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transformation problem - (Jun/16/2009 )

Hi!
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 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!! :)

-laith-

laith on Jun 17 2009, 02:39 PM said:

Hi!
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 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 can try transforming your plasmid in E. coli BL21(DE3) strains which can overcome this problem- protein toxicity to the cell- hope this helps you.

-spanishflower-

You should have little or no expression in Rosetta or Rosetta pLys unless the T7 promoter is induced. You should never get expression in DH5a. Have you done a control transformation of empty PET vector to test transformation efficiency? Transforming directly into Rosetta will likely not work due to low transformation efficiency. Instead clone into DH5a, Top10 or another cloning strain, isolate plasmid, and then transform that plasmid into Rosetta.

-phage434-

If you want to express the toxic protein, have you considered in vitro expression? Like in vitro transcription and translation? That will solve the problem of transformation.

-jiajia1987-