Simple question that is hard to answer

Duncan Clark via methods%40net.bio.net (by blackhole from abuse.plus.com)
Mon Feb 19 06:52:41 EST 2007


Historians believe that in newspost 
<1171648654.333893.138780 from m58g2000cwm.googlegroups.com> on Fri, 16 Feb 
2007, peter <peter.ianakiev from gmail.com> penned the following literary 
masterpiece:
>On Feb 16, 11:34 am, Duncan Clark <blackh... from abuse.plus.com> wrote:
>> Historians believe that in newspost
>> <a59bt2dap5kc69sqqjd0cd00f9p01pv... from 4ax.com> on Fri, 16 Feb 2007, ChenHA
>> <h... from freeuk.com> penned the following literary masterpiece:
>>
>> >The Duet series are plasmids with
>> >different replicons, so are different as far as the original question
>> >is concerned.  The question would be one about plasmids with the same
>> >replicon, and that is problematic.  If there are different antibiotics
>> >used, then it may be possible to maintain the plasmids, but do
>> >remember that antibiotics like ampicillin are broken down fairly
>> >quickly, and when the antibiotics are gone, there is in fact no
>> >selection, and one of the plasmids will most probably be lost.
>>
>> 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.
>>
>> Duncan
>> --
>> I love deadlines. I especially like the whooshing noise they make as
>> they go flying by.
>>
>> Duncan Clark
>> GeneSys Ltd.
>
>Duncan,
>Your input is always appreciated, just my question was different and I
>don't know how better to describe  - can one have two alleles of the
>SAME Plasmid in the SAME cell, not different ori, not different
>antibiotic resistances, everything is the same, just point muataion
>that comes from cloning of heteroduplex DNA that has a mutation.
>Peter

Well a long long time ago before PCR, one made mutants using M13 ssDNA, 
annealed a mutagenic primer and extended around the M13 with Klenow, T7 
DNA pol or T4 DNA pol. Alternatively one used pEMBL vectors which were 
pUC18 plus an M13 F1 ori.

I never heard of anyone getting out a heteroduplex at the end. If they 
had they would have got both mutant and wt protein expressed, which 
never happened.

So I don't think you will be able to maintain a heteroduplex plasmid.

I think you would get one homozygote plasmid per E.coli cell.

There must be papers from years ago confirming or denying this, surely?

Duncan
-- 
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.


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