Dominic-Luc Webb molmed wrote:
> On Thu, 20 Dec 2001, Duncan Clark wrote:
>> > >In most of the places varying concentration of IPTG has been suggested
> > >for induction depending upon the expression system and other things.
> > >Most of these are for an low ODs like 0.5-1.
> > >
> > >In case we need to work on high ODs like 50-100 or 150 would one
>>> You will never get that far for the simple reason that
> spectrophotometers peak out at about 3. They will give
> values up to about 4, which are meaninglessly beyond
> the linear range of the spectrophotometer. OD600 are
> absorbance values.
Yes and no. I also think bugs cannot grow at so high OD600 (at least E. coli or
similar gram negative bacteria). However, it's very common to get OD600 near 6
or 7, depending on the media. To measure it, the sample must be diluted in fresh
media to avoid the saturation of the spectrophotometer. Usually, we dilute the
culture to get a OD 600nm between 0.1 and 1.5
Your bugs will already be dying
> at about OD600 of about 0.7.
>
AFAIK, 0.7 is usually consider middle-late log phase (once again, depending on
the media). Even in LB, a non-buffered media, E.coli grows as much as 2.0
>>> > >require much higher concentration of IPTG and if in what range is
> > >generally used for such cultures.
>> No. This will be useless. There will be so much antibiotic
> resistance in the medium that you can clear out nearly 100%
> of the antibiotic within minutes! Your soup could quickly
> be overrun by bugs spitting out these lethal concentrations
> of IPTG.
That's the reason it's better to work with non degradable antibiotics, like
tetracycline, or to add fresh atibiotic at the same time you add the IPTG.
They are quite good at beating this induction
> system if given need and opportunity. You give both need
> and opportunity with this protocol.
>>>> > I've always used the same concentration regardless of OD.
>> This ends up making sense, but it was not very intuitive to
> me at first. I think 100 µM is quite enough for most
> systems. I have several different T7 expression bugs. All
> of the working ones are killed by only 100 µM IPTG. In fact,
> if your bugs still grow well in 1 mM IPTG, your system is
> not optimal. Consider a different bug, or subculturing.
>>> > The more critical bit is stage of growth rather than OD itself i.e.
> > culture must be in logarithmic growth and not late log or stationary.
>> I think I would agree, from my own experience. In general, it is
> better to have lower OD, below 0.5. Even at 0.5, ampicillin can
> be consumed within minutes in a typical T7 system, like BL21(DE3)
> and HMS174(DE3) lysogens and proper preparations of these
> are pretty much completely killed by 100 to 500 µM IPTG. My
> best bugs, are totally killed by this treatment, with no
> growth whatsoever if suspensions with this treatment are seeded
> onto plates.
>>
I'd say the protein you are expressing could be toxic. With multicopy plasmids,
carrying a Ptac promoter, we use to induce with IPTG 1mM and only in one case we
detect toxic effects. I know that most probably 1mM is overconcentrated and we
could get the same induction level at lower concs, but we never tried to do a
tritaion curve since we got good yields at the first try.
Sergio
