Re: [ccp4bb] Codon Optimized Expression

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

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Subject: Re: Codon Optimized Expression
From: mjvdwoerd {- at -} NETSCAPE {- dot -} NET mjvdwoerd {- at -} NETSCAPE {- dot -} NET
Date: 2008-02-02

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Subject: Re: Codon Optimized Expression
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Date: 2008-02-02

Subject: Re: Codon Optimized Expression
From: Artem Evdokimov artem {- at -} XTALS {- dot -} ORG
Date: 2008-02-02

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 -
leading to an increased fraction of 'empty' cells in the resulting culture.
Furthermore, even after induction the 'expression-less' cells will continue
to grow, thus depriving the expression-competent ones from nutrients
necessary to build your protein of interest.

There is a good deal of precedent regarding lack of expression from
previously expressing cells (specifically E. coli, even more specifically -
the DE3 cryptic lysogens) after glycerol stocks are used to start cultures
rather than single colonies. There is a recent article in Microbial Cell
Factories journal that analyzes this phenomenon - and the authors insist
that the cells do not lose the plasmid, but rather mutate the lambda
polymerase to become inefficient. This obviously does not happen to ALL the
cells in the glycerol stock - the proportion of mutant must be relatively
low, but repeated re-culturing brings them to the forefront so to speak, by
allowing the mutants to win over their competitors.

Therefore, in practical terms - if you just transform a bunch of cells with
a pure plasmid - it's perfectly OK to use the entire transformation for
growth. On the other hand, 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:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Andreas Förster
Sent: Saturday, February 02, 2008 11:51 AM
To: CCP4BB@JISCMAIL.AC.UK
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 culture from them? This way I'll be
more certain to get the clones that express.

The clones that don't express will dilute the yield, but I doubt they'd
outcompete the rest, would they? For me, the start-with-plenty
procedure has always worked well, but some insist on starting from
single colonies.

Andreas

James Stroud wrote:
> 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 a low but folded expression is better than a
>> high expression to inclusion body.
>
> Anecdotal evidence also suggests to try picking several colonies from
> the original cloning procedure and doing test expressions with each.
> Some clones express better than others even though they should in
> principle express identically. I have no idea what the mechanism for
> this differential expression could be.
>
> --
> James Stroud
> UCLA-DOE Institute for Genomics and Proteomics
> Box 951570
> Los Angeles, CA 90095
>
> http://www.jamesstroud.com
>

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: Codon Optimized Expression
From: mjvdwoerd {- at -} NETSCAPE {- dot -} NET mjvdwoerd {- at -} NETSCAPE {- dot -} NET
Date: 2008-02-02

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Subject: Re: Codon Optimized Expression
From: price {- at -} UCHICAGO {- dot -} EDU price {- at -} UCHICAGO {- dot -} EDU
Date: 2008-02-02