in vitro alpha complementation

Chris Boyd chrisb at hgu.mrc.ac.uk
Tue Aug 3 05:27:15 EST 1999


John Rebers (jrebers at nmu.edu) wrote:
: 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.

I'm sceptical that SURE would make much of a difference as F's have a
copy number of one so the gene dosage is unchanged. I can't see why
DH5alpha shouldn't work.  The following reference (I have only the
abstract) may be of some help:

   Dunn, I. S. (1996) `In vitro alpha-complementation of
   beta-galactosidase on a bacteriophage surface.' Eur. J. Biochem.,
   242, 720-726.

Best wishes,
-- 
Chris Boyd                      | from (but not \  MRC Human Genetics Unit
Christopher.Boyd at hgu.mrc.ac.uk  | on behalf of) /      Crewe Rd, Edinburgh
http://www.hgu.mrc.ac.uk/Users/Christopher.Boyd          EH4 2XU, SCOTLAND



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