tight regulated vector construction

Philip Heacock pheacock at bmb.med.uth.tmc.edu
Wed Dec 30 12:11:51 EST 1998


In article <367FAC27.1BFDF7C7 at email.hanyang.ac.kr>, Young Chai
<ygchai at email.hanyang.ac.kr> wrote:

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> Dear all,
> I have a cloning problem which is new to me, though hopefully someone
> can help.
> Could anyone tell me that there are any promoters which completely block
> "leaking" of inserted gene in prokaryotic system (such as Bacillus)
> without using lac promoter.
> I appreciate your helps.
> Young Chai

You ask a question that is dear to me, we have for years looked for a
regulatable OP that totaly shuts off. The genes we are trying to regulate
are required for cell viability and just a few copies allow the cell to
retain a wild phenotype.

If you need tight regulation you will not get it in most plasmids as there
will always be some leakage in the promotor. You will need to go to a low
copy number plasmid or even better a single copy in the chromosome (or F'
ect.).

Check out:

Lutz and Bujard, Nuc acids Res, 1997, Vol 25, No. 6 1203-1210

They describe E.coli alternitives that might work in Bacillus.



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