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From: pell@boulder.UUCP
Newsgroups: sci.bio
Subject: Re: Cloning/Expression vector copy numbers
Message-ID: <1465@sigi.Colorado.EDU>
Date: Mon, 29-Jun-87 15:32:52 EDT
Article-I.D.: sigi.1465
Posted: Mon Jun 29 15:32:52 1987
Date-Received: Tue, 30-Jun-87 04:43:58 EDT
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Reply-To: pell@boulder.Colorado.EDU (Anthony Pelletier)
Organization: University of Colorado, Boulder
Lines: 24
Keywords: Plasmids; Gene amplification; Protein overexpression

(Dan Davison) writes:
  (Dizzy Dan) writes:
>>Does anyone know of any plasmids with even higher copy numbers? If so,
>>where are they available from, and what is their selection?
>
>Have you considered and rejected chloramphenicol amplification?  I've heard
>it can get 1000s (!) per cell.  
 
How is one going to do protein overexpression in the presence of a protein
synthesis inhibitor???
 
>The plasmid must be descended from ColE1,though.

Not quite, the plasmid must have a means of replication that does not
rely on continued protein synthesis--as the coli chromosome requires new
synthesis  for each round of initiation.  ColE1 replication is 
one such dnaA-independant replicon.

For those of you using pACYC or pBR-types that have CAT on them, spectinomycin
works fairly well for amplification, by the same mode of action.

tony 

"don't pull that; you never know what it might be connected to." -BB