Dear all,
I am trying to reproduce results from this paper:
http://www.ncbi.nlm.nih.gov/pubmed/16233057
When I try to express the construct (PhoC - I cloned in a different vector, pRSET A) the transformed cells are growing extremely slow on LB-Plates (100 µg/ml Ampicillin) - they need 48 h to be pickable.
Cultures inoculated with these colonies stay clear even after incubation over the weekend at 37 °C.
I suppose the protein might be toxic? But event then there should be some growth as pointed out in the paper??
The funny thing is, that this expression worked once (nice colonies, good OD600 for the subsequent cultures) but never again.
Where is the "bug"?
Regards,
Peter
Hi Peter,
as you've had good expression of the protein previously, it is hard to think it is now toxic, although there could be an additive that isn't precent there this time. Is the media the same batch / source as before?
I'd suggest that you at least remake all the components to exclude any contamination that's causing your problem.
- Richard
If you suspect toxicity,
Replace Tryptone in in LB with Soypeptone.
Add 1% glucose to LB plates.
Use glucose containing media throughout you experiments. Starting from immediately after transformation to induction.
Autoinduction is another good option.
I am sure you sequence construct and check all possible and impossible mutations, also incubation temperature must be checked and of course induction recommended
You can have a contamination with bacteriophages. When the protein is highly toxic, it can also be affected transformation frequency. You must discriminate between these two possible causes in their next experiments.
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