Chris Kafer :Plasmid incompatibility and BL21(DE3)

Aawara Chowdhury via methods%40net.bio.net (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 
> frustrating!

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.

AC
-- 
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