How to exchange antibiotic resistance markers on plasmids for E. coli

[Yoram Gerchman]

DK and David

Indeed it is the dilution factor, not an active mechanism (if we ignore the copy
control). I must say though I tried same origin + different markers and vise
verse combinations in the past and many times ended up loosing one of the
plasmid (not always, but enough).
It really is a question of how lucky you feel, if you use BOTH different origin
AND different markers you are on the safe side.
Just my 2 cents
Yoram

> Date: Tue, 16 Jun 2009 23:51:19 GMT
> From: dk from no.email.thankstospam.net (DK)
> Subject: Re: How to exchange antibiotic resistance markers on plasmids
>
> In article <mailman.423.1245184382.21502.methods from net.bio.net>, Yoram Gerchman
> <gerchman from research.haifa.ac.il> wrote:
> >
> >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)
> >AND
> >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 origin.
>
> It's *either* 1) or 2) above, not *and*!
>
> We routinely co-transform pET31 and pET24, which are essentially
> 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 cloned
> into the plasmids.
>
> DK
>
> 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.
>
> David
>
> --- 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>
> > wrote:
> > >
> > >
> > >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)
> > >AND
> > >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
> > origin.
> >
> > It's *either* 1) or 2) above, not *and*!
> >
> > We routinely co-transform pET31 and pET24, which are
> > essentially
> > 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 cloned into the plasmids.
> >
> > DK
>




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