How to exchange antibiotic resistance markers on plasmids for
(by amanosz from yahoo.com)
Wed Jun 17 10:38:30 EST 2009
Actually, just started reading about the issue of origin of replications as well, and will end up moving the gene to a different plasmid with separate marker and ori, so original problem is solved. Thanks!
But for the sake of discussion, wouldn't it be more ideal to have both different resistance marker *and* origin? Sure it sounds doable to just have different resistance marker, but wouldn't asymmetric plasmid doubling potentially dilute one plasmid to effectively low copy number? For example, two plasmids sharing an ori with copy number, say 40. If you're unlucky at picking clones, you could conceivably result in a cell with 39 copies of one plasmid and 1 copy of the other. Both resistance markers are present to satisfy the selection requirements but you'd have uneven protein expression, promoter differences notwithstanding.
--- On Tue, 6/16/09, DK <dk from no.email.thankstospam.net> wrote:
> From: DK <dk from no.email.thankstospam.net>
> Subject: Re: How to exchange antibiotic resistance markers on plasmids for E. coli
> To: methods from magpie.bio.indiana.edu
> Date: Tuesday, June 16, 2009, 4:51 PM
> In article <mailman.423.1245184382.21502.methods from net.bio.net>,
> Yoram Gerchman <gerchman from research.haifa.ac.il>
> >Unfortunately it is not that simple.
> It is. E.coli does not have an active mechanism of plasmid
> incompatibility. The loss of one plasmid in the case of
> two plasmids carrying the same ori and selection marker
> is simply a dilution effect.
> >To co-transform E. coli, and have stable transformation
> you have to have:
> >1. Two different selection markers on the plasmids
> (e.g. resistance genes)
> >2. Two different origin of replication markers on the
> plasmids- e.g. pA15 (from
> >pACYC184) with ColE1 (pBR322) or another combination.
> Mind you, pUC19 origine
> > IS
> >NOT compatible with ColE1 as they basicaly the same
> It's *either* 1) or 2) above, not *and*!
> We routinely co-transform pET31 and pET24, which are
> the same plasmids carrying different resistance. Stable
> enough to
> get a large colony, grow an overnight culture and expand
> into 12 liters
> of culture - without losing expression of both of the genes
> into the plasmids.
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