pET and ampicillin

Clemens Suter-Crazzolara un691cs at genius.embnet.dkfz-heidelberg.de
Sun Nov 20 09:15:01 EST 1994


> 	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.
> 
> 
> Todd Miller
> tmiller at newssun.med.miami.edu

High !
I have the same problem. My protein is so toxic, that within hours
the plasmid disappears from the culture (less than 2 % !).
However, in my opinion it is not necessary to switch to more 
expensive ampicillin variants: I have developed the following method,
which appears to solve all problems.
1. transform the pLysS strain with your plasmid.
2. pick a single colony, dissolve cells in 600 ul water (vortex)
and plate on LB with ampiciline (standard concentration)
3. incubate over night. The next day you will have a lawn with e.g.
1000-10000 colonies.
4. wash the cells off with 5 ml LB. Carry out the novagen stability test.
In my hands, about 95-98 % of the cells still have the plasmid. This is
probably because the cells on the plate cannot be overgrown by plasmid less
cells (a rather theoretical explanation, I guess).
5. add the 5 ml to 300 ml LB/IPTG/Amp (standard concentration).
6. induce at 37C, 275rpm, 3-6 h.
7. purify protein.

I hope this method will work as well in your hands as it did in mine. 
Please let me know the outcome ! I have already send this protocol to
Novagen, but they didn't tell me whether they have tested it also.

clemens suter-crazzolara, PhD
heidelberg




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