pET and ampicillin
Paul A Bucciaglia
bucc0003 at gold.tc.umn.edu
Tue Nov 15 20:25:51 EST 1994
tmiller at newssun.med.miami.edu (Todd Miller - Pharmacology) writes:
> Our lab is using the pET system to express eukaryotic pro-
>teins. We seem to be experiencing a problem with the BL21 host cells
>losing their plasmid. Does anyone have suggestions on how to avoid
>this? We're thinking of trying carbenicillin but its about 10 times
>as expensive as ampicillin. Also, have inhibitors of beta lactamase
>ever been used (like clavulanic acid)?
> I'm aware that overnight cultures should be highly diluted and
>we've also played around a bit with the pLys S and E containing hosts.
>We're trying to use this system in a fermentor, but I fear that if we
>grow to the high densities that can be achieved in a fermentor, none
>of the bacteria will have the plasmid when we induce expression.
>Thanks for any suggestions.
>tmiller at newssun.med.miami.edu
The people at Novagen reccomended to me not to grow overnight cultures if
your construct encodes something unstable in ecoli. They reccomend
growing cultures to the point where bacteria become visible and then
storing this overnight at 4 C. Use this to innoculate your culture the
next day. It won't help to dilute an overnight that has been overrun by
bugs which have lost the plasmid or found a way not to express your protein.
I assume you have tried using Bl21(DE3)pLysS , which has a plasmid
expressing T7 lysozyme, a bifunctional protein which inhibits the T7 RNA
polymerase, which can increase the stability of some plasmids. Another
thing to try is using glucose (0.4%) when plating out the BL21
transformants, when growing starter cultures and perhaps in cultures to
be induced (its generally gone by the time you add IPTG to induce). Like
everything else in protein expression, its very empiracle :-(
If all else fails try the newer Novagen vectors which carry a Kanamycin
resistance gene and have the lac operator downstream of the T7
promoter; next to using a phage to deliver the polymerase this is as
tight as it gets.
Hope things work out,
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