Invitrogen's pZERO & E.coli with ccdA

Mikhail Alexeyev malexe at biost1.thi.tmc.edu
Tue Aug 13 12:12:50 EST 1996


In article <4uq75r$hrh at frazier.backbone.uoknor.edu>,
bsellman at microbiology.bmsb.uokhsc.edu (Bret Sellman) wrote:

> I have been using the pZERO vector from invitrogen with great success. 
> The only problem is that the vector is not cheap to buy and I cannot
> propagate the plasmid without first cutting it and ligating in DNA. 
> Does anyone know how to propagate this vector?  Are there any E. coli
> strains available which overexpress ccdA. Thanks.

In the original article authors used some gyrA (gyrA 401?) mutant to
propagate plasmids (gyrA96 mutation common to many laboratory strains will
not do the job). The article has been published in Gene, so strain should
be available from authors.  I am not aware of any ccdA -overexpressing
strains, but it should be possible to construct one (the gene is encoded
by F plasmid -just PCR it out, place under the strong promoter in the
transposon and shoot it into the chromosome). On the other hand, if lac
promoter is repressed well on the plasmid, one should be able to propagate
plasmid in a strain bearing F'. 
Does Invitrogen still require to sign a peper saying that you are not
going to attempt to propagate vector without an insert? 

M.A.



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