Question about lac operon and blue/white screening - (Dec/06/2006 )
Hello everyone,
I'm new to working with bacteria, and I have a very basic question.
I'm trying to transform bacteria with a plasmid that contains 'lacI' according to my vector map...I don't see 'lac operon' or 'lacZYA' anywhere on the map....does this mean that I can't use X-Gal for blue/white screening? or does the presence of 'lacI' imply the presence of the relevant genes for this type of selection?
If not....then what is the point of having a plasmid with lacI without the other genes?
Thanks,
H
Only the lacI ORF in the plasmid? Or is it the lacI binding site?
In most cases, the lacY, lacI, lacA remains functional in e coli. Only lacZ is inactivated. Thus one will only see the lacZ gene on an empty plasmid vector.
Well if the lacZ is not around, then you don't really have a colour testing system.
Who knows why the lacI ORF is present, maybe the plasmid you have, was suppose to work in conjunction with a lacI minus strain?
what plasmid is that? can u post the image/pic of its map?
Sure, here it is
H
E. coli strains intended for blue/white screening typically have a complete lac operon knockout and an insertion (either chromosomal or on the F plasmid) of a beta lactamase (lacZ) gene with an N terminal deletion. The genotype of these strains will typically read lacZ<delta>M15. The beta lactamase produced is inactive, and to become active must be complemented with a short protein product, lacZ<alpha>, contained typically in a cloning plasmid. Versions of this short gene have been engineered to have a multiple cloning site within the gene. With no insertion, the gene is active, and in the presense of X-Gal the colonies are blue. With an insertion (usually) the gene is interrupted, and the colonies remain white. The presence or absence of the lacI gene should have little or no effect on this process.
Makes sense,
so am I to infer that my plasmid (see above picture) won't provide the necessary component for blue/white screening to a bacterial strain that is intended for that purpose?
H
Not unless there is an unannotated lacZ<alpha> gene on the plasmid surrounding the MCS. If you have the plasmid sequence, this is easy to look for. Incidentally, if you are planning on using blue/white screening, i can recommend the Sigma S-Gal as a superior substrate which makes differentiating the colonies much easier. Of course, the colonies are black, so it is black blue/white screening. Or something.