opinions: plasmid loss, protein expression differences

Scott Coutts scott.coutts at med.monash.edu.au
Fri Jun 14 06:16:43 EST 2002


Hi Everyone,

I'm a PhD student that's been working in the lab now for 3 years 
(including honours). So I know what I'm doing, but I dont have a _lot_ 
of experience. Anyhow, when I do protein expression work in E. coli, I 
first transform, then maintain a glycerol stock at -70oC of DH5a that 
contains my plasmid (usually pET.15b). For the expression part, I 
transform BL21. My question is:

How many people here, if any, store their BL21 strains for protein 
expression in glycerol stocks? I have always done this. Whenever I need 
to do the expression, I take some BL21 from the glycerol, grow it on a 
plate and pick a colony for a culture for expression.

Recently, I was told that for every protein expression experiment, I 
should re-transform my BL21s with a plasmid prep from DH5a, because they 
will lose the plasmid or will not express as well when BL21 is kept in 
the freezer. So they're suggesting that I do a transformation every time 
I want to express protein... is the the normal way of doing things?

If they do lose the plasmid from storage in glycerol stocks - how does 
this occur? What does the glycerol do?

Also, does anybody have any explanation for the differences that can be 
see in proein expression amounts when BL21s are transformed and screened 
for expression? For example, say I clone something, do a plasmid prep 
and sequence it - everything's fine. Then I take some of our competent 
cells, which have been prepared from a broth originating from a single 
colony. Transforming them, growing them and inducing them shows they 
they express the protein - but some of them will have almost 
undetectable (by eye on a gel) amounts, whereas others will produce huge 
quantities... it's the same strain and the same plasmid in each case... ?

Thanks for any advice,


-- 
Scott J. Coutts
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Bacterial Pathogenesis Research Group
Monash University, Australia
Phone: +61 3 9905 4838
Email: scott.coutts at med.monash.edu.au
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