SUMMARY:Plasmids losing inserts
IBR100E at ODUVM.BITNET
IBR100E at ODUVM.BITNET
Mon Feb 7 11:34:47 EST 1994
Many thanks to all the people who took the time to reply to
my question. Unfortunately I have been unable to read the newsgroups or
post to the net for the last 5 days due to technical problems. If anyone
posted during this time I am sorry to say it has gone from my screen forever.
I have summarised the replies below, including e-mail addresses which I
hope no one objects to and have paraphrased rather than include direct quotes.
To recap: on Friday 28 Jan I posted a question about how to prevent plasmids
losing their inserts during culture. We have cloned a reverse transcriptase
gene into an E.coli plasmid system and the insert was proving to be quite
unstable particularly in large grow-up volumes, even under uninduced
conditions. We believe this is most likely due to a toxicity effect although
we have no actual proof that any protein is being expressed.
Mark Garfinkel <garfinkl at iitmax.acc.iit.edu> suggested the problem may have
been due to a clonally impure culture. This is definitely not the case as it
has happened with four independent clones, all carefully isolated.
Raj Shankarappa <bsh at med.pitt.edu> and Sean Moore <baldy at brahms.udel.edu>
both suggested lowering the temperature to 30C may have a beneficial effect.
Raj also suggested using minimal media and not growing the cultures to sta
saturation. If the problem is culture running out of antibiotic and bacteria
which have lost the plasmid overrrunning the culture ( which my case does not
seem to be the problem) Gregg Wells <pathology at a1.mscf.upenn.edu> recommends
using freshly grown colonies to inoculate cultures and not growing to
Sean Moore also suggested using a lower copy number plasmid or a different
strain. Stratagene does have two strains, ABLE C and ABLE K, which can
reduce the copy number of ColE1 based plasmid (e.g. pUC, Bluescript) by four
and 10 fold respectively. We intend to try these strains.
Several people also suggested using a different promoter system. The one we are
using has the gene downstream of a T7 promoter. Exrpression is induced by
transfection with an M13 which expressess the T7 polymerase. We could switch
with a minimum of inconvenience to another vector from Invitrogen which relies
on the lacIq repression system. Rafael Maldonado <Rafael at genetics.med.utah.edu
> suggested that if we were to use a lacIq system we should grow the cultures
in minimal medium + 0.3% casaminoacids and glucose as the carbon source to
allow the lacIq product to give more repression . He also suggested that using
a vector that had the lacIq gene included would give more product and therefore
Angelo Gunasekera <angelo at phoenix.Princeton.EDU> suggests that if using a
system where T7 is in the host and is under IPTG control it may be necessary
to do fresh transformations each time as lethal proteins can cause host to
activate recombinations systems (hosts carrying T7polymerase are usually
recA+) and thereby delete the lethal gene from the plasmid.
Any further correspondance or postings are welcome. Please let me know if
I have said anything that is inaccurate. I hope this summary may be useful
Once again, many thanks to all those who replied,
Email : ibr100e at oduvm.cc.odu.edu
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