Chris Kafer :Plasmid incompatibility and BL21(DE3)

Aawara Chowdhury via (by aawara from
Thu Feb 8 23:02:11 EST 2007

In <mailman.1117.1170951549.19683.methods from>,
 marlis eisenkolb <marlis.e from> 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.

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