I have some genes that have been synthesised in pET-9a however the expression in these has not worked out well at all and they seem quite toxic (low or no expression on SDS-PAGE gel!). Even making plasmids from these things seems to be troublesome with low yield!
So I'm thinking of changing the vector to something else.
The other proteins we have are in a different vector pET 47b (+) and seem to be well behaved.
I've seen the pET manual and there seems to be heaps and heaps of different vectors which do similar things. We don't need anything fancy like tags or whatnot. Is there a relationship between vector and expression?
I am looking around at different synthetic gene suppliers; We usually use GeneArt but I have looked also at "DNA2.0"
Their website has some claims that I'd like to ask around about:
"Inserts are flanked by transcriptional terminators to allow cloning of inserts encoding proteins that are toxic to E. coli. DNA2.0 can even guarantee that your gene will express in E. coli."
Exactly how does this work?
"Inserts are cloned nondirectionally to minimize potential toxicity of inserts."
What does this "directionality" mean?
"We also have vectors with low-copy origins for cloning genes that are problematic despite the transcriptional terminators."
I strongly suspect that my proteins are toxic. What is a low copy origin and why does it matter?
Finally some of their vectors have T5 promoters. I know T7 promoters are IPTG inducible, but T5? What's the difference there and can it be used interchangeably with other cell lines such as BL21 (DE3)?
Sorry if this might sound basic to some!!
Thanks in advance for your help!
Edited by Luria Bertani, 19 May 2011 - 01:41 AM.