'Leaky' IPTG inducible promoters...
Simon Twigger
spaces!)
I have a gene cloned into plasmid pKK233-2 which I assume is an earlier
version of pKK233-3 that Pharmacia sell at present. Gene expression is
inducible using IPTG and I get about 1mg of product/litre of bacteria
following induction.
I did an analysis of bacterial extract before and after induction and found
that the actual induction seemed to make little difference. Using
antibodies to detect the product, there was a large amount of product prior
to induction and not a great deal more after induction.
My induction is as follows: Grow up 10ml overnight, add to 990ml of fresh
media, grow for 2hrs (mid-log) add IPTG to 0.1mM, leave to induce for
4hrs then lyse by sonication. If i sample the 10ml overnight and also
after the induction step there is little difference in product levels.
The total protein levels in each sample appear the same on coomassie
gels.
The promoter used in pKK233-2 is Ptrc. I know other promoters, especially
the T7 promoters, are known to be leaky and you get certain amounts of
background expression without IPTG.
Has anyone had similar problems with pKK vectors?
Does anyone know of any literature discussing these leaky promoters so I
can mention it in my thesis to prove its not just me!
Any help would be most appreciated.
Thanks in advance,
Simon.
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Simon Twigger =
University of Nottingham Biochemistry Dept. =
E-MAIL mbx...@unicorn.nott.ac.uk =
==============================================================================
Bernard Heymann
mbx...@unicorn.nott.ac.uk (Simon Twigger) wrote:
>
> Does anyone know of any literature discussing these leaky promoters so I
> can mention it in my thesis to prove its not just me!
>
> Any help would be most appreciated.
>
> Thanks in advance,
>
> Simon.
Hi
I have experienced leaky promoters in every one of the three expression
systems (lac, tac, T7) I have used. Despite the rosy reports in the
literature of completely repressable promoters, I have yet to see one in
real life. I guess the common wisdom is: Live with it! I also haven't seen
anybody making a big deal of it in the literature, so I assume it is the
kind of irritating, almost embarrasing thing that commonly occurs but
nobody wants to admit it.
--
Bernard Heymann
bhey...@bragg.bio.purdue.edu
John McDougall
mbx...@unicorn.nott.ac.uk (Simon Twigger) wrote:
>
> My induction is as follows: Grow up 10ml overnight, add to 990ml of fresh
> media, grow for 2hrs (mid-log) add IPTG to 0.1mM, leave to induce for
> 4hrs then lyse by sonication. If i sample the 10ml overnight and also
> after the induction step there is little difference in product levels.
> The total protein levels in each sample appear the same on coomassie
> gels.
>
> The promoter used in pKK233-2 is Ptrc. I know other promoters, especially
> the T7 promoters, are known to be leaky and you get certain amounts of
> background expression without IPTG.
>
> Has anyone had similar problems with pKK vectors?
You can reduce a leaky Tac and Ptrc promoter by growing on minimal media
(M63, M9, etc). A good deal of the leaky nature is due to small amounts of
other compounds in LB that can take the place of IPTG for induction. A
minimal defined media eliminates a lot of this problem, although the
promoter is still a little leaky. For leathal genes in E. coli, I still
prefer T7 because the T7 promoter is almost dead when you do not carry the
T7 polymerase, so it is easier to do your cloning work. However, I still
get mutations that inactivate my leathal genes even though no T7 polymerase
is present, suggesting that there is still some expression.
> Does anyone know of any literature discussing these leaky promoters so I
> can mention it in my thesis to prove its not just me!
>
Studier may have discussed some aspect of this in his Meth in Enz. paper on
the T7 pET vectors- but I am not sure.
Bye for now
John M
--
John McDougall / TEL: 47-776-44479
NFH / FAX: 47-776-71832
Univ. of Tromsoe / E-MAIL: joh...@fagmed.uit.no
N-9037 Tromsoe /
Norway /
Duncan Clark
presenting a modified vector to get around this. Basically put laciq on
the vector and a terminator just upstream of the trc promoter. I
think it isn't just that the promoter is leaky but there is read
through from another plasmid encoded promoter that causes the
problem. You will also find that yeast extract contains an inducer.
Glucose in the media will help repression.
Duncan
-----------------------------------------------------------------------------
Duncan Clark | Internet: dun...@genesys.demon.co.uk
GeneSys Ltd. | Compuserve: 10001...@compuserve.com
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Tracy Aquilla
mbx...@unicorn.nott.ac.uk (Simon Twigger) wrote:
...(stuff deleted)...
>
>Has anyone had similar problems with pKK vectors?
>
>Does anyone know of any literature discussing these leaky promoters so I
>can mention it in my thesis to prove its not just me!
>
>Any help would be most appreciated.
>
> Thanks in advance,
>
>
> Simon.
What is the source of the gene you have cloned? If you are cloning
prokaryotic genes, it is possible that you have cloned part of a prokaryotic
promoter (and/or Shine-Dalgarno sequences) that is active in your expression
system. This has been known to happen occasionally.
Tracy Aquilla, Ph.D.
Dept. of Molecular Physiology and Biophysics
University of Vermont, College of Medicine
aqu...@salus.med.uvm.edu