You may also want to try 0.5 - 2% (w/v) glucose in your plates alongside
transformation into the NEB T7 Express strains:
http://www.neb.com/nebecomm/products/productC3013.asp
http://www.neb.com/nebecomm/products/faqproductC3013.asp
LysY gives you higher final ODs [no lysozyme activity] than using
pLysS/E and is compatible with the T7 promoter in your pET20b.
Renos
On 14/07/2011 03:33, Dima Klenchin wrote:
In this case, pGEX4T3 vector also expressed inserts constitutively.
<http://www.biovisualtech.com/bvplasmid/pGEX-4T-3.htm>http://www.biovisualtech.com/bvplasmid/pGEX-4T-3.htm
Not quite. pGEX vectors carry a copy of lacI^q, resulting in very low
leak expression from PTAC promoter. Definitely lower than the relatively
leaky T7 promoter in pET20, which does not express laqI^q. Switching to
a plasmid from higher pET series that do have laqI^q (e.g. pET31) should
reduce the leakiness.
I also thought that target protein may have tocicity on host strain.
But, why pGEX4T3-target protein was not show the same phenomena.
Now, we are trying to change the host BL21(DE3) to Rosetta(DE3) and
BL21(DE3)pLysS.
pLysS should help with repression under non-inducing conditions.
If it is true toxicity issue, you might need to switch to plates with
synthetic medium that does not contain any traces of lactose.
- Dima
