Why do colonies containing insert get blue?

[David F. Spencer]

Andrei V. Evteev wrote:
> My question:
> 
> Why do colonies containing inserts sometimes get blue?
> 
> When using blue/white selection I usually get dark blue, pale blue
> and white colonies. Pale blue colonies actually contain inserts (so do
> white ones).
> 
> I suppose that in pale blue colonies insert does not completely disrupt
> beta-galactosidase function (though I can hardly imagine that
> this is true for 14 kb insert). In white colonies beta-gal is inactive
> probably due to frame shift or presence of nonsense codones within
> insert sequence. (I am cloning sonicated DNA so inserts can differ).
> 
> Are my suppositions true? Maybe there is some other explanation?
> 

It is not as unlikely as it first appears to get lacZ production (and
hence blue colonies) with large inserts.  First, unless you're working
in a tightly repressed system (lacIq) or your insert happens to contain
transcription termination sites, there will be a transcript starting
upstream of the polylinker going through the insert and the lacZ
fragment.  Alternatively, it is possible that the insert happens to 
have the appropriate promoters, etc.  Then you would need a ribosome 
binding site (Shine-Dalgarno sequence) somewhere in the insert, probably 
towards its right end, a 'met' not too far away, and an open reading 
frame, without stop codons, through the lacZ.  And remember that most of 
the host cells used for cloning are _supE_ , and hence amber suppressed, 
so that will also improve the odds.  It is still possible to get blue 
colonies (more likely late blue or "bullseye") even with a fair chunk of 
foreign protein fused to the lacZ protein. The critical DNA sequences 
required for E.coli transcription and translation are not that long and 
there is some sloppiness so they will occur by chance in some large 
inserts and with spacings acceptable to the E.coli expression systems.

Dave
 
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David F. Spencer, PhD
Dept. of Biochemistry
Dalhousie University
Halifax, Nova Scotia
Canada

dspencer at is.dal.ca
dspencer at rsu.biochem.dal.ca

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