Hi,

I only read the topic this morning -

In some cases, I have to wait 30+ hours to see colonies when I transform BL21 DE3 Star while normally they should appear after 14 hours.

Dianfan

ar...@xtals.org wrote:
Hello

In order to know *exactly* what the reason for poor transformation outcome
was one has to do all sorts of experiments. This is not likely to be your
goal, right? Leaky expression, overload of DNA, somehow compromised cells,
or even plain old user error - and numerous other reasons can be proposed
(including some esoteric ones like incompatibility of your gene product
with a specific strain, for reasons going beyond protocol errors and
simple considerations).

This can take a couple of years to sort out.

If you're talking about using M15[pREP4] cells then the reason why you
failed earlier is likely to be toxicity - pREP4 plasmid supplies lacI
in-trans, which represses 'stray' transcription reasonably well. The cells
that you used before and did not succeed with must have had low intrinsic
levels of lacI and thus permitted leakage.

To test this theory you could streak a couple of colonies of the
M15[pREP4] cells on a plate that has a small amount of IPTG in the agar
(say 0.1 mM) - if the colonies don't grow at all, or appear to be very
tiny then you know it's your protein toxicity that's the issue here. Keep
in mind that bacteria are extremely survival-oriented and therefore you
will eventually generate 'safe' mutants and recombinants that will be able
to grow even on IPTG. Likely those mutants will be useless to you from
practical standpoint.

Once you induce the M15[pREP4] cells - the toxicity will manifest itself
again. You will likely observe total cessation of growth (for a long
period of time) and possibly even lysis of the culture. Therefore you may
have to adjust your fermentation protocol to take this into account. One
of the common adjustments is deliberate induction at higher OD, another is
to include 0.8% glucose in the growth medium; there are other options
available to you. Hopefully once you induce transcription, the gene
products do not shut down protein synthesis - which would be a disaster
since it would likely shut down its own synthesis as well. Since I don't
know what you're growing, this is just one of the possibilities :)

Artem

now i have transformed pqe 30 clone into the m15 host successfully, can
someone let me know exactly what was reason behind the problem into
transformation??is it leaky expression into the other host that is toxic
for the cells,if it so then will i would be  able to get good expression
into this host???

atul
-----Original Message-----
From: CCP4 bulletin board on behalf of ar...@xtals.org
Sent: Tue 5/5/2009 6:24 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] problem in transformation of pqe 30 clone

To clarify: I am not implying that I've worked with many proteins that
express better in XL1-blue cells than they would express in BL21(DE3) or
such if cloned into a pET vector or similar. In fact I can probably recall
only one or two that expressed *better* in XL1 cells. However in the good
old days pQE series of vectors was quite commonly used and I had things in
that were inherited from others - these 'things' were fairly simple to
express in XL1-blue whereas they gave me loads of trouble in other strains
and I was too busy/lazy to re-clone them.

Artem

Hello Artem,

     We express almost all our proteins in BL21 derivatives. It sounds
like you've worked with many proteins that express/behave better in
XL1-Blue?


ho
UC Berkeley

-------------------------------------------------------------------------------------------------
XL1-Blue is a strain of E. coli. Whether it is or isn't an expression
host
depends on the definition, and I am not going to argue semantics.

The T5 promoter works with regular garden variety RNApol of E. coli.
Therefore ANY E. coli strain is an 'expression host' for vectors that
contain this promoter.

I've expressed many proteins in XL1-Blue and I see no reason why you
can't
express yours, either.

Artem

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