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
al the expression down (I wish
it was as easy to dial it up!).
Happy expressionizing.
Artem
_
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Looney, Andrea Lynn (Andrea Hevrdeys)
Sent: Friday, February 01, 2008 10:29 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [c
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
, aged glycerol stocks made of such transformations
(i.e. without purifying single colonies) may pose a problem later.
Artem
-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Andreas Förster
Sent: Saturday, February 02, 2008 11:51 AM
To: CCP4BB@JISCMAIL.AC.UK
Su
TED]>
To: CCP4BB@JISCMAIL.AC.UK
Sent: Sat, 2 Feb 2008 9:51 am
Subject: Re: [ccp4bb] Codon Optimized Expression
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 c
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
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
<http://www.accelagen.com/>
_
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Looney, Andrea Lynn (Andrea Hevrdeys)
Sent: Friday, February 01, 2008 7:29 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Codon Optimized Expression
Dear All,
I am getting very litt
[EMAIL PROTECTED] On Behalf Of Looney, Andrea
Lynn (Andrea Hevrdeys)
Sent: Friday, February 01, 2008 7:29 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Codon Optimized Expression
Dear All,
I am getting very little (not zero) expression of my protein. I am curious to
know if it is worthwhile to co
Dear All,
I am getting very little (not zero) expression of my protein. I am curious to
know if it is worthwhile to codon optimize my gene, which is ~1200bp, for
E.coli. Can you have "too much of a good thing"? Can codon optimizing
overwhelm the cell or in some way cause death or lowered e
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