Chris Kafer :Plasmid incompatibility and BL21(DE3)
(by aawara from FEMA-trailer.org)
Thu Feb 8 23:02:11 EST 2007
In <mailman.1117.1170951549.19683.methods from net.bio.net>,
marlis eisenkolb <marlis.e from gmx.at> wrote:
> Hi Chris or anyone who has an answer!
> We have the same problems with transforming BL21(DE3) with an pSC101 ori. As your request is from 1998 -- i hope you solved this problem and can give me an explanation .... !!?
> you wrote:
> Hi. We are having a devil of a time transforming a plasmid into BL21(DE3)
> (The competent cells were purchased from Stratagene and the appropriate
> controls done). The plasmid goes into DH5alpha w/o problem.
> The only thing we can think of is that maybe the origin of replication,
> psc101, is incompatible for some reason, though I've not found any references.
> Anybody out there have any insights? If so, please share as this is quite
This is reconstructed from a rather distant memory. pSC101 requires
dnaA for its replication, and there are differences in the dnaA sequence
between E. coli K (DH5alpha), and E. coli B (BL21).
There are also E. coli K dnaA alleles that don't support replication
of pSC101 at 44oC, but do support it at 30oC. So you might try growing
your BL21 transformants at 30oC. It may just work.
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