How to get a plasmid into WT E.coli

Tom Dougherty Doughert at synapse.bms.com
Fri Mar 15 09:17:13 EST 1996


In article <4h9r4g$rjn at info.usuhs.mil>, jim rogers <rogers at usuhs.usuhs.mil> says:
>
>Need some help.
>We are trying to get a plasmid into a clinical isolate of E coli.
>Transformation does not work, so we have tried electroporation,
>conjugation using different methods.  We can get pBR328 in but not
>a RK6 based plasmid.  The control using DH5 work.  My first thought
>was that maybe it had to do with the oriR of the second plasmid
>
>Any ideas?
>Jim
Have you considered that it might be a restriction/modification problem?
It's conceivable that the clinical isolate might have an active system that
is degrading the plasmid, particularly if it is large.  The DH5 strains are 
restriction minus, and this may allow the second plasmid to establish
itself.  The other possibility is plasmid incompatibility.  Have you checked
the clinical isolate to see if it carries a natural plasmid?  If it is an R6K
based replicon, then your incoming plasmid would not be able to
establish itself.
			Tom Dougherty



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