In article <1996Oct7.193816.6102 at alw.nih.gov>, bernard at elsie.nci.nih.gov
(Bernard Murray) wrote:
> 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
Just to add my $0.02, you can also try to minimize the leakage from Plac
by adding some glucose to the growth media. We've found around 0.5% to be
adequate. Not something you want to do if you intend to package the
phagemid into M13, glucose cuts down the efficiency of phage replication
(in some fashion unknown to me). We've figured out largely from
discussions on this group and in our own experiments, that it can be a big
mistake to screen by blue/white when you're looking for something you're
eventually going to express. We've started changing our protocols to
avoid expression and trying to keep it shut off until we want to actually
University of California - Davis