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
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