In article <goldberg-0810960859350001 at 188.8.131.52>, goldberg at bms.com
(Steven Goldberg) wrote:
> In article <01bbb38f$3e926f60$8c91fccc at ------>, "Takbum Ohn"
> <ahnss at hitel.kol.co.kr> wrote:
>> > Hello!! netters...
> > I have cloned a cell wall hydrolase (probably toxic to E. coli host) from
> > Moraxella sp. and now, I subcloned the insert as short as 1 kb.
> > The subclone expressed the gene very highly but, oddly, the copy of the
> > subclone plasmid became very low.
> > I don't know what make my plasmid like that.
> > The mother plasmid is pT7T3 19u, a high-copy number phasimid vector.
>> You said the product of your cloned gene was toxic to E. coli...so I would
> guess the subclones you isolated were plasmids with mutant (low) copy
> number. E. coli probably cannot survive if expression is from a high copy
> number plasmid in this instance.
I have the whole collection of clones that behave like that, but I think that
copy number is low not necessarily because certain replicon mutants survived
after transformation. The reasons to say so are as follows:
1. Some clones with a low copy number contain only introns with no apparent
ORFs inside ( the sequence is known).
2. With some such clones when you remove a certain piece, the copy number is
back to reasonable. When you cut the insert into two and clone them into
another plasmid, one set of clones has a low copy number, and the other
set a normal one. It looks like the reduction is the property of a certain
fragment but not
necessarily of its protein product.
3. With such clones as the ones containing a protease gene in a lac-vector,
the ORF is sitting backwards relative to the lac transcription ( it can be
expressed from a read-through, though).
Typically, with such clones, a higher yield is obtained when initial miniprep
is transformed back into the same host and many ( 20 ) small scale cultures
are grown in parallel. Pooling these after growth and isolating a plasmid
usually gives a better yield than growing a single large volume culture.
Strange enough, though, is the fact that all classical manuals avoid discussing
this problem, and it is not so rare...Does anyone know anything published about
Anyway, do cheer up, you are not the only one suffering from it.
Alexander Kraev, PhD
Biochemie III, ETHZ Zurich
e-mail kraev at bc.biol.ethz.ch