. As mentioned above, transformed BL21(DE3) tends to be genetically unstable. But I would not advise to prepare glycerol stock as the unstability will take over at the end which means the clone will be eventually unproductive.
Try working with freshly transformed BL21(DE3), as these tend to work more reliably than glycerol stocks for protein expression. What is the source of your competent cells? Is it possible that these are BL21 and not BL21(DE3)?
- Badges
- Science topic