basal expression - Is it possible in DH5a or in Rosetta pLYS? - (Apr/28/2006 )
Hi!
I have a toxin gene and its antitoxin gene together as an insert (~500bp) and I ligate this in pET 28a. Vector and insert are digested with Bam HIin one end and the other is blunt end.
I checked insert, vector and competent cells and they are all ok but after transformation I have no colonies! Can the toxin gene be expressed alone and kill the host cells without any induction? 
?
how toxic is toxic?
the pET vectors are driven by T7 promoters, right? you should need T7 RNA Pol (provided separately, I'm thinking you know about that one)...however, T7 promoters are supposed to be 'leaky', meaning some expression occurs with no induction at all. So I suppose it could be that your protein is killing them off...how much protein is required for toxicity?
Alternatively, it could be a problem with the cloning. If all your bits and pieces and cells are OK, have you checked your ligase? Are you certain the ligase buffer's OK? old ATP in the buffer can make your ligation flaky...what exactly is your ligation protocol? If you have checked your comp cells, does this mean you have done an efficiency check with your vector alone? what I'm getting at, are you certain your antibiotic selectivity is OK....it's easy enough to add the wrong antibiotic, or too much...
anyhow, good luck, it sounds as though you are quite frustrated! 
oh, hey, and I read your other post too....is there a way to dose the cells with the antidote prior to transformation?
I have a toxin gene and its antitoxin gene together as an insert (~500bp) and I ligate this in pET 28a. Vector and insert are digested with Bam HIin one end and the other is blunt end.
I checked insert, vector and competent cells and they are all ok but after transformation I have no colonies! Can the toxin gene be expressed alone and kill the host cells without any induction?
?HI. I think the pET series uses cells that have way to inhibit induction. Use the BL21 pLysS or pLysE cells (they have T7 pol inhibition built in). If you're trying to transform a friendly strain like DH5a, you might need to get hold of the plasmid. Amikins' idea of dopiong the system with antitoxin sounds good...can you express/buy the antitoxin, and add it to the recovery medium, or even to the plates???
Another, really funky way around you problem is to clone into a so-called "intein" expression plasmid. You will need to Google to get all of the details, but my understanding is that these little beauties are great for very toxic products.
Good luck.
Thank´s for the tips! They seem very good! I´ll try them 