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From: pell@boulder.UUCP
Newsgroups: sci.bio
Subject: Re: Cloning/Expression vector copy numbers
Message-ID: <1362@sigi.Colorado.EDU>
Date: Mon, 15-Jun-87 11:40:09 EDT
Article-I.D.: sigi.1362
Posted: Mon Jun 15 11:40:09 1987
Date-Received: Wed, 17-Jun-87 01:28:06 EDT
References: <1137@aecom.YU.EDU>
Sender: news@sigi.Colorado.EDU
Reply-To: pell@boulder.Colorado.EDU (Anthony Pelletier)
Organization: University of Colorado, Boulder
Lines: 29
Keywords: Plasmids; Gene amplification; Protein overexpression

(Dizzy Dan) writes:
>
>A post-doctoral fellow here at Einstein informed me recently that the
>Gemini series of plasmids marketed by Promega Biotech (pGEM1, 2, 3, 4,
>and blue) have copy numbers on the order of 400 per cell. This is one of
>the highest copy numbers I have heard of. We may try to clone an E. coli
>gene into one of these plasmids in order to see whether we can get
>increased levels of an enzyme we're studying just on the basis of copy
>number. 

Check me on this one, but I believe the "400/cell" number is the amount
after 12 hours or so of amplification with chloramphenicol.
I have gotten in excess of 6 mgs of twice-banded plasmid from a liter
of cells with this vector, which, in molecular bio terms, is "tons."
I know of no plasmids that amplify better.

I think the copy number in healthy cells is about an order of magnitude
down.  But, since the pGems are deleted for all copy-number control regions
of ColE1, I can't imagine you could do much better (let me hedge a bit, i don't
think the pGems make the regulatory RNA, at least not an intact one. BTW the
promoters for RNA1 and RNA2 are close and transcribe in opposite directions.
I think the two transcripts are complimentary for a hundred or so bases).

As for other replicons altogether, i don't think any are higher
in copy number than ColE1.

happy cloning

tony