Methods Digest, Vol 21, Issue 18

Virash Gupta via methods%40net.bio.net (by virashkgupta from gmail.com)
Sun Feb 18 12:09:25 EST 2007


Simple question that is hard to answer
Yes I know one E.coli strain (MTCC 131 with Institute of Microbial
Technology, Chandigarh, India) which harbors seven to nine plasmids of
different sizes in range of about 2 Kb upto 40 Kb. Replicaons in different
plasmids can be compared through elaborate project.
 Dr V K Gupta, Mole Biology Lab Dept of Entomology, PAU, Ludhiana-Pb- India


On 2/18/07, methods-request from oat.bio.indiana.edu <
methods-request from oat.bio.indiana.edu> wrote:
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> Today's Topics:
>
>   1. Re: Simple question that is hard to answer (hzhen)
>   2. Re: Simple question that is hard to answer (hzhen)
>   3. Isozyme staining protocol (arun anoo)
>   4. Re: Simple question that is hard to answer (hzhen)
>   5. Re: Simple question that is hard to answer (hzhen)
>   6. Re: Simple question that is hard to answer (peter)
>   7. Re: Simple question that is hard to answer (Peter Ellis)
>   8. Re: Simple question that is hard to answer (Peter Ellis)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Sat, 17 Feb 2007 02:50:53 +0000
> From: hzhen <hzhen from freeuk.com>
> Subject: Re: Simple question that is hard to answer
> To: methods from net.bio.net
> Message-ID: <1171680647.95013.0 from iris.uk.clara.net>
> Content-Type: text/plain; charset=us-ascii; format=flowed
>
> Duncan Clark wrote:
>
> >
> > I was always curious about the replicons used in some clinical E.coli's
> > that carry umpteen different plasmids. I remember many many years ago
> > that one such E.coli was used for plasmid markers and had may 10
> > different plasmids in it. They didn't appear to lose those plasmids
> > despite no selection. May have been published in "Plasmid" back in the
> > late 70's or early 80's.
>
>
> It all depends on the replication control mechanism of the replicons I
> suppose.  ColE1 replicon is the standard one taught to students and I
> haven't really look into others, so I'm speaking in complete ignorance
> here, but there is no reason why a plasmid with different replicon would
> necessary result in plasmid incompatibility (because ColE1 has an
> unusual replication control mechanism?), although I understand that many
> would.
>
>
>
> > Duncan
>
>
> ------------------------------
>
> Message: 2
> Date: Sat, 17 Feb 2007 03:00:10 +0000
> From: hzhen <hzhen from freeuk.com>
> Subject: Re: Simple question that is hard to answer
> To: methods from net.bio.net
> Message-ID: <1171681205.96275.0 from iris.uk.clara.net>
> Content-Type: text/plain; charset=us-ascii; format=flowed
>
> peter wrote:
>
>
> >
> > Thanks Chen,
> > I am not trying to clone anything. I am doing development work, in
> > which I wonder if I have mixed population of different point mutant
> > plasmids in same bacteria or one plasmid with different point
> > mutations. Simple question - hard to answer without doing experiment.
> > I thought someone might have the answer, but...
> >
>
> I think similar kind of work has probably been done before (maybe 20-30
> years ago), you should just do a literature search for plasmid
> incompatibility.  My understanding is that the great majority of cells
> will have one or the other of the plasmids, a tiny percentage will have
> both.  It is all about statistics and probability.
>
>
> ------------------------------
>
> Message: 3
> Date: Sat, 17 Feb 2007 21:47:02 +0530
> From: "arun anoo" <arunjugran from gmail.com>
> Subject: Isozyme staining protocol
> To: methods from magpie.bio.indiana.edu
> Message-ID:
>        <5e916de0702170817l62af5f98of36a2b284c042c00 from mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1; format=flowed
>
> Hi myself working on isozymes but i dont have good Staining protocol for
> it
> .If  anyone have aur know send me .thanking you very much.
