Screening for DNA binding protein

[Daniel Pereira]

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
Dan Pereira