Clarification for LacZ gene in expression vectors

Charles Fracchia via methods%40net.bio.net (by charlesfracchia from gmail.com)
Sun Jan 4 21:28:02 EST 2009


On Jan 2, 10:27 am, "vedha giri" <vedha... from gmail.com> wrote:
> Dear Sir,
>
> If I Ligate my gene of interest in to the expression vector that do not have
> the LacZ gene, what will be result of Blue /white selection test?
>
> How should I conformed my insert result?
>
> Regards
>

> --
> K.Vedhagiri
> Research Scholar,
> Dr K.Natarajseenivasan Lab,
> Department of Microbiology,
> Bharathidasan University,
> Tiruchirappalli - 620 024.
> India.
> Mobile +91 99446-83728

The LacZ gene from the lactose operon (http://en.wikipedia.org/wiki/
Lac_operon) encodes from the enzyme Beta-galactosidase. The point of
Blue/White screening is to test for the activity of Beta-
Galactosidase.
In the Blue/White screening what you do is feed the bacterium X-Gal
and if the enzyme is active it will create -as a product of the
reaction- an insoluble blue compound resulting in the formation of
blue colonies.

The results will vary depending on what you want to do. It looks like
you want to test for the uptake of your expression vector by the
bacterium.

First you need to make sure that the bacterial strain you are going to
use does not have a functional lacZ gene in it (you could do that with
a White/Blue screening). At this point your bacteria should all be
white.
You can subculture one of the white colonies and incubate to use for
your transfection.
Once you have created your ligated the gene of interest to your
expression vector containing a functional copy of the LacZ gene -which
itself is linked to a promoter-, transfect your cells.
Now you can perform a valid White/Blue test providing you are under
conditions that promote the expression of lacZ (depends on the
promoter you link it to)
The colonies that have succesfully uptaken your expression vector
should be blue.

Hope this answers your questions.


More information about the Methods mailing list