alpha-complementation, host strains

Scott Craig s.craig at student.unsw.edu.au
Tue Aug 24 00:52:48 EST 1999


Dear John,
You've forgotten about the f' episome which does in fact provide the ZdeltaM15
mutation of B-gal in JM109. You might be better off with HB101 or BL21 which
are both deficient in B-gal, have a look in the back reference section of a New
England Biolabs catalogue where they have a list of many strains with the
genotype and availability
Another method of tracking your protein might to use anti-Bgal antibody in a
dot blot or something similar to track the protein. I know Roche (boehringer)
have one which I think detects both fragments of B-gal and therefore fusion
made with them

John Rebers wrote:

> I am planning to make a clone a small fragment of a gene I'm working on
> into the alpha fragment of lacZ in pUC19. I would then like to track
> purification of the fusion protein by in vitro alpha complementation, by
> making a cell extract from cells transformed with the fusion plasmid,
> mixing this with a separate extract made from cells that have the
> lacZ[delta]M15 deletion, and assaying beta-galactosidase.
>
> Would JM109 be a suitable host for transformation with the fusion plasmid?
> It's genotype is recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi
> [delta](lac-proAB). If I'm reading the genotype correctly (I'm not an E.
> coli geneticist...), this means that these cells are deleted for the lac
> operon, and thus won't provide either lacZ omega fragment. Most of the
> common host strains for transformation do supply that fragment, but for the
> assay of protein at different stages of purification I have in mind, I want
> to do the complementation in vitro by mixing the extracts, rather than
> having the lacZ omega fragment already in the transformed cells.
>
> If JM109 would not work well, can anyone suggest an alternative strain and
> source for the cells? (The E. coli genetics stock center strain number
> would be fine, but I've had problems figuring out how to look for strain
> names like CSH18 or whatever, on their web page).
>
> Finally, does the strategy outlined above seem like a rational way to track
> purification of my fusion protein? I can also run protein gels and check by
> size, but I thought the alpha-complementation assay might be more sensitive
> and faster once I get the initial assay worked out.
>
> 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)
> 706-583-9618 (home)
>
> (On sabbatical leave from Northern Michigan University until May 2000;
> please use contact information above to reach me.)

--
Scott Craig
Dept. of Biotechnology
University of New South Wales
Sydney, Australia





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