Screening for DNA binding protein
mgs6145 at student.health.ufl.edu
Thu Apr 4 12:00:45 EST 1996
kernan at uga.cc.uga.edu (Rich Kernan) wrote:
> I wonder if anyone can suggest a good method or refer me to a paper to
> accomplish my task:
> I am working with a promoter which seems to be positively regulated by
> some transacting factor. I would like to screen the rest of the
> chromosome for the gene (or genes) encoding proteins which bind my
> promoter. Previously this was done by sonicating chromosomal DNA and
> constructing a library in pUC19. The promoter was cloned in front of LacZ
> in a plasmid with a compatible origin and different selectable marker.
> This cell line was then made competent and electroporated with the plasmid
> library. Clones containing genes encoding a transacting factor which
> binds and activates the promoter will drive the expression of LacZ and
> those colonies will be blue on X-gal plates.
> What I want to know is if there is a convenient method to directly select
> for clones which contain the transacting factor. The promoter is a little
> leaky in E.coli so I dont think it is possible to put the promoter in
> front of an antibiotic resistance gene and use that method of selection.
> Any comments would be greatly appreciated as I am a lowly graduate student
> whose committee meeting is rapidly approaching.
> Rich Kernan
> Dept of Microbiology
> University of GA
I am doing something very similar to you but I am using the yeast
one-hybrid system, from Clontech. The principal is the same as you
have described except you can select for His growth and blue colonies.
They also have a pretty long list of libraries availible for this
system. I can't tell you exactly how well it works because I am in the
middle of it, but everything looks good so far. Good Luck
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