gal4 bait and pB42ad combination?

Ashok Aiyar aiyar at ebv.oncology.wisc.edu
Wed Jan 28 10:08:11 EST 1998


On 28 Jan 1998 04:51:56 -0800,
Jessie Theuns (theuns at uia.ua.ac.be) wrote:

>has anybody tried to perform a two-hybrid screen using a GAL4 bait construct
>and a cDNA library cloned in pB42AD (the LexA prey plasmid)?

I think that it would be difficult to use GAL-DBD-bait constructs as
bait for cDNA libraries constructed in pB42AD.  

I believe that the GAL1 promoter is used to express the B42-cDNA 
fusion protein in pB42AD.  Thus to ensure expression of the library fusion
proteins, the transformed strain has to be be GAL4-positive and be grown
in the presence of galactose (to overcome repression).

The endogenous wild-type GAL4 protein will likely compete with the 
GAL4-DBD-bait construct for binding GAL4 sites upstream of the reporter.
Further it will activate the reporter in the absence of any library
plasmid.

later,
Ashok
-- 
Ashok Aiyar, Ph.D.
McArdle Laboratory for Cancer Research
aiyar at ebv.oncology.wisc.edu






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