>
>
> ------------------------------
>
> Message: 4
> Date: Sat, 17 Feb 2007 03:04:16 +0000
> From: hzhen <hzhen from freeuk.com>
> Subject: Re: Simple question that is hard to answer
> To: methods from net.bio.net
> Message-ID: <1171681451.96720.0 from iris.uk.clara.net>
> Content-Type: text/plain; charset=us-ascii; format=flowed
>
> Peter Ellis wrote:
>
> >
> >
> > the whole *point* of the transformation is that you only get one
> > plasmid into any given bacterium.
> >
>
> No, you can have different plasmids transformed into a cell.  That you
> usually get only one is neither here nor there.
>
>
>
>
>
> ------------------------------
>
> Message: 5
> Date: Sat, 17 Feb 2007 03:08:45 +0000
> From: hzhen <hzhen from freeuk.com>
> Subject: Re: Simple question that is hard to answer
> To: methods from net.bio.net
> Message-ID: <1171681719.96881.0 from iris.uk.clara.net>
> Content-Type: text/plain; charset=us-ascii; format=flowed
>
> Peter Ellis wrote:
>
>
> >
> > It would seem unlikely.  Even assuming the heteroduplex molecule got
> cloned
> > in the first place, my suspicion is that the first time it gets
> replicated,
> > the mismatch would be "corrected"
>
> I'm not really sure about this, do you think it will happen to any great
> extent with our normal cloning strains?
>
>
> ------------------------------
>
> Message: 6
> Date: 17 Feb 2007 05:35:51 -0800
> From: "peter" <peter.ianakiev from gmail.com>
> Subject: Re: Simple question that is hard to answer
> To: methods from net.bio.net
> Message-ID: <1171719351.008226.321260 from p10g2000cwp.googlegroups.com>
> Content-Type: text/plain; charset="iso-8859-1"
>
> On Feb 17, 3:34 am, "Peter Ellis" <p... from cam.ac.uk> wrote:
> > peter wrote:
> > > On Feb 16, 6:45 pm, "Peter Ellis" <p... from cam.ac.uk> wrote:
> > >> peter wrote:
> >
> > >>> Peter,
> > >>> Mismatch will be corrected indeed, but using what template - it
> > >>> seems that it will be corrected again 50/50 and still end up as
> > >>> mixed population?
> >
> > >> Doesn't matter what template.  When you do the transformation,
> > >> you'll only have *ONE* copy of the plasmid in each bacterium.  Then,
> > >> when it copies that for the first time, it'll correct using one
> > >> strand or the other, and you'll have two identical copies of one
> > >> allele or the other.
> >
> > >>> If that is the case and I isolate DNA and re-transform, I
> > >>> should be able to get 50/50 of homozygous plasmids in the cells and
> > >>> have colonies that have one or the other allele.
> >
> > >> Um, yes.  If you have a mixed population and you transform it, some
> > >> of the colonies will bear one allele and some will bear the other.
> > >> What you will *not* find are single colonies that carry both
> > >> alleles, or single bacteria that carry both alleles, or bacteria
> > >> carrying uncorrected heteroduplexes.
> >
> > >> I'm not quite certain why you expect to have heteroduplex DNA in your
> > >> transformation mixture in any case: how are you generating your
> > >> plasmid for the transformation?
> >
> > >> Peter
> >
> > > Making heteroduplex on purpose...
> >
> > Why?  What's the goal of your experiments?  Why is it important to you
> to
> > have both alleles present in one bacterium, and why can't you simply
> > transform first with one allele and then the other?  Because that's the
> only
> > way I can think of reliably getting both into the one cell, and even
> then
> > it's hit and miss as to whether you'll retain both.
> >
> > It doesn't look to me as though cloning heteroduplex DNA is going to get
> you
> > where you want to go.  It may be that in a tiny minority of cells you
> don't
> > repair the mismatch, and end up heteroplasmic for the two alleles.  Or,
> if
> > you just transform the mixture using a large excess of plasmid, a
> minority
> > of cells will pick up more than one plasmid, and of that minority a
> small
> > proportion will end up with both alleles.
