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Raji,
Two plasmids bearing same origin of replication will not co-exist in
the
cell for very long, which is why one has to use different replication
origins to stably co-transform multiple plasmids. Luckily, there are
quite a
few plasmids available commercially for that specific reason.
Once you've engineered two compatible plasmids, you may wish to
transform
them together or in sequence. Simultaneous transformation is best done
via
electroporation (since the success of co-transformation depends on the
efficiency of single transformation). You can chemically co-transform
in a
single step but efficiency is much lower.
although the above is correct, using plasmids that are in principle not
compatible
but have different resistance to antibiotics, works well in a number of
cases and is easy to do.
Co-transformation in 'standard' cells work well in some cases;
otherwise one can make competent cells with the first plasmid and
transform these with the second plasmid.
Tassos
Commonly used compatible replication origins include:
cloDF
pACYC
pBR/pUC/ColE1
etc...
Artem
-----Original Message-----
From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On Behalf Of
Peter S. Horanyi
Sent: Saturday, July 08, 2006 5:26 PM
To: Raji Edayathumangalam; 'CCP4 Bulletin Board'
Subject: Re: [ccp4bb]: Co-transforming two different plasmids
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Dear Raji,
I have gotten similar stuff to work also, but the right tool to this
is out
there from Novagen. They sell plasmids that would allow for you to
co-transform and have separate replicons, like pCDF and pACYC along
with
pCOLA or pRSF to use with the pET vectors.
But if you want to stick with your two plasmids, then I would
transform with
1/2 of the "usual volume" of DNA from each plasmid and plate both on
AMP
only and KAN only plates as well as the double antibiotic AMP+KAN
plates. I
always use the usual dose of each the antbiotics even when I use 4
antibiotics for the strain (for 4 different plasmids). If you get each
of
the plasmids to work alone on each of the control single antibiotic
plates
and the co-transformed ones on the double antibiotic plates do not
work,
then I would presume that something about the two plasmids are not
compatible. Then you can try and see if you can transform one plasmid
and
then make that strain competent and then transform the second plasmid.
Hope
this helps
Peter
----- Original Message -----
From: "Raji Edayathumangalam" <[EMAIL PROTECTED]>
To: "'CCP4 Bulletin Board'" <[email protected]>
Sent: Friday, July 07, 2006 4:45 PM
Subject: [ccp4bb]: Co-transforming two different plasmids
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Hi Y'all,
Non-xtallo query.
I have the foll. plasmids:
1) a pET21a vector (AmpR) expressing a single protein
2) a pET28 vector (KanR) containing a polycistronic construct that
co-expresses four proteins
While we contemplate sticking the fifth protein and making a single
multi-expression pET28 vector, I tried to co-transform the two
plasmids by
bumping up the two antibiotics and that did not work.
If you have had success with co-transforming two plasmids that share
the
same replication origin (for e.g., two different pET vectors) into
E.coli
expression strains, could you pl. share your tricks and/or
suggestions. Do
you have favourite strains/vectors when it works?
Many thanks.
Raji
--
Raji Edayathumangalam
Postdoctoral Associate
The Rockefeller University
Box 224. 1230 York Avenue
New York, NY 10021
