Hi, everyone - long time reader, first time poster:
I'm also among the "strongly opposed" camp regarding mixing colonies for
expression. Namely, when expressing proteins which might be slightly toxic to
their host cells - either as a direct toxin, or by somehow altering the host's
gene expressio
Hi,
For E. coli, as others already pointed out - optimizing codon usage of a
gene 'does not usually hurt'. I can attest to the fact that it can boost
expression quite a bit - at least in the modest number of cases where I have
had a chance to compare the expression levels under the same conditi
Rumor also has it that more than one "bad" codon in a row, particular
near the beginning, can be extra-bad. For one small, A/T rich
protein, we simply "fixed" a pair of bad Arg codons near the
N-terminus at the same time as recloning it, by using the N-terminal
cloning primer to do the mutagen
In my humble opinion starting with a mixed culture is a bad idea, because:
Successful expressors have heightened metabolic load even in the absence of
inducing agents. Therefore, during cultivation their 'expression-less'
companions will progress more rapidly and undergo more cell divisions -
lead
I have seen procedures in a reputable lab where they do indeed pick multiple
colonies and mix them; the people with the most experience claim(ed) that this
MUST be done for the particular prep they were doing or else they get very low
yields. The concern would be (in my mind) that if the diffe
Yes, it might be the old quotes problem.
Later versions of ccp4i add in quotes around the filenames (to deal with
WIndows paths with spaces) and later versions of Phaser interpret quoted
arguments correctly. But a new gui with an old phaser will cause problems.
Martyn
-Original Message-
F
There has been a somewhat related discussion in the lab the other day.
If some colonies might express well and other not so well, why don't I
just scoop up loads and start my culture from them? This way I'll be
more certain to get the clones that express.
The clones that don't express will di
Is it possible that you have phenix (or some old version of phenix)
installed and that the PATH variable from the CCP4i gui finds that phenix
first?
Check if ccp4_first_in_path is set to 1 in your $CCP4/include/ccp4.setup
script.
Tim
--
Tim Gruene
Institut fuer anorganische Chemie
Tammannst
Hi Andrea,
We have seen examples where it can make a dramatic difference, no
expression even with codon plus strains, but really good expression
with the daughter constructs as already pointed out. It is
particularly true for parasite proteins where the percentage of AT is
higher than bacteria. Al
On Feb 2, 2008, at 2:15 AM, M T wrote:
One classical way to optimize expression level is to screen culture
conditions.
For my proteins, I solved my expression problems by changing the
expression vector to a pET or changing a pET 20 to a pET 30 (if the
protein is toxic).
But keep in mind that
One classical way to optimize expression level is to screen culture conditions.
For my proteins, I solved my expression problems by changing the
expression vector to a pET or changing a pET 20 to a pET 30 (if the
protein is toxic).
But keep in mind that a low but folded expression is better than
On Feb 2, 2008, at 1:36 AM, Gerard DVD Kleywegt wrote:
Do you mind if we call you Bruce, just to keep it clear?
No need for the formalities, Lenny will be fine.
--
Sig wisely sitting this one out.
Python 3.0 should have just been named something like "Anaconda" or "Boa"
Actually (and etymologically), Python is an eponym, named after Monty.
Hence, any spin-off should be called Basil or Brian or, a trifle
presumptuously perchance, Holy Grail or Meaning of Life.
--Bruce
Professor of Hege
On Feb 1, 2008, at 5:40 PM, Gerard DVD Kleywegt wrote:
Python 3.0 should have just been named something like "Anaconda" or
"Boa" so
Actually (and etymologically), Python is an eponym, named after
Monty. Hence, any spin-off should be called Basil or Brian or, a
trifle presumptuously perchan
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