Hello researchers,
I am currently trying to clone the toxic ccdB gene under tac promoter into a pBAD vector, using classic restriction-ligation method. The ccdB gene was amplified from a Gateway pDONR vector, and tac promoter added by PCR via floating ends. I then transformed JM109 competent cells with my ligation product to obtain the clones, as this strain still contains the F plasmid and the ccdA gene expressing the antitoxin counteracting ccdB.
However, as a negative control, I also transformed a strain from the Keio collection (BW25113). I thought this strain would be sensitive to ccdB toxicity, but I had several colonies on the plate the next day. Here is the genotype of the strain :
F- DE(araD-araB)567 lacZ4787(del)::rrnB-3 LAM- rph-1 DE(rhaD-rhaB)568 hsdR514
The "F-" means the bacteria do not contain the F plasmid (nor the ccdA gene), and should therefore die in case of ccdB expression...
Do you have any idea why this BW25113 strain is growing despite ccdB presence?
Thanks !
It should not carry the F plasmid. If you wish to confirm you can see if M13 or other filamentous phages will plaque on it.
It may be that your ccdB plasmid has picked up a mutation. You might try and transform it into some other strains to test like DH5 or some wild type strains.
But when you say you got several colonies upon transformation, do you mean you saw a few where you should have gotten thousands of colonies with a wild type plasmid, if so then this may just be some background noise.
- Badges
- Science topic