in vitro alpha complementation

Michael Benedik benedik at uh.edu
Tue Aug 3 14:30:50 EST 1999


The Stratagene Sure strain does not overproduce the lacZ M15 peptide
(omega fragment) as far as I can discern from the genotype, it does
overproduce the lacI repressor like many other cloning strains. I doubt
you will see much different between it and DH5alpha or any JM host.

alpha complementation is fairly weak. If you put pUC18 into DH5alpha
and measure B-gal activity it will be failry low.  Before you try
something complicated, why don't you attempt a reconstruction
experiment. Put pUC18 or pBluescript or something similar in one strain
deleted for all of lacZ and prepare extracts and mix with DH5 and see
if you can measure anything. 



In article <4.1.19990802183643.0097ad80 at pop.mail.nmu.edu>
jrebers at nmu.edu (John Rebers) writes:

> I would like to set up an in vitro alpha complementation assay, mixing a
> fusing protein made using the alpha fragment of lacZ with a cell-free
> extract containing an alpha-donor (aka omega) fragment, and then assaying
> for beta-galactosidase activity. Will a standard host strain for alpha
> complementation, such as DH5 alpha, work for this, or would it be better to
> use a strain overproducing the lacZ(delta) M15 deletion? Stratagene sells a
> strain,  E. coli SURE, with an F' plasmid with this gene, but they charge
> $80 for it - I'd rather not shell out that much if an extract from DH5
> alpha would work, or if a similar strain is freely available elsewhere.
> 
> Thanks,
> 
> John Rebers
> Department of Cellular Biology
> University of Georgia
> Athens, GA  30602
> jrebers at nmu.edu
> 706-542-0802 (voice)
> 706-542-4271 (FAX)



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