Help: mutations in pATH vectors?

[Curtis L. Ashendel]

On Wed, 16 Feb 1994 15:30:32 GMT, Loren Joseph wrote:

>How well should DH5 alpha carrying pATH vectors (10 or 11 so far for me)
>grow in LB  media NOT supplemented with tryptophan. My transformants grow
>quite happily.  I initially grew the bacteria in LB +W, did the plasmid
>prep, showed appropriate vector size and restriction patterns, then looked
>for induction with and without a cloned insert.  I didn't see any
>dramatically increased bands, although the control vector did give a very
>modest band c/w appropriate size. I tried growing the bacteria with the
>vector in the absence of tryptophan, and the bacteria grew quite well. 
>Does LB really have enough tryptophan to make the bacteria grow well. Could
>this be why even in LB-W media the induced band is so modest? Is there moe
>than one formulation of LB? The original article describing the pATH
>vectors noted that mutations in the promoter can be selected for by growth
>in tryp free media, but I assumed it wouldn't be so easy.  
>
>Can anyone comment on the frequency of such mutations, make suggestions
>w.r.t. media, and/or suggest (if thought necessary) a simple way to
>reselect the appropriate plasmid.
On a even more mundane note, I have been making the tryptophan fresh
>regularly per the original article. Why can one not freeze aliquots (and
>save on the cost of sterile filters (I'd test this myself, if I could get
>the vector to work).
>
>Loren Joseph 
>University of Chicago/Dep't opf Pathology/ljlj at midway.uchicago.edu

According to my references on the pATH vectors they do not 
express well in recA cells such as DH5alha, HB101, and JM109.  We use RR1 
cells.  As was already pointed out, LB is too rich to get the background 
expression down.  Addition of the inducing agent (IAA) in addition to 
tryptophan deprivation boosts expression markedly. In RR1 cells with M9+CAA+
thiamine, +W vs -W+IAA (indole 3-acrylic acid), the induction is superb in 
all cases with correct constructs.  Apparently, the low level of expression 
in recA cells does not place a large enough selective pressure on the cells 
to yeild mutations with significant freqency.  Since this could occur in 
RR1 cells, we always subclone and amplify the plasmid using DH5 alpha then 
transform RR1 for expression.  It is recommended to store the plasmid as 
DNA rather than in RR1 cells, because of the potential for mutations.

Send E-Mail if you need help finding references with detailed expression 
protocols.  The main reference is Meth. Enz. 194: 477-490 (1991).
Curt Ashendel
Purdue University
West Lafayette, IN
ashendel at aclcb.purdue.edu