In article <1995Nov21.174013.13424 at alw.nih.gov>, bernard at elsie.nci.nih.gov
(Bernard Murray) wrote:
> Just wondering...
> I hope you are using the bacteria they supply with the vector (one of their
> TOP10 derivatives, I think) as this has the necessary gyr+ phenotype.
> The reason I ask is that one of their reps told me that I could still use
> DH5alpha as for other vectors and was surprised to hear that it wouldn't work.
> As was discussed in the group earlier, most of the normal host strains are
> gyrA96 and will not work with this positive selection protocol.
> If its not as simple as this then I hope you find your answer soon.
> Bernard Murray, Ph.D.
>bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
Dna-gyrase is required for DNA replication, so all common laboratory
E.coli strains should have it. gyrA96 mutation, if I am not mistaken, does
not eliminate gyrase protein, but makes it relatively insensitive to
inhibition by nalidixic acid (similar to rpoB - resistance to rifampicin
inhibition of beta (beta'?) subunit of RNA-polymerase). Vector itself
codes for some sort of DNA-gyrase inhibitor protein, that should be toxic
to cells because of interference with DNA replication. Therefore, vector
cannot be propagated without insert in any common strain (in the presence
of IPTG). Once gene for inhibitor protein is disrupted by insert, plasmid
no longer confers lethal phenotype and can be propagated in any strain.
The 'naked' vector should be propagated in a strain whose DNA-gyrase for
some reason is insensitive to inhibition by plasmid product or has very
efficient suppression of inhibitor protein expression or (?) overexpresses
Regarding previous discussion that I missed: is that true tha gyrA96
mutation makes DNA gyrase less succeptible to inhibition by ccd?