Sorry Dominic,
I also disagree with you on this is much as Duncan does.
Dominic-Luc Webb molmed <domweb at mbox.ki.se> wrote in message news:<Pine.GSO.4.43.0112211529240.20977-100000 at mbox.ki.se>...
> On Thu, 20 Dec 2001, Duncan Clark wrote:
>> > >In most of the places varying concentration of IPTG has been suggeste
> d
> > >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. Your bugs will already be dying
> at about OD600 of about 0.7.
>
Reaching higher OD's with E.coli is neither difficult nor novel any
more. People have reached dry weights of 100 g/ L ( that will make
the OD to be close to 250). In a simple fermenter with pH and oxygen
control one can reach
reach an OD of 20-40 very easily ( in less than 24 hrs). Even in
shake flasks they can reach OD of 15 or so over 48 hrs ! Here we are
talking about plain old BL 21 strain
>> > >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. They are quite good at beating this induction
> system if given need and opportunity. You give both need
> and opportunity with this protocol.
>
Quite true if you are using B-lactam based antibiotics. Though I am
not sure how significant role does it play in actual fermentation.
There are still lot's of guys using B-lactam with out any major hitch
!
>> > 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 ?M is quite enough for most
> systems. I have several different T7 expression bugs. All
> of the working ones are killed by only 100 ?M IPTG. In fact,
> if your bugs still grow well in 1 mM IPTG, your system is
> not optimal. Consider a different bug, or subculturing.
>>
Well some researchers use upto 2mM to induce. I have read one
reference where ~55 mM of IPTG !!! ( yes milli molar) was used to
induce a high cell density fermentation ( done by Covance biotech)
> > 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 ?M IPTG. My
> best bugs, are totally killed by this treatment, with no
> growth whatsoever if suspensions with this treatment are seeded
> onto plates.
>>> Cheers,
>>> Dominic
>> North 59 37' 30"
> East 17 48' 10"
