Case of the Rearranging Plasmid?

[Mike Poidinger]

Graham Dellaire <popa0206 at PO-Box.McGill.CA> wrote:

>I have prepared a plasmid based vector that contains a large terminal fragment of
>the adenovirus-2 genome.  The vector is 22 kbp and has the odd and frustrating
>ability to recombine and rearrange when grown in bacteria in large culture volumes
>(greater than 5 ml!!)  When I grow the bacteria (Dh5 or10) in 5 ml cultures I get no
>rearrangment but as soon as I try to grow a larger culture volume I end up with
>a ladder of plasmids or a new one all together!!!

>Is it perhaps a problem with adeno DNA in bacteria... perhaps expression of one
>of the adeno genes etc?

>ANyone have similar problems? and most importantly what was your solution???<g>

Researchers trying to make a full-length infectious copy of flavivirus RNA have
had similar problems.  Partial genome sequence is fine, but when you put the
whole genome together it causes plasmid rearrangement. Two solutions that I know
of.

1)  For some viruses the problem was never solved, and the infectious clone
remains in two halves, and is ligated in vitro and infectious transcript
produced.

2)  Use a low copy number plasmid

Mike


------------------------------------------------------------------------------
Dr Mike Poidinger        
Microbiology, UQ       ==> Animal Welfare NOT Animal Rights       
Australia              ==> 668, the neighbour of the beast                 
mikep at biosci.uq.oz.au  ==> Time flies like an arrow, fruit flies like a banana
-----------------------------------------------------------------------------