toxic gene ligation/transformation URGENT HELP! - (Apr/28/2006 )
I'm trying to express a toxic protein.
I made the insert with an operon containing the toxin gene and also the antidote gene. This insert is 500 bp. I'm trying to ligate in pET28a
The vector and the insert are blunt-end and BamHI.
The problem is that after ligation I tried to transform DH5a, rosetta and Rosetta pLys but I had no colonies. I know that everything is OK with this vector because with another insert it works well.
What can be happening?? Somebody tolds me that a basal expression of the toxic gene is preventing the cells' growing. Is it possible? What can I do? Help me please!!
you may try to culture at lower temperatures (30 but less may be appicable (for ex: RT))