> >
> > In each case, while you may get a very few colonies that carry both
> plasmids
> > (but with no guarantee of preserving them both), you'll have a massive
> > background level of colonies that only carry one or other of the alleles
> -
> > and there won't be ably way or telling which is which, short of
> genotyping a
> > gazillion colonies.
> >
> > Peter
>
> Can't tell sorry....
>
>
>
> ------------------------------
>
> Message: 7
> Date: Sat, 17 Feb 2007 08:28:08 -0000
> From: "Peter Ellis" <pjie2 from cam.ac.uk>
> Subject: Re: Simple question that is hard to answer
> To: methods from net.bio.net
> Message-ID: <53nskoF1tgb0uU1 from mid.individual.net>
>
> hzhen wrote:
> > Peter Ellis wrote:
> >
> >
> >>
> >> It would seem unlikely.  Even assuming the heteroduplex molecule got
> >> cloned in the first place, my suspicion is that the first time it
> >> gets replicated, the mismatch would be "corrected"
> >
> > I'm not really sure about this, do you think it will happen to any
> > great extent with our normal cloning strains?
>
> I can't see why not.  Cloning strains aren't (to my knowledge) deficient
> in
> any of the normal DNA repair mechanisms.  If they were, you couldn't rely
> on
> the integrity of the sequence of whatever you clone into them!
>
> Peter
>
>
>
>
> ------------------------------
>
> Message: 8
> Date: Sat, 17 Feb 2007 08:34:54 -0000
> From: "Peter Ellis" <pjie2 from cam.ac.uk>
> Subject: Re: Simple question that is hard to answer
> To: methods from net.bio.net
> Message-ID: <53nt1dF1sq19uU1 from mid.individual.net>
>
> peter wrote:
> > On Feb 16, 6:45 pm, "Peter Ellis" <p... from cam.ac.uk> wrote:
> >> peter wrote:
> >>
> >>> Peter,
> >>> Mismatch will be corrected indeed, but using what template - it
> >>> seems that it will be corrected again 50/50 and still end up as
> >>> mixed population?
> >>
> >> Doesn't matter what template.  When you do the transformation,
> >> you'll only have *ONE* copy of the plasmid in each bacterium.  Then,
> >> when it copies that for the first time, it'll correct using one
> >> strand or the other, and you'll have two identical copies of one
> >> allele or the other.
> >>
> >>
> >>
> >>> If that is the case and I isolate DNA and re-transform, I
> >>> should be able to get 50/50 of homozygous plasmids in the cells and
> >>> have colonies that have one or the other allele.
> >>
> >> Um, yes.  If you have a mixed population and you transform it, some
> >> of the colonies will bear one allele and some will bear the other.
> >> What you will *not* find are single colonies that carry both
> >> alleles, or single bacteria that carry both alleles, or bacteria
> >> carrying uncorrected heteroduplexes.
> >>
> >> I'm not quite certain why you expect to have heteroduplex DNA in your
> >> transformation mixture in any case: how are you generating your
> >> plasmid for the transformation?
> >>
> >> Peter
> >
> > Making heteroduplex on purpose...
>
> Why?  What's the goal of your experiments?  Why is it important to you to
> have both alleles present in one bacterium, and why can't you simply
> transform first with one allele and then the other?  Because that's the
> only
> way I can think of reliably getting both into the one cell, and even then
> it's hit and miss as to whether you'll retain both.
>
> It doesn't look to me as though cloning heteroduplex DNA is going to get
> you
> where you want to go.  It may be that in a tiny minority of cells you
> don't
> repair the mismatch, and end up heteroplasmic for the two alleles.  Or, if
> you just transform the mixture using a large excess of plasmid, a minority
> of cells will pick up more than one plasmid, and of that minority a small
> proportion will end up with both alleles.
>
> In each case, while you may get a very few colonies that carry both
> plasmids
> (but with no guarantee of preserving them both), you'll have a massive
> background level of colonies that only carry one or other of the alleles -
> and there won't be ably way or telling which is which, short of genotyping
> a
> gazillion colonies.
>
> Peter
>
>
>
>
> ------------------------------
>
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