In article <3255510E.6808 at compuserve.com>, 102055.606 at compuserve.com says...
>>If this repeats itself, I apologize, but my mail got a little
>funky on me. I am trying to clone a cDNA in Bluescript and I found that
>all of my clones with the right orientation were very poor in growing and
>that the others all grew well. Now I take this to mean that it is a
>toxic gene (an extracellular protein with a single TM domain). Now, why
>does this occur? Is it because the protein is being produced somehow or
>is it because a cryptic bacterial promoter is being used that is encoding
>a toxic gene product? Any opinions outh there? And next, what to do
>about it. Do I try to use ABLE or TOPP cells from Stratagene that limit
>plasmid levels and hence toxicity? And last, do I assume that any
>positive clones I receive are screwed up, rearranged?
There was a thread on this subject a week or so ago.
Just to summarise a few thoughts;
Since the ill effects are orientation dependent it would appear
that it is a transcription/translation product that is the
culprit rather than some problem inherent in the DNA sequence.
pBluescript has a lac promoter upstream of the MCS to allow
prokaryotic expression of fusions between LacZ and the inserted
gene. To reduce expression from this promoter you should move
it to a bacterial host containing LacIq. There are a variety
available including JM109, XL-1 Blue F' derivatives, TOP10F'
etc. and you should select for the presence of the LacIq to'
ensure that it is present (this will require antibiotics or
minimal media). This will *reduce* rather than eliminate spurious
expression so if yields are very low you may want to shift to a
lower incubation temperature or low glucose medium.
When screening for positive colonies, do not use
blue/white screening as the IPTG will permit expression from
the promoter. Instead, make minipreps and screen by restriction
analysis. I assume that the insert is in a single site in
pBluescript so you should be able to increase the possibility
of picking up the clone that you want by cutting an aliquot
of the reverse and immediately ligating which would give you 1:1
forward/reverse (if the forward wasn't toxic).
As you mention above, Stratagene sell ABLE cells which
are supposed to maintain plasmids at a lower copy number and so
reduce the toxicity. I have no experience of this.
You could possibly either eliminate the LacP from
the vector or clone a transcription terminator upstream of the
insert. I have used the former approach successfully.
Finally, the most obvious solution. If you are only cloning
the insert then use the other version of pBluescript - either
move from SK to KS or vice versa as this puts the MCS in the
opposite orientation with respect to the lac promoter.
I hope that something works. Personally I think that
at times biotech companies try and put too much into one vector
and this is an unfortunate consequence.
[no commercial affiliations]
Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
postdoc for hire: will create transgenic mice in exchange